NFATc3 in chronic hypoxic pulmonary hypertension

描述（由申请方提供）：尽管起源于肺动脉高压（PAH），但由于肺血管收缩、动脉重塑和红细胞增多导致肺血管阻力升高，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。PAH发展的复杂过程部分由基因表达的变化驱动。在PAH中，平滑肌细胞内Ca 2+增加，内皮素1（ET-1）表达上调。Ca 2+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。NFATc 3同种型特别涉及血管系统的发育和平滑肌分化表型的维持。该提案的总体目标是确定NFATc 3在与CH-PAH相关的血管变化的分子机制中的作用。假设CH激活PASMC中的NFATc 3介导肥大并增强肺动脉（PA）的收缩力，从而导致PAH。具体目的1：确定NFATc 3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中肥大标志物α-肌动蛋白和肌球蛋白重链的mRNA和蛋白质，NFATc 3结合1-肌动蛋白和肌球蛋白重链启动子，并确定暴露于常氧和低压CH的野生型+/-钙调磷酸酶/NFAT抑制剂和NFATc 3敲除小鼠的肺血管结构变化。确定NFATc 3对CH诱导的Kv通道表达下调和肺血管收缩反应性增加的作用。我们将使用aim 1中提出的相同动物模型，并确定mRNA和蛋白质Kv亚型; NFATc 3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结合; PASMC膜电位和孤立加压PA中激动剂诱导的血管收缩。具体目标3：确定CH增加PASMC中NFATc 3转录活性的机制。我们将使用NFAT-荧光素酶报告小鼠和与NFATc 3 KO小鼠杂交的NFAT-荧光素酶来确定CH-增加的NFAT活性的介质（ET-1、Ca 2+、钙调磷酸酶和Rho-激酶）。从拟议的研究结果将提供新的信息信号机制调节PAH的基因转录的变化。更好地理解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.