Pressure in lung development and congenital diaphragmatic hernia

肺部发育压力与先天性膈疝

• 批准号: 9918958
• 负责人: Jason Paul Gleghorn
• 金额: $ 38.34万
• 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/9918958
• 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.

摘要 先天性横隔性疝气(CDH)是一种破坏性的结构性出生缺陷，导致显著 围产期发病率和死亡率。在CDH中，如果隔膜不能完全关闭，就会导致腹部 器官进入胸腔，压迫发育中的肺，导致通常是致命的 肺发育不全。由于CDH的高发病率和死亡率与结构缺陷(腹部)有关 压迫肺的器官)，没有一致的遗传缺陷，识别靶向的信号通路 在治疗上是很困难的。到目前为止，治疗策略主要集中在外科手术闭塞气管和 增加肺部的液体积累，这与动物模型中肺的加速生长有关。 然而，这些策略对新生儿存活率的改善微乎其微，特别是在光照下。 任何产前手术的风险。成功地将这些发现转化为动物的一个主要挑战 模型对机械信号的理解很差，比如流体引起的压力升高 积聚，被转化为加速的肺生长和分支。这方面的几个方面 机械转导系统已经确定。气道平滑肌(ASM)很早就被认为是 肺的蠕动，这被假设为提供必要的动态刺激来诱导 呼吸道的分支和生长。为了支持这一点，我们最近已经表明，呼吸道压力直接 调节分支事件的时间，这取决于ASM功能。 在这项建议中，我们关注的是下游的分子力学转导途径。 肺部压力。具体地说，我们假设了新的机械转导通路将压力与 肺生长的三个截然不同的方面。首先，我们测试了机械敏感的TRPV4离子通道和 肌球蛋白轻链激酶在连接呼吸道平滑肌功能中的作用。其次，我们测试了TRPV4和K-的作用。 RAS在介导呼吸道上皮细胞增殖和分支中的作用。第三，我们测试了一个正反馈 系统，其中压力激活的Fgf7的表达导致液体分泌增加，并进一步 加压。为了测试这些目的，我们使用了我们的新型微流控培养的小鼠肺的体外培养。 设备，使我们能够直接控制发育中的肺内的压力。此外，我们将聘用 我们建议的通路的药理抑制和激活。为了延长和验证我们的体外实验 此外，我们还将在体外培养模型中使用siRNA和质粒转染法。 通过确定基于压力的肺形态发生的分子机制，这项工作将 为未来的研究提供了一个框架，以探索两个正常肺的中枢机械转导事件 发育和CDH中发生的失调。此外，这项工作将确定潜在的治疗方法 可作为当前外科CDH治疗的辅助或替代的靶点。