Pressure in lung development and congenital diaphragmatic hernia

摘要 先天性腹股沟疝（CDH）是一种毁灭性的结构性出生缺陷， 围产期发病率和死亡率。在CDH中，横膈膜不能完全闭合， 器官进入胸腔，压迫发育中的肺，导致通常致命的 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， 器官压迫肺），没有一致的遗传缺陷，识别信号通路，以靶向 治疗是困难的。迄今为止，治疗策略集中在手术阻塞气管， 增加肺中的液体积聚，这与动物模型中的肺生长加速有关。 然而，这些策略对新生儿存活率的改善微乎其微，特别是在光照条件下。 产前手术的风险成功地将这些发现从动物身上翻译出来的主要挑战是 模型对机械信号的理解很差，例如流体引起的压力升高， 累积，被转换成加速的肺生长和分支。几个方面进行 mechanictransduction系统已经确定。气道平滑肌（ASM）长期以来一直被认为表现出 肺中的肺水肿，并且这已被假设为提供基本的动态刺激以诱导 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。 在这个建议中，我们专注于下游的分子机械转导途径， 肺压具体来说，我们假设新的机械转导途径连接压力， 肺生长的三个不同方面。首先，我们测试了机械敏感性TRPV 4离子通道的作用， 肌球蛋白轻链激酶与气道平滑肌功能的关系其次，我们测试TRPV 4和K- Ras介导气道上皮细胞的增殖和分支。第三，我们测试一个正反馈 系统，其中压力激活的FGF 7表达导致液体分泌增加，并且进一步 加压。为了测试这些目标，我们利用我们的新型微流体培养物对小鼠肺进行离体培养 这是一个新的设备，允许我们直接控制发育中的肺内的压力。此外，我们将采用 药理学抑制和激活我们提出的途径。为了扩展和验证我们的体外 研究结果，我们将另外使用siRNA和质粒转染与体外培养模型。 通过确定压力肺形态发生的分子机制，这项工作将 为将来的研究提供了一个框架，以探索对正常肺和肺组织都至关重要的机械转导事件。 CDH中发生的发育和失调。此外，这项工作将确定潜在的治疗 这些靶点可以被开发作为当前手术CDH治疗的替代或替代。

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.