Mechanisms of Repair of the Alveolar Epithelium after Lung Injury

 描述(申请人提供)：意义。急性呼吸窘迫综合征(ARDS)是导致严重发病率和死亡率的主要原因。除了肺保护性机械通气策略和保守的液体管理外，还没有针对ARDS的特殊治疗方法。ARDS的一个特点是肺泡上皮损伤，包括细胞死亡，主要是肺泡型(AT)I型细胞，导致通透性和EDMA液内流，这反过来又导致难治性低氧血症。这一损伤阶段之后是修复反应，在此期间存活的ATII细胞扩散到剥脱的基底膜上，增殖并转分化为ATI细胞，从而恢复屏障的完整性。重要的是，上皮修复是ARDS康复的主要决定因素。创新。从历史上看，基础的ARDS研究一直集中在损伤阶段；不幸的是，旨在减轻损伤的治疗干预通常都不成功。对修复机制的研究已经探索了ATII细胞的增殖。在这个方案中，我们关注ATII细胞在ATI细胞脱落后扩散到裸露的基底膜上。自20世纪70年代以来，这种现象已经被认识到，但还没有在动物模型中得到很好的研究。我们将扩散定义为表面积的增加，导致裸露的基底膜的覆盖率增加。我们开发了一种新的方法，使用严格的体视学技术严格测量基因标记的ATII细胞的扩散。我们将描述轻度和严重损伤后ATII细胞的扩散与增殖、转分化和屏障完整性恢复的关系。假设。根据我们的初步数据，我们假设缺氧诱导因子1α通过CXCR4/SDF1信号通路促进血管紧张素Ⅱ细胞的扩散和屏障完整性的重建。研究计划。目的1检验在局限性肺损伤中，有丝分裂前ATII细胞的快速扩散是否与屏障重建有关；此外，在严重损伤后，需要增殖来取代丢失的细胞，有丝分裂后扩散是否与屏障重建相关。目标2将确定HIF1α是否对传播至关重要。目的3验证CXCRSDF1信号通路促进和介导HIF1α在血管紧张素Ⅱ细胞扩散中的作用。我们将同时使用获得和丧失功能的技术，包括ATII细胞特异性基因缺陷小鼠。我们的主要读数将是使用体视学技术对扩散(牙槽表面覆盖的面积)进行形态测量。我们将直接将体内研究与原代培养的ATII细胞中的机械扩散试验联系起来。结论。在这里，我们研究通过ATII细胞扩散修复受损的肺泡上皮，重点是恢复上皮屏障的完整性，这是一个高度临床相关的结果。这些研究将确定新的治疗靶点，以加速ARDS的上皮修复，最终促进肺水肿的缓解和临床恢复。

 DESCRIPTION (provided by applicant): Significance. The Acute Respiratory Distress Syndrome (ARDS) is responsible for significant morbidity and mortality. Aside from lung protective mechanical ventilation strategies and conservative fluid management, there are no specific therapies for ARDS. A hallmark of ARDS is injury to the alveolar epithelium, including cell death, primarily of alveolar type (AT) I cells, resulting in permeability and the influx of edma fluid, which in turn leads to refractory hypoxemia. This injury phase is followed by a reparative response during which surviving ATII cells spread onto the denuded basement membrane, proliferate, and transdifferentiate into ATI cells, thus restoring barrier integrity. Importantly, epithelial repair is a primary determinant of recovery from ARDS. Innovation. Historically, basic ARDS research has focused on the injury phase; unfortunately, therapeutic interventions designed to mitigate injury have generally been unsuccessful. Investigations into reparative mechanisms have explored ATII cell proliferation. In this proposal, we focus on ATII cell spreading onto the denuded basement membrane after the sloughing of ATI cells. This phenomenon has been recognized since the 1970s but has not been well studied in animal models. We define spreading as an increase in surface area resulting in increased coverage of the denuded basement membrane. We have developed a novel method to rigorously measure spreading of genetically-labeled ATII cells using stringent stereologic techniques. We will characterize ATII cell spreading after mild and severe injury in relationship to proliferation, transdifferentiation, and the restitution of barrier integrity. Hypothesis. Based on our preliminar data, we hypothesize that Hypoxia Inducible Factor (HIF) 1α promotes ATII cell spreading and the restitution of barrier integrity via CXCR4/SDF1 signaling. Research Plan. Aim 1 will test whether in limited lung injury, rapid, pre-mitotic ATII cell spreading is associated with barrier restoration; in addition, after severe injury, requiring proliferation to replace lost cells, whethr post-mitotic spreading correlates with barrier restoration. Aim 2 will determine whether HIF1α is critical for spreading. Aim 3 will test the hypothesis that CXCR/SDF1 signaling promotes and mediates the role of HIF1α in ATII cell spreading. We will use both gain- and loss-of-function techniques, including ATII cell specific gene deficient mice. Our primary readout will be morphometric measurement of spreading (area of alveolar surface covered) using stereologic techniques. We will directly link in vivo studies to mechanistic spreading assays in cultured primary ATII cells. Conclusion. Here, we study repair of the injured alveolar epithelium by ATII cell spreading with an emphasis on the restitution of epithelial barrier integrity, a highly cliniclly relevant outcome. These studies will identify novel therapeutic targets to accelerate epithelial repair in ARDS, ultimately promoting the resolution of pulmonary edema and clinical recovery.