Hypoxia-mediated protective estrogen receptor signaling in pulmonary hypertension

低氧所致的肺动脉高压(HPH)是退伍军人慢性肺病的严重临床问题。 疾病。17β-雌二醇(E_2)可减轻HPH，但其机制尚不清楚。我们的预赛 资料表明：1)E_2‘S的抗增殖作用仅在实际缺氧或化学缺氧时发生； 2)低氧上调肺动脉内皮细胞雌激素受体(ER)转录和/或表达 细胞(PAECs)和右室(RV)。我们假设雌二醇衰弱的机制是 PH的形成和改善RV功能是通过低氧和低氧诱导因子(HIF)1α- 使PA-RV单位ER表达增加，随后诱导细胞自噬和 改善轮状病毒线粒体的生物发生。由于在HPH和非缺氧性PH中RV衰竭都是 以整体或局部(细胞)缺氧为特征，并激活已知的HIF-1α诱导剂，我们建议 E_2-ER轴对低氧诱导的(适应性)RV重构的RV保护作用将延长 以不适应重塑为特征的右室衰竭。我们的机械实验方法利用 综合体内临床相关的PH终点，辅以对原发PAECs的研究。我们 提出以下具体目标： SA#1：确定增强ER信号是否可以预防PH的发生。 1A：建立缺氧和HIF1β诱导的大鼠脑内ER表达增加的模式和时间进程 肺血管和RV。 1B：研究转基因条件下组织特异性增强ER信号是否增强E_2‘S 在HPH中的保护作用，甚至在非缺氧性PH中。 1C：询问增强ER信号是否足以保护HPH或非缺氧性PH 在没有外源E2进一步刺激的情况下 SA#2：建立E2抑制RV重塑和改善RV功能的机制 PH患者的适应性和非适应性RV反应。 2A：阐明E_2是否通过ER依赖的机制预防病理性RV重构 适应性和非适应性RV肥厚模型中的自噬。 2B：研究雌激素2是否通过优化线粒体底物利用来改善RV功能 PH患者的适应性和非适应性RV反应。 体内实验将在两种不同的高血压啮齿动物模型上进行：a)高血压和b)血管内皮生长因子受体 阻断加低氧诱导的PH。我们将主要使用转基因小鼠模型来研究机械 实验，并使用大鼠进行需要对功能终点进行全面分析的研究。端点 活体评估包括血流动力学、超声心动图右室形态/功能、运动能力和PA/RV 重建，辅之以体内和体外ER表达、细胞增殖、 存活、凋亡和自噬，以及HIF-1α的激活和线粒体底物的利用。这个 提出的研究具有新颖性，因为它们将1)首次建立内质网保护机制 心肺系统；2)首次研究将E2的保护作用扩大到更严重 RV失败的形式；以及3)证实了对自噬和线粒体关键作用的新认识 底物在失败房车内质网介导的保护中的利用。提出的研究具有重要意义，因为 1)结果将有助于确定针对晚期RV衰竭的新的治疗靶点； 2)这项工作将是朝着我们开发有针对性的非激素疗法的长期目标迈出的关键一步 对患有PH的男性和女性退伍军人有利；3)这一结果可能解释了为什么女性更倾向于 特发性肺动脉高压的发展，但一旦受到影响，表现出较轻的疾病。

Hypoxia-induced pulmonary hypertension (HPH) is a serious clinical problem in veterans with chronic lung disease. 17beta-estradiol (E2) attenuates HPH, but the mechanisms are poorly understood. Our preliminary data demonstrate that 1) E2's anti-proliferative effects occur exclusively during actual or chemical hypoxia and 2) hypoxia upregulates estrogen receptor (ER) transcription and/or expression in pulmonary artery endothelial cells (PAECs) and the right ventricle (RV). We hypothesize that the mechanism through which E2 attenuates PH development and improves RV function is through hypoxia- and hypoxia-inducible factor (HIF) 1alpha- enabled increases in ER expression in the PA-RV unit, with subsequent induction of cellular autophagy and improved RV mitochondrial biogenesis. Since RV failure in both HPH as well as non-hypoxic forms of PH is characterized by global or local (cellular) hypoxia with activation of known HIF-1alpha inducers, we propose that the RV-protective effects of the E2-ER-axis against hypoxia-induced (adaptive) RV remodeling will extend to RV failure characterized by maladaptive remodeling. Our mechanistic experimental approach utilizes comprehensive clinically relevant PH endpoints in vivo, complemented by studies in primary PAECs. We propose the following specific aims: SA#1: To determine if enhancing ER signaling protects against development of PH. 1a: To establish the pattern and time course of hypoxia- and HIF1¿-induced increases in ER expression in the pulmonary vasculature and RV. 1b: To investigate if transgenic conditional tissue-specific enhancement of ER signaling potentiates E2's protective effects in HPH and even in non-hypoxic forms of PH. 1c: To interrogate whether enhancing ER signaling is sufficient to protect against HPH or non-hypoxic PH even in the absence of further stimulation by exogenous E2 SA#2: To establish the mechanisms by which E2 inhibits RV remodeling and improves RV function during both adaptive and maladaptive RV responses in PH. 2a: To elucidate if E2 protects against pathological RV remodeling by a mechanism involving ER¿-dependent autophagy in both adaptive and maladaptive RV hypertrophy models. 2b: To investigate if E2 improves RV function by optimizing mitochondrial substrate utilization during both adaptive and maladaptive RV responses in PH. In vivo experiments will be performed in two distinct rodent models of PH: a) HPH and b) VEGF-receptor blockade plus hypoxia-induced PH. We will primarily employ transgenic mouse models for mechanistic experiments, and use rats for studies requiring comprehensive analysis of functional endpoints. Endpoints assessed in vivo include hemodynamics, RV form/function by echocardiography, exercise capacity, and PA/RV remodeling, complemented by in vivo and in vitro measurements of ER expression, cellular proliferation, survival, apoptosis and autophagy, as well as HIF-1alpha activation and mitochondrial substrate utilization. The proposed studies are novel because they will 1) for the first time establish the mechanisms of ER protection in the cardiopulmonary system; 2) be the first investigations to extend the protective effects of E2 to more severe forms of RV failure; and 3) substantiate the novel appreciation of the key role of autophagy and mitochondrial substrate utilization in ER-mediated protection in the failing RV. The studies proposed are significant because 1) the results will facilitate the identification of new therapeutic targets directed at advanced forms of RV failure; 2) the work will be a critical step towards our long-term goal of developing targeted non-hormonal therapies to benefit male and female veterans with PH; and 3) the results may explain why women are more prone to idiopathic pulmonary arterial hypertension development, yet - once affected - exhibit less severe disease.