Pressure in lung development and congenital diaphragmatic hernia

肺部发育压力与先天性膈疝

• 批准号: 9311116
• 负责人: Jason Paul Gleghorn
• 金额: $ 36.94万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311116
• 关键词: Abdomen; Address; Affect; Animal Model; Animals; Biophysical Process; Biophysics; Calmodulin; Chest; Coculture Techniques; Congenital Abnormality; Congenital diaphragmatic hernia; Data; Development; Devices; Electrophysiology (science); Engineering; Event; Exhibits; Failure; Feedback; Fetal Lung; Fetal Mortality Statistics; Fluids and Secretions; Future; Grant; Growth; Guanine Nucleotide Exchange Factors; Human; Hypoxemia; In Vitro; Ion Channel; KRAS2 gene; Light; Link; Liquid substance; Live Birth; Lung; Mechanics; Mediating; Microfluidics; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Mus; Muscle Contraction; Muscle function; Mutation; Myosin Light Chain Kinase; Neonatal; Newborn Infant; Operative Surgical Procedures; Organ; Organ Culture Techniques; Pathway interactions; Perinatal mortality demographics; Peristalsis; Pharmacology; Plasmids; Play; Proteins; Pump; Regulation; Replacement Therapy; Respiratory Diaphragm; Respiratory Insufficiency; Risk; Role; Severities; Signal Pathway; Signal Transduction; Small Interfering RNA; Stimulus; Stretching; Structural Congenital Anomalies; Structural defect; Structure of parenchyma of lung; Survival Rate; System; Techniques; Testing; Thoracic cavity structure; Trachea; Transfection; Translating; Work; airway epithelium; base; clinical translation; cost; embryo surgery; fetal; in vitro Model; keratinocyte growth factor; lung basal segment; lung development; lung pressure; malformation; mechanotransduction; medical specialties; mortality; new therapeutic target; novel; perinatal morbidity; pressure; pulmonary hypoplasia; ras Proteins; respiratory smooth muscle; response; stem; success; targeted treatment; therapeutic target; treatment strategy

ABSTRACT Congenital diaphragmatic hernia (CDH) is a devastating structural birth defect, resulting in significant perinatal morbidity and mortality. In CDH, a failure of the diaphragm to completely close allows abdominal organs to move into the thoracic cavity, compressing the developing lung and resulting in often lethal pulmonary hypoplasia. As the high morbidity and mortality of CDH is linked to a structural defect (abdominal organs compressing the lung) with no consistent genetic defect, identifying signaling pathways to target therapeutically is difficult. To date, treatment strategies have focused on surgically occluding the trachea and increasing fluid accumulation in the lung, which has been linked to accelerated lung growth in animal models. However, these strategies have resulted in minimal improvement to neonatal survival rates, especially in light of the risks of any prenatal surgery. A major challenge in successfully translating these findings from animal models is a poor understanding of how mechanical signals, such as the elevated pressure caused by fluid accumulation, are transduced into accelerated lung growth and branching. Several aspects of this mechanotransduction system have identified. Airway smooth muscle (ASM) has been long known to exhibit peristalsis in the lung, and this has been hypothesized to provide an essential dynamic stimulus to induce branching and growth of the airway. In support of this, we have recently shown that airway pressure directly regulates the timing of branching events, and that this depends on ASM function. In this proposal, we focus on the molecular mechanotransduction pathways downstream of lung pressure. Specifically, we hypothesize novel mechanotransduction pathways connecting pressure to three distinct aspects of lung growth. First, we test the role of the mechanosensitive TRPV4 ion channel and myosin light chain kinase in linking airway smooth muscle function. Secondly, we test the role of TRPV4 and K- Ras in mediating the proliferation and branching of the airway epithelium. Third, we test a positive feedback system, where pressure activated expression of FGF7 leads to increased fluid secretion and further pressurization. To test these aims we utilize ex vivo culture of mouse lungs using our novel microfluidic culture device, allowing us to directly control pressures within the developing lung. Further, we will employ pharmacological inhibition and activation of our proposed pathways. To extend and validate our ex vivo findings, we will additionally use siRNA and plasmid transfection with in vitro culture models. By identifying molecular mechanisms that underlie pressure-based lung morphogenesis, this work will provide a framework for future studies to explore mechanotransduction events central to both normal lung development and the dysregulation that occurs in CDH. Further, this work will identify potential therapeutic targets that can be exploited as adjuncts to or replacements for current surgical CDH treatments.

抽象的 先天性膈疝 (CDH) 是一种破坏性的结构性出生缺陷，可导致严重的 围产期发病率和死亡率。在 CDH 中，膈肌无法完全关闭，导致腹部 器官移入胸腔，压迫正在发育的肺部，导致致命的后果 肺发育不全。由于 CDH 的高发病率和死亡率与结构缺陷（腹部 压缩肺部的器官）没有一致的遗传缺陷，识别目标信号通路 治疗上是困难的。迄今为止，治疗策略主要集中在手术阻塞气管和 肺部积液增加，这与动物模型中肺部生长加速有关。 然而，这些策略对新生儿存活率的改善甚微，尤其是在轻度情况下。 任何产前手术的风险。成功地将这些发现从动物身上转化过来是一个重大挑战 模型对机械信号（例如流体引起的压力升高）的理解很差 积累，转化为加速的肺部生长和分支。这几个方面 机械传导系统已确定。众所周知，气道平滑肌 (ASM) 会表现出 肺部蠕动，这被假设提供了必要的动态刺激以诱导 气道的分支和生长。为了支持这一点，我们最近表明气道压力直接 调节分支事件的时间，这取决于 ASM 功能。 在本提案中，我们重点关注下游的分子力转导途径 肺压力。具体来说，我们假设新的机械传导途径将压力与 肺部生长的三个不同方面。首先，我们测试了机械敏感 TRPV4 离子通道的作用， 肌球蛋白轻链激酶连接气道平滑肌功能。其次，我们测试TRPV4和K-的作用 Ras 介导气道上皮的增殖和分支。第三，我们测试正反馈 系统中，压力激活 FGF7 的表达导致液体分泌增加，并进一步 加压。为了测试这些目标，我们利用新型微流体培养物对小鼠肺部进行离体培养 设备，使我们能够直接控制发育中的肺部内的压力。此外，我们将聘请 我们提出的途径的药理学抑制和激活。扩展和验证我们的离体 根据研究结果，我们将另外在体外培养模型中使用 siRNA 和质粒转染。 通过确定基于压力的肺形态发生的分子机制，这项工作将 为未来的研究提供一个框架，探索正常肺的机械转导事件 CDH 中发生的发育和失调。此外，这项工作将确定潜在的治疗方法 可以用作当前 CDH 手术治疗的辅助或替代的目标。