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

摘要/摘要 支气管肺发育不良(BPD)是早产儿最常见的慢性肺部疾病。 这些婴儿的肺动脉高压(PH)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 与BPD和PH的发病机制有关，这是一种没有特殊治疗方法的疾病。肾上腺髓质素(Adrenomedullin) 是一种内源性多肽，调节血管生成和内皮细胞的生存和功能，使这一点 多肽是开发这种疾病治疗方法的理想靶点。AM通过其同源受体发出信号， 降钙素受体样受体(Calcr1)和受体活性修饰蛋白(RAMP)-2。最新研究 提示AM信号在新生啮齿动物肺发育和减轻肺损伤中是必需的。 然而，AM是否能改善BPD相关的肺和肺血管功能障碍尚不清楚。 动物。此外，AM信号保护新生儿肺损伤的细胞和分子机制 受伤情况尚不清楚。因此，我们建议使用一只已建立的新生小鼠来解决这些知识差距 高氧性肺损伤模型。我们的初步研究表明AM信号对健康的肺是必要的 发展和减轻高氧诱导的新生小鼠肺损伤。此外，我们观察到AM 调节细胞外信号调节激酶(ERK)1/2的激活和内皮型一氧化氮合酶 内皮型一氧化氮合酶(ENOS)在新生小鼠肺组织和胎鼠肺内皮细胞中的表达。基于 这些数据，我们将检验中心假设，内皮特异性AM信号激活ERK1/2和 ENOS促进新生肺血管生成和防止内皮细胞功能障碍，进而 减轻高氧诱导的实验性BPD和PH。我们将使用独特的分子、细胞、 功能和翻译方法来检验这一假说。在目标1中，我们将使用转基因小鼠 确定内皮细胞特异性AM信号是否必要且足以保护新生小鼠免受 高氧性肺和肺血管损伤及功能障碍。在目标2中，我们将使用双转基因 小鼠研究AM、ERK 1/2信号和eNOS在发育肺中的相互作用 暴露在高氧环境中。目标3有两个子目标，旨在审查我们的翻译潜力 求婚。在子目标1中，我们将检查AM信号是否调节高氧诱导的人肺损伤 内皮细胞。在次目标2中，我们将测定AM及其受体在婴儿肺中的表达 有BPD和没有BPD。我们预计，这些研究的成功完成将提供一种机制 以AM、Calcr1或RAMP2为靶点开发有意义的BPD和PH疗法的理论基础。此外，这些 研究可为黄连素治疗BPD合并PH的临床试验提供科学前提。我们的研究 也可能对其他与血管生成和PH相关的研究领域产生积极影响，如先天性 横隔疝和先天性肺发育不全。

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.