Hypoxia-mediated protective estrogen receptor signaling in pulmonary hypertension

缺氧性肺动脉高压是退伍军人慢性肺功能不全的严重临床问题 疾病17 β-雌二醇（E2）可减轻HPH，但其机制尚不清楚。我们的初步 数据表明，1）E2的抗增殖作用仅发生在实际或化学缺氧期间， 2)缺氧上调肺动脉内皮细胞雌激素受体转录和/或表达 细胞（PAEC）和右心室（RV）。我们假设E2减弱的机制 PH的发展和改善RV功能是通过缺氧和缺氧诱导因子（HIF）1 α- 能够增加PA-RV单位中的ER表达，随后诱导细胞自噬， 改善RV线粒体生物合成。由于HPH和非缺氧形式的PH中的RV衰竭， 其特征是全球或局部（细胞）缺氧与已知的HIF-1 α诱导剂的激活，我们提出， E2-ER轴对缺氧诱导的（适应性）RV重塑的RV保护作用将延长 以适应不良重塑为特征的RV衰竭。我们的机械实验方法利用 体内全面的临床相关PH终点，由原发性PAEC研究补充。我们 提出以下具体目标： SA#1：确定增强ER信号传导是否可防止PH的发展。 图1a：为了确定缺氧和HIF 1 α诱导的ER表达增加的模式和时间过程， 肺血管和RV。 1b：研究ER信号的转基因条件性组织特异性增强是否增强E2的表达。 在HPH中甚至在PH的非缺氧形式中的保护作用。 1c：为了询问增强ER信号传导是否足以保护免受HPH或非缺氧PH， 在缺乏外源性E2进一步刺激的情况下， SA #2：确定E2抑制RV重塑和改善RV功能的机制， 适应性和适应不良的RV反应。 2a：阐明E2是否通过涉及ER ²依赖性的机制来防止病理性RV重塑 适应性和适应不良RV肥大模型中的自噬。 2b：研究E2是否通过优化线粒体底物利用来改善RV功能， 适应性和适应不良的RV反应。 体内实验将在两种不同的PH啮齿动物模型中进行：a）HPH和B）VEGF受体 阻断加缺氧诱导的PH。我们将主要采用转基因小鼠模型， 实验，并使用大鼠进行需要功能终点综合分析的研究。端点 体内评估包括血流动力学、超声心动图RV形式/功能、运动能力和PA/RV 重塑，辅以ER表达，细胞增殖， 存活、凋亡和自噬，以及HIF-1 α激活和线粒体底物利用。的 提出的研究是新颖的，因为它们将1）首次建立ER保护机制， 心肺系统; 2）是第一个研究，以扩大E2的保护作用，以更严重的 RV失败的形式;和3）证实了自噬和线粒体的关键作用的新认识 在失败的RV中ER介导的保护中的底物利用。所提出的研究是重要的，因为 1)这些结果将有助于识别针对晚期RV衰竭的新治疗靶点; 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， 有利于男性和女性退伍军人与PH;和3）结果可以解释为什么女性更容易 特发性肺动脉高压发展，但一旦受到影响，疾病严重程度较低。

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.