Potential Protective Mechanisms of Tissue Factor in Acute Lung Injury

急性呼吸窘迫综合征（ARDS）是急性呼吸衰竭的常见原因， 死亡率高，没有有益的靶向治疗。这是一个紧迫的问题，退伍军人和目前的 估计表明每年死于ARDS的退伍军人比死于肺癌的退伍军人多。尽管 目前还没有治疗ARDS的药物。需要对发病机制有新的认识， 加深我们对导致ARDS的潜在机制的理解， 治疗学ARDS的一个主要病理特征是组织因子（TF）途径的激活， 凝固在空气中。TF是一种整合的膜蛋白，启动外源性凝血， 级联并提供几种非凝血功能，包括通过 与整合素蛋白的相互作用。几十年来，促凝血途径，包括TF途径，一直是 与ARDS损伤机制有关。然而，一些抗凝剂的临床试验还没有 显示了这种方法的好处。一个可能的解释是，TF在空气中是保护性的ARDS。 我们初步的体内和体外数据表明，肺上皮细胞的损失，但不是巨噬细胞，TF引起的损失 上皮屏障完整性、细胞表面β1整合素减少和细胞粘附异常。相反地， 肺泡上皮II型细胞中TF的过表达恢复屏障完整性。此外，治疗与 重组因子VII（rFVII），主要的TF配体，进入空气空间减弱小鼠的渗透性 ARDS模型。总之，这些观察结果代表了对TF作用的理解的重大转变 这是一种几乎普遍被认为有害的途径。本提案的目标 是为了检验新的假设，即（1）肺上皮TF的丧失导致上皮屏障完整性的丧失， 通过破坏整合素蛋白和（2）在空气空间中上调TF维持上皮屏障 在直接肺损伤模型中的完整性。因此，rFVII向空域的局部递送可以代表独特的 预防和治疗ARDS的治疗方法。使用新的转基因小鼠， 上皮II型细胞特异性缺失或过度表达TF，我们将定义体内细胞特异性机制 TF对肺上皮屏障完整性的影响。我们还将使用一种新的TF敲除肺上皮细胞系， 研究TF与β1整合素之间的分子相互作用及TF在调节上皮屏障中的作用 通过表达新型TF突变体文库来实现完整性。最后，我们将使用一个临床相关的人类模型， ARDS（离体灌注的人肺）以测试rFVII收紧肺上皮屏障的治疗潜力。 人类ARDS。我们的实验设计是机械和翻译，并利用新的转基因 小鼠模型、细胞系以及人肺损伤模型，以了解TF在 以及测试rFVII的治疗潜力。这项提案利用了 以及范德比尔特现有的资源，包括最先进的计算建模和粘附力 复杂的动力学测量，从而确保这些研究的结果将推动该领域的发展， 为退伍军人ARDS的靶向治疗提供必要的基础。

Acute Respiratory Distress Syndrome (ARDS) is a common cause of acute respiratory failure that carries a high mortality rate and has no beneficial targeted therapies. It is a pressing problem for Veterans and current estimates suggest that more Veterans will die from ARDS each year than will die from lung cancer. Despite this, there are no pharmacologic therapies for ARDS. New insights into the pathogenesis are needed to deepen our understanding of the underlying mechanisms that lead to ARDS as well as to develop novel therapeutics. A major pathologic feature of ARDS is activation of the Tissue Factor (TF) pathway of coagulation in the airspace. TF is an integral membrane protein that both initiates the extrinsic coagulation cascade and serves several non-coagulant functions including promoting cell adhesion and migration through interactions with integrin proteins. For decades, procoagulant pathways, including the TF pathway, have been implicated as a mechanism of injury in ARDS. However, a number of clinical trails of anti-coagulants have not shown a benefit with this approach. One potential explanation is that TF in the airspace is protective in ARDS. Our preliminary in vivo and in vitro data show that loss of lung epithelial, but not macrophage, TF causes loss of epithelial barrier integrity, decreased cell surface β1 integrin and abnormal cell adhesion. Conversely, overexpression of TF in alveolar epithelial type II cells restores barrier integrity. In addition, treatment with recombinant factor VII (rFVII), the primary TF ligand, into the airspace attenuates permeability in a mouse model of ARDS. Together, these observations represent a major shift in understanding the role of the TF pathway in ARDS, a pathway that has almost universally been considered harmful. The goals of this proposal are to test the novel hypotheses that (1) loss of lung epithelial TF leads to loss of epithelial barrier integrity through disruption of integrin proteins and (2) upregulation of TF in the airspace maintain epithelial barrier integrity in a direct lung injury model. Therefore, local delivery of rFVII to the airspace could represent a unique therapeutic approach for the prevention and treatment of ARDS. Using novel transgenic mice with alveolar epithelial type II cell specific deletion or overexpression of TF we will define the in vivo cell specific mechanism of TF effects on lung epithelial barrier integrity. We will also use a novel TF knockout lung epithelial cell line to study the molecular interactions between TF and β1 integrin and the role of TF in regulating epithelial barrier integrity by expressing a library of novel TF mutants. Finally we will use a clinically relevant human model of ARDS (ex vivo perfused human lung) to test the therapeutic potential of rFVII to tighten the epithelial barrier in human ARDS. Our experimental design is both mechanistic and translational and utilizes novel transgenic mouse models, cell lines as well a human lung injury model to both understand the mechanism of TF effects in the lung as well as to test the therapeutic potential of rFVII. This proposal takes advantage of the unique skills and resources available at Vanderbilt including state of the art computational modeling as well as adhesion complex dynamics measurements thus ensuring that results from these studies will advance the field and provide the necessary foundation for development of targeted therapeutics for Veterans with ARDS.