Angiopoietin/Tie signaling regulation of vascular leakage in lung inflammation

Angiopoietin/Tie信号通路对肺部炎症血管渗漏的调控 项目摘要 作为急性肺损伤和急性呼吸窘迫综合征（ARDS）的一个中心特征，血管渗漏并不 不仅损害气体交换，而且还引发可能具有致命后果的级联事件。没有 目前可以通过稳定肺脉管系统来减少渗漏。该项目将 确定血管生成素-2（Ang 2）、孤儿受体Tie 1和VE-PTP（也称为 血管内皮蛋白酪氨酸磷酸酶，调节Tie 2（Tek）受体信号传导， 肺损伤中增加的血管渗透性（“渗漏”）。基于已知的防渗漏效果， 血管生成素-1（Ang 1）和其他Tie 2激动剂，该项目将针对最近描述的 控制血管渗漏严重程度的正反馈回路。触发Tie 1的炎性细胞因子 内皮细胞中的受体胞外域脱落和失活以及Tie 2信号传导抑制也 增加Foxo 1转录活性和Ang 2表达，其维持Tie 2失活和渗漏。 目标1将探索减少肺损伤中渗漏和促进血管稳定性的新方法， 逆转肺泡毛细血管内皮细胞中Tie 2信号传导的抑制。实验将 确定Ang 2过表达、Tie 1胞外域脱落和失活以及VE-PTP- 介导的Tie 2去磷酸化导致肺损伤中Tie 2失活和渗漏。遗传学和药理学 将使用功能获得和丧失策略来激活Tie 2、抑制Ang 2、抑制Tie 1胞外域 在脓毒症的预防和逆转试验中， 小鼠流感病毒感染。Tie 1、Tie 2磷酸化、Ang 2、VE-PTP和渗漏将评估为 读数渗漏将通过新的方法进行定位和测量，以可视化外渗的纤维蛋白原 以及在单个肺泡毛细血管水平上的20-nm荧光微球。目标#2将检查 可溶性Tie 1（sTie 1）循环水平升高的功能和预后意义， 肺损伤中的内皮功能障碍。在小鼠中的研究将确定内皮细胞Tie 2信号传导的变化， 导致血液中sTie 1水平升高，以及这些与肺毛细血管渗漏严重程度的关系。 对危重患者的平行研究将探讨sTie 1血浆水平升高在危重患者中的临床意义。 脓毒症，伴有或不伴有ARDS的发展。这项工作将建立在试点研究的数据基础上， 23例入院时血浆sTie 1和Ang 2水平升高的重症患者的死亡率， sTie 1和Ang 2正常者仅为8%。总之，这些实验将确定 在肺损伤中激活Tie 2、抑制Ang 2、抑制Tie 1胞外域脱落和抑制VE-PTP， 并将确定sTie 1作为ARDS预后生物标志物的临床意义。

Angiopoietin/Tie signaling regulation of vascular leakage in lung inflammation PROJECT SUMMARY As a central feature of acute lung injury and acute respiratory distress syndrome (ARDS), vascular leakage not only impairs gas exchange but also initiates a cascade of events that can have fatal consequences. No treatments are currently available to reduce leakage by stabilizing the lung vasculature. This project will determine the contributions of angiopoietin-2 (Ang2), orphan receptor Tie1, and VE-PTP, also known as vascular endothelial protein-tyrosine phosphatase, to the regulation of Tie2 (Tek) receptor signaling and increased vascular permeability (“leakage”) in lung injury. Building on known anti-leakage effects of angiopoietin-1 (Ang1) and other Tie2 agonists, the project will target components of a recently described positive feedback loop that governs the severity of vascular leakage. Inflammatory cytokines that trigger Tie1 receptor ectodomain shedding and inactivation and Tie2 signaling suppression in endothelial cells also increase Foxo1 transcriptional activity and expression of Ang2, which sustains Tie2 inactivation and leakage. Aim #1 will explore new approaches for reducing leakage and promoting vascular stability in lung injury by reversing the suppression of Tie2 signaling in endothelial cells of alveolar capillaries. Experiments will determine the contributions of Ang2 overexpression, Tie1 ectodomain shedding and inactivation, and VE-PTP- mediated Tie2 dephosphorylation to Tie2 inactivation and leakage in lung injury. Genetic and pharmacologic gain- and loss-of-function strategies will be used to activate Tie2, inhibit Ang2, suppress Tie1 ectodomain shedding, and inhibit VE-PTP activity in the lung vasculature in prevention and reversal trials of sepsis and influenza virus infection in mice. Tie1, Tie2 phosphorylation, Ang2, VE-PTP, and leakage will be assessed as readouts. Leakage will be localized and measured by novel methods for visualizing extravasated fibrinogen and 20-nm fluorescent microspheres at the level of individual alveolar capillaries. Aim #2 will examine the functional and prognostic significance of elevated circulating levels of soluble Tie1 (sTie1) as a biomarker of endothelial dysfunction in lung injury. Studies in mice will identify changes in endothelial cell Tie2 signaling that lead to increased blood levels of sTie1 and how these relate to the severity of leakage from lung capillaries. Parallel studies of critically ill patients will probe the clinical significance of elevated plasma levels of sTie1 in sepsis, with or without the development of ARDS. The work will build on data from pilot studies showing 67% mortality in 23 critically ill patients with elevated plasma levels of sTie1 and Ang2 at admission, compared to only 8% in those with normal sTie1 and Ang2. Together, the experiments will define the functional benefit of activating Tie2, inhibiting Ang2, suppressing Tie1 ectodomain shedding, and inhibiting VE-PTP in lung injury, and will determine the clinical significance of sTie1 as a prognostic biomarker in ARDS.