Molecular regulation of the capillary barrier in acute critical illness

急性危重症毛细血管屏障的分子调控

基本信息

批准号：
10683786
负责人：
RICHARD W PIERCE
金额：
$ 41.85万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10683786
关键词：
Acute Adherent Culture Agonist Animal Model Antibodies Biological Assay Blood Vessels Blood capillaries Capillary Endothelial Cell Cell Culture Techniques Cessation of life Child Clinical Clinical Trials Coupled Critical Illness Critically ill children Cytoskeletal Modeling Data Dermal Development Edema Electrical Resistance Endothelial Cells Endothelium Engineering Etiology Extravasation Failure Female Foundations Functional disorder GTPase-Activating Proteins Gene Expression Gene Expression Profile Genetic Transcription Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Health Heart Arrest Human Immunodeficient Mouse In Situ Individual Inflammation Mediators Kinetics Liquid substance Lung MAP Kinase Gene MAPK8 gene Mediating Mediator of activation protein Mitogen-Activated Protein Kinase Kinases Modeling Molecular Molecular Target Monomeric GTP-Binding Proteins Morbidity - disease rate Morphology Organ Pathologic Pathway interactions Pharmacology Phase Phosphotransferases Process Protein Isoforms Regulation Research Resolution Rest Role Shock Signal Pathway Signal Transduction Signaling Molecule Skin Skin Tissue Stereotyping TNF gene Testing Tight Junctions Tissue Grafts Tissue Sample Transcription Factor AP-1 disability effective therapy endothelial dysfunction formoterol gene product hemodynamics human disease improved in vivo in vivo Model inhibitor lung microvascular endothelial cells male monolayer mortality novel therapeutics oncostatin M p38 Mitogen Activated Protein Kinase prevent receptor response rho single-cell RNA sequencing solute targeted treatment tool transcription factor transcriptomics transcytosis vesicle transport

项目摘要

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.

项目摘要以前健康的儿童毛细血管泄漏与与之相关的电击的初始阶段发生心脏停搏。毛细血管泄漏有助于血流动力学不稳定，器官功能障碍，最终增加发病率和死亡率。尽管如此临床重要性，但尚无已知的治疗或逆转疗法毛细管泄漏是因为底层机制未知。重要器官中的毛细管内皮细胞（EC）通过形成控制控制的内部紧密连接（TJ），形成连续的，允许的屏障细胞细胞通量和精确调节跨介症。毛细管泄漏是由于其中一个或两个的破坏而导致的过程。但是，即使没有建立反式和细胞细胞泄漏的相对贡献。我们的总体假设是，尽管产生代偿性冲击的临床损伤可能是可变的，并且多余的，它们聚集以激活EC中最终的共同机制，以防止或反向毛细管泄漏。这种冗余性在最初的震动状态下，心脏骤停状态缺乏针对单个介体的临床益处。通过比较从一般健康的孩子那里收集的单个EC的转录曲线，而在早期的早期阶段与心脏骤停相关的休克，鉴定培养的微血管EC中的候选分子负责用于特定的结构变化，产生跨细胞泄漏。我们将测试这些功能培养模型中的分子，由正常供体（男性和女性）人类微血管EC组成皮肤和肺形成TJ，使用跨内皮电阻和大分子通量测定法形态分析，分子工程和免疫化学工具。在AIM 1中，我们将确定角色在早期冲击中与儿童分离的EC中增加的小型GTPases及其调节剂心脏骤停：ARHGEF12,15，ARHGAP21,26和Rhoa-C，J，U。我们利用肿瘤坏死因子（TNF）诱导健康供体皮肤毛细血管EC中TJ的细胞细胞泄漏。我们还将调查甲他醇抑制TNF诱导的泄漏的机制。在AIM 2中，我们研究了OnCostatin-M（OSM），如何在我们的转录组分析中，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会扰动TJ。具体而言，我们将测试OSM的假设激活JAK/STAT/P38-MAPK信号传导，从而导致AP-1依赖性基因表达和增加囊泡运输。我们还将探讨如何在药理上抑制OSM的作用 Formoterol。最后，在AIM 3中，我们将确定目标1和2的发现是否概括为完整的血管使用离体灌注的人体器官和体内的网络，与人类的免疫缺陷小鼠皮肤。成功完成拟议的研究可以从根本上促进我们对如何的理解毛细管泄漏发生在严重冲击的最早阶段，并评估针对内皮的疗法防止或逆转泄漏及其病理后果。