Angiopoietin/Tie signaling regulation of vascular leakage in lung inflammation

血管生成素/Tie信号通路在肺部炎症血管渗漏中的调节作用 项目总结 作为急性肺损伤和急性呼吸窘迫综合征(ARDS)的中心特征，血管渗漏不是 这不仅会损害气体交换，还会引发一系列可能造成致命后果的事件。不是 目前有治疗方法可通过稳定肺血管来减少渗漏。这个项目将 确定血管生成素-2(Ang2)、孤儿受体Tie1和VE-PTP的作用 血管内皮细胞蛋白酪氨酸磷酸酶对Tie2(Tek)受体信号转导的调节作用 肺损伤时血管通透性增加(“渗漏”)。基于已知的防渗漏效果 血管生成素-1(Ang1)和其他Tie2激动剂，该项目将靶向成分最近描述 控制血管渗漏严重程度的正反馈回路。触发Tie1的炎性细胞因子 内皮细胞受体胞外结构域脱落失活和Tie2信号抑制 增加Foxo1的转录活性和Ang2的表达，从而维持Tie2的失活和泄漏。 目的#1将探索减少肺损伤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Tie2信号的抑制。实验将会 确定Ang2过表达、Tie1胞外结构域脱落和失活以及VE-PTP- 介导Tie2去磷酸化导致肺损伤中Tie2失活和渗漏。遗传学和药理学 将使用增益和功能损失策略来激活Tie2、抑制Ang2、抑制Tie1胞外结构域 在脓毒症的预防和逆转试验中，清除并抑制肺血管中VE-PTP的活性 小鼠感染流感病毒。Tie1、Tie2磷酸化、Ang2、VE-PTP和渗漏将被评估为 读数。渗漏将通过可视化渗出的纤维蛋白原的新方法来定位和测量 单个肺泡毛细血管水平的20 nm荧光微球。目标#2将检查 循环中可溶性Tie1(STie1)水平升高作为血吸虫病生物标志物的功能和预后意义 肺损伤中的内皮功能障碍。对小鼠的研究将确定内皮细胞Tie2信号的变化 导致血液中sTie1水平的增加，以及这些与肺毛细血管渗漏的严重程度之间的关系。 危重病患者的平行研究将探讨血浆sTie1水平升高的临床意义 脓毒症，有或没有ARDS的发展。这项工作将建立在来自试点研究的数据基础上，显示67% 入院时血浆sTie1和Ang2水平升高的23名危重患者的死亡率 STie1和Ang2正常者中仅有8%。总而言之，这些实验将定义 激活Tie2，抑制Ang2，抑制Tie1胞外结构域脱落，抑制VE-PTP， 并将确定sTie1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.