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，它携带 死亡率高，而且没有有益的靶向治疗。这是退伍军人和当代人面临的紧迫问题 据估计，每年死于ARDS的退伍军人比死于肺癌的退伍军人更多。尽管 这个，没有治疗ARDS的药物疗法。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，以及开发新的 治疗学。ARDS的一个主要病理特征是组织因子(TF)途径的激活 空域内凝结。Tf是启动外源性凝血的一种完整的膜蛋白 级联作用，具有多种非凝血功能，包括通过 与整合素蛋白的相互作用。几十年来，促凝血途径，包括转铁蛋白途径，一直是 被认为是ARDS的一种损伤机制。然而，许多抗凝血剂的临床试验并没有 显示了这种方法的好处。一种可能的解释是，空域中的转运蛋白在ARDS中具有保护性。 我们在体内和体外的初步数据表明，肺上皮细胞的丢失，而不是巨噬细胞，转铁蛋白的丢失导致了丢失 上皮屏障完整性降低，细胞表面β-1整合素减少，细胞黏附异常。相反， 肺泡II型上皮细胞过度表达转铁蛋白可恢复肺屏障的完整性。此外，还可以使用 重组因子VII(RFVII)是主要的转铁蛋白配体，进入空气中可减弱小鼠的通透性 ARDS的模型。总而言之，这些观察结果代表着对工作组作用的理解发生了重大转变 ARDS中的一种途径，一种几乎普遍被认为有害的途径。这项提案的目标是 是为了检验新的假设，即(1)肺上皮细胞因子的丢失会导致上皮屏障完整性的丧失 通过破坏整合素蛋白和(2)上调Tf维持上皮屏障 在直接肺损伤模型中的完整性。因此，本地向空域交付rfvii可能代表着一种独特的 ARDS防治的治疗途径。利用新型肺泡转基因小鼠 上皮型II型细胞特异性缺失或过度表达转铁蛋白我们将确定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型的TF基因敲除的肺上皮细胞株来 Tf与β-1整合素的分子相互作用及其在调节上皮屏障中的作用 通过表达新的Tf突变体文库来整合。最后，我们将使用一个与临床相关的人类模型 ARDS(体外灌流人肺)，以测试rFVII对血管内皮细胞屏障的治疗作用 人类ARDS。我们的实验设计既是机械式的，也是平移式的，并使用了新的转基因技术 小鼠模型、细胞系以及人肺损伤模型，以了解TF对大鼠肺损伤的作用机制 并测试rFVII的治疗潜力。这项提议充分利用了 和范德比尔特可用的资源，包括最先进的计算建模和粘合 复杂的动力学测量，从而确保这些研究的结果将推动领域和 为发展针对患有ARDS的退伍军人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.