Molecular regulation of the capillary barrier in acute critical illness

项目摘要 毛细血管渗漏在先前健康的儿童中发生，与休克的初始阶段同时发生， 心脏骤停毛细血管渗漏导致血流动力学不稳定、器官功能障碍，并最终增加 发病率和死亡率。尽管有这样的临床重要性，但没有已知的治疗或逆转的疗法 毛细血管渗漏，因为其潜在机制尚不清楚。重要器官中的毛细血管内皮细胞（EC） 通过细胞间紧密连接（TJ）的形成，形成一个连续的，选择性透过的屏障， 并精确调节胞吞转运。毛细血管泄漏是由于上述一个或两个因素的破坏造成的 流程.然而，即使如此，跨细胞渗漏和旁细胞渗漏的相对贡献也没有建立。 我们的首要假设是，虽然临床损伤产生失代偿性休克可能是可变的， 它们是多余的，它们汇聚在一起，激活EC中的最终共同机制，这些机制可以有针对性地预防或逆转 毛细血管渗漏在心脏骤停的休克初始状态下，信号的这种冗余解释了缺乏 从针对个体介质的临床获益。我们的假设是通过比较 从一般健康儿童收集的单个EC的转录谱与在早期阶段的那些相比， 休克与心脏骤停，确定候选分子在培养的微血管EC负责 用于产生跨细胞或旁细胞渗漏的特定结构变化。我们将测试这些功能 在由正常供体（男性和女性）人微血管EC组成的培养模型中， 使用跨内皮电阻和大分子通量测定， 形态学分析、分子工程和免疫化学工具。在目标1中，我们将确定 小GTP酶及其调节剂在从早期休克儿童分离的EC中增加， 心脏骤停：ArhGEF 12，15，ArhGAP 21，26和RhoA-C，J，U。我们利用肿瘤坏死因子（TNF）诱导 健康供体真皮毛细血管EC中的细胞旁渗漏伴TJ破坏。我们还将调查 福莫特罗抑制TNF诱导渗漏的机制。在目标2中，我们研究了抑瘤素-m（OSM）， 在我们的转录组学分析中受体和下游信号分子也被上调， 在我们的模型中诱导跨细胞泄漏而不干扰TJ。具体来说，我们将测试OSM的假设， 激活JAK/STAT/p38-MAPK信号传导，导致AP-1依赖性基因表达增加， 增加囊泡运输。我们还将探讨如何行动的OSM可能被抑制 福莫特罗最后，在目标3中，我们将确定目标1和2的结果是否重现完整血管 使用离体灌注的人器官和在体内用移植有人的免疫缺陷小鼠的网络 皮肤成功完成拟议的研究可以从根本上推进我们对如何 毛细血管渗漏发生在严重休克的早期阶段，并评估针对内皮的治疗 以防止或逆转渗漏及其病理后果。

Project Summary Capillary leak develops in previously healthy children concurrent with the initial phase of shock associated with cardiac arrest. Capillary leak contributes to hemodynamic instability, organ dysfunction and ultimately, increased morbidity and mortality. Despite such clinical importance, there are no known therapies to treat or reverse capillary leak because the underlying mechanisms are unknown. Capillary endothelial cells (ECs) in vital organs form a continuous, permselective barrier through the formation of intercellular tight junctions (TJs) that control paracellular flux and precisely regulate transcytosis. Capillary leak results from disruption of one or both of these processes. However, even so much as the relative contributions of trans- and paracellular leak is not established. Our overarching hypothesis is that while clinical insults producing decompensated shock may be variable and redundant, they converge to activate final common mechanisms in ECs that can be targeted to prevent or reverse capillary leak. Such redundancy in signaling in the initial state of shock with cardiac arrest accounts for the lack of clinical benefits from targeting individual mediators. Our hypothesis is supported by comparing the transcriptional profiles of single ECs collected from generally healthy children vs. those in the early stage of shock associated with cardiac arrest, identifying candidate molecules in cultured microvascular EC responsible for specific structural changes producing either trans- or paracellular leak. We will test the functions of these molecules in culture models consisting of normal donor (both male and female) human microvascular ECs from skin and lung that form TJs, using transendothelial electrical resistance and macromolecular flux assays, morphological analyses, molecular engineering, and immunochemical tools. In Aim 1, we will determine the role of small GTPases and their regulators that are increased in ECs isolated from children in early-stage shock with cardiac arrest: ArhGEF12,15, ArhGAP21,26, and RhoA-C,J,U. We utilize tumor necrosis factor (TNF) to induce paracellular leak with disruption of TJs in healthy donor dermal capillary ECs. We will also investigate the mechanism by which formoterol inhibits TNF-induced leak. In Aim 2, we investigate how oncostatin-m (OSM), for which the receptor and downstream signaling molecules are also upregulated in our transcriptomic analyses, induces transcellular leak without perturbing TJs in our models. Specifically, we will test the hypothesis that OSM activates JAK/STAT/p38-MAPK signaling that results in increased AP-1-dependent gene expression and increased vesicular transport. We will also explore how the actions of OSM may be pharmacologically inhibited formoterol. Finally, in Aim 3 we will determine if the findings of Aims 1 and 2 are recapitulated intact vascular networks using ex vivo perfused human organs and in vivo with immunodeficient mice engrafted with human skin. Successful completion of the proposed research can fundamentally advance our understanding of how capillary leak occurs in the earliest stages of severe shock and evaluate therapies targeted to the endothelium to prevent or reverse leak and its pathologic consequences.