Mechanistic Roles of Adrenomedullin and its Signaling Receptors in Experimental Bronchopulmonary Dysplasia and Pulmonary Hypertension

摘要/概要 支气管肺发育不良（BPD）是早产儿最常见的慢性肺部疾病; 肺动脉高压（PH）的发生增加了BPD相关的死亡率和发病率。中断 血管生成和肺泡化、内皮细胞功能障碍和肺血管重塑 BPD和PH的发病机制，一种没有特异性治疗方法的疾病。肾上腺髓质素（AM） 是一种调节血管生成和内皮细胞存活和功能的内源性肽， 肽是开发治疗这种疾病的理想靶点。AM通过其同源受体发出信号， 降钙素受体样受体（Calcrl）和受体活性修饰蛋白（RAMP）-2。最近的研究 表明AM信号传导对于新生啮齿动物肺发育和改善肺损伤是必需的。 然而，目前尚不清楚AM是否能改善这些患者中BPD相关的肺和肺血管功能障碍， 动物此外，AM信号传导保护新生儿肺损伤的细胞和分子机制， 受伤情况不明。因此，我们建议使用已建立的新生小鼠来解决这些知识缺口 高氧肺损伤模型。我们的初步研究表明，AM信号是健康肺所必需的 发育和减轻新生小鼠中高氧诱导的肺损伤。此外，我们还观察到， 调节细胞外信号调节激酶（ERK）1/2激活和内皮型一氧化氮合酶 在新生小鼠肺和胎儿人肺内皮细胞中的eNOS表达。基于 通过这些数据，我们将检验内皮特异性AM信号转导激活ERK 1/2和 eNOS促进新生儿肺血管生成和防止内皮细胞功能障碍，这反过来将 减轻高氧诱导的实验性BPD和PH。我们将使用分子，细胞， 功能和翻译的方法来测试这一假设。在目标1中，我们将使用转基因小鼠， 确定内皮特异性AM信号传导是否是保护新生小鼠免受 高氧诱导肺和肺血管损伤和功能障碍。在目标2中，我们将使用双转基因 小鼠来检测AM、ERK 1/2信号传导和eNOS活性在发育中的肺中的相互作用 暴露在高氧环境中目标3有两个子目标，旨在检查我们的翻译潜力。 提议在子目标1中，我们将研究AM信号是否调节高氧诱导的人肺损伤。 内皮细胞在子目标2中，我们将确定AM及其受体在婴儿肺中的表达 不管有没有波士顿警局我们希望这些研究的成功完成将提供一个机制， 本文描述了靶向AM、Calcrl或RAMP 2以开发针对BPD和PH的有意义的疗法的基本原理。 本研究为AM治疗BPD合并PH患者的临床试验提供了科学依据。 也可能对其他血管生成和PH相关的研究领域产生积极影响，例如先天性 疝与先天性肺发育不全。

Abstract/Summary Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants; development of pulmonary hypertension (PH) in these infants increases BPD-associated mortality and morbidity. Interrupted angiogenesis and alveolarization, endothelial cell dysfunction, and pulmonary vascular remodeling contribute to the pathogenesis of BPD and PH, a disease for which there are no specific therapies. Adrenomedullin (AM) is an endogenous peptide that regulates angiogenesis and endothelial cell survival and function, making this peptide an ideal target to develop therapies for this disease. AM signals through its cognate receptors, calcitonin-receptor like receptor (Calcrl) and receptor activity-modifying protein (RAMP)-2. Recent studies indicate that AM signaling is necessary for lung development and to ameliorate lung injury in neonatal rodents. However, it is unclear if AM improves BPD-associated lung and pulmonary vascular dysfunction in these animals. Further, the cellular and molecular mechanisms by which AM signaling protects against neonatal lung injury are unknown. So, we propose to address these knowledge gaps using an established neonatal mouse model of hyperoxic lung injury. Our preliminary studies indicate that AM signaling is necessary for healthy lung development and to mitigate hyperoxia-induced lung injury in neonatal mice. Further, we observed that AM regulates extracellular signal-regulated kinase (ERK) 1/2 activation and endothelial nitric oxide synthase (eNOS) expression in the lungs of neonatal mice and in fetal human pulmonary endothelial cells. Based on these data, we will test the central hypothesis that endothelial-specific AM signaling activates ERK 1/2 and eNOS to promote angiogenesis and prevent endothelial cell dysfunction in neonatal lungs, which in turn will mitigate hyperoxia-induced experimental BPD and PH. We will use a unique combination of molecular, cellular, functional, and translational approaches to test this hypothesis. In Aim 1, we will use transgenic mice to determine if endothelial-specific AM signaling is necessary and sufficient to protect neonatal mice against hyperoxia-induced lung and pulmonary vascular injury and dysfunction. In Aim 2, we will use double transgenic mice to examine the interactions between AM, ERK 1/2 signaling, and eNOS activity in the developing lungs exposed to hyperoxia. Aim 3, which has two sub-aims, is designed to examine the translational potential of our proposal. In sub-aim 1, we will examine if AM signaling regulates hyperoxia-induced injury in human pulmonary endothelial cells. In sub-aim 2, we will determine the expression of AM and its receptors in the lungs of infants with and without BPD. We expect that successful completion of these studies would provide a mechanistic rationale for targeting AM, Calcrl, or RAMP2 to develop meaningful therapies for BPD and PH. Further, these studies could provide a scientific premise for clinical trials with AM to treat BPD patients with PH. Our studies could also positively impact other angiogenesis- and PH-related research areas, such as congenital diaphragmatic hernia and congenital lung hypoplasia.