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：
10718721
负责人：
RICHARD W PIERCE
金额：
$ 58.51万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10718721
关键词：
Acute Adrenergic Agonists Agonist Antibodies Area Biological Assay Blood Blood Vessels Blood capillaries Capillary Endothelial Cell Capillary Leak Syndrome Cardiovascular system Caring Cell Culture Techniques Cell Separation Cells Cessation of life Child Clinical Clinical Trials Complement Confusion Critical Illness Critically ill children Data Data Set Dermal Development Electrical Resistance Endothelial Cells Endothelium Engineering Equipment Extravasation FDA approved Female Foundations Functional disorder GTPase-Activating Proteins Gene Expression Gene Expression Profiling Genes Genetic Transcription Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Health Heart Arrest Human Immunodeficient Mouse Implant In Situ In Vitro Individual Inflammatory Investigation JAK3 gene Learning Liquid substance Lung Mediating Mediator Modeling Molecular Molecular Target Morphology Multiple Organ Failure Mus Organ Organ failure Pathway interactions Patient-Focused Outcomes Patients Perfusion Phase Phosphotransferases Plasma Process Proteins Regulation Research Respiratory Failure Rest Resuscitation Rodent Model STAT3 gene Sampling Serum Shock Signal Pathway Signal Transduction Skin Stereotyping Supportive care Surface Synaptic Vesicles TNF gene TNFRSF1A gene Testing Tight Junctions Time Tissue Sample Tracer Vascular Endothelial Cell Vesicle body system candidate identification cytokine disability effective therapy ex vivo perfusion formoterol human disease human model improved in vivo in vivo Model inhibitor male monolayer novel oncostatin M pharmacologic prevent response rho rho GTP-Binding Proteins single-cell RNA sequencing skin xenograft therapeutic target tool transcription factor

项目摘要

Project Summary Capillary leak is common in acutely critically ill children. Although no gold standard definition exists, it is clinically recognized as new or worsening organ failure despite appropriate cardiovascular resuscitation. Unfortunately, little has been learned of the pathophysiologic processes despite decades of struggling at the bedside of volume overloaded children with multiple organ dysfunction syndromes. Treatment is limited to intensive supportive care for failing organ systems. Less confusion exists in vitro, where leak around cultured human microvascular endothelial cells (EC) is identified as disruption of intercellular tight junctions (TJs) with functional changes in monolayer permselectivity. Such changes may be modeled in the EC response to cytokines, including those known to be elevated in the plasma of critically ill children. However, targeting specific cytokines has repeatedly failed to improve patient outcomes. Our overarching hypothesis is that while a great many leak-producing cytokines may be elevated in acute critical illness, there are only limited EC responses and that final common signaling pathways result in leak either between (para-) or through (trans-) ECs are therapeutic targets. However, the relative contributions of trans- and paracellular leak to the clinical manifestations of leak (i.e., organ dysfunction) and the pathways that cause them are incompletely understood. We will focus on targets we identified upregulated in ECs isolated from critically ill children (collected from vascular access insertion equipment and immediately analyzed by single-cell RNA-sequencing). This unique data set has identified candidate regulatory molecules associated with paracellular leak and oncostatin M (OSM) as a novel mediator of transcellular leak. We will test the contribution of these targets to leak in our culture models of TJ-forming human microvascular ECs from a healthy donor (both male and female) skin and lung using trans-endothelial electrical resistance, macromolecular flux assays, morphological analyses, molecular engineering, and immuno- chemical tools. Aim 1 will utilize tumor necrosis factor (TNF) to model paracellular leak to test the hypotheses that ArhGEF15, ArhGAP21, and -26 regulate RhoB activity, promoting junctional disassembly via downstream kinases directly acting on TJs and amplified new gene transcription. We have also discovered that formoterol, but not other β2-adrenergic agonists, inhibits TNF-induced leak and will investigate potential mechanisms. Aim 2 will test the hypothesis that OSM induces vesicle-associated JAK3/STAT3 signaling resulting in transcellular leak, which also depends on new gene expression, and investigate how formoterol reduces OSM-induced leak. Finally, in Aim 3, we will determine if the specific pathways identified in Aims 1 and 2 are recapitulated in intact human capillaries in vivo using human skin xenografts in mice and ex vivo using machine-perfused human lungs. The proposed research will advance our fundamental understanding of how capillary leak occurs in acutely critically ill children and evaluate an FDA-approved therapy re-targeted to the endothelium to prevent or reverse capillary leak, greatly improving the care of our sickest patients.

项目摘要毛细血管泄漏在急性危重儿童中很常见。尽管没有黄金标准的定义，但它在临床上是被认为是新的或恶化的器官故障目的地适当的心血管复苏。很遗憾，几乎几乎了解到病理生理过程目的地数十年来挣扎在体积的床边患有多个器官功能障碍综合征的过载儿童。治疗仅限于密集的支持护理用于故障器官系统。体外存在较少的混乱，其中培养的人类微血管周围泄漏内皮细胞（EC）被识别为具有功能变化的细胞间紧密连接（TJ）的破坏单层允许选择性。这种变化可以在EC对细胞因子的反应中建模，包括已知在重症儿童的血浆中被抬高。但是，靶向特定的细胞因子已经反复无法改善患者的预后。我们的总体假设是，虽然有很多泄漏产生急性危重疾病的细胞因子可能会升高，EC反应有限，最终通用信号通路会导致（Para-）或通过（Trans-）EC之间泄漏目标。但是，跨细胞和细胞细胞泄漏对叶片的临床表现的相对贡献（即器官功能障碍）以及导致它们的途径未完全理解。我们将专注于目标我们确定了从重症儿童隔离的EC中上调（从血管通道插入收集设备并立即通过单细胞RNA测序分析）。这个独特的数据集已确定与细胞细胞泄漏和oncostatin M（OSM）相关的候选调节分子作为新型介体跨细胞泄漏。我们将测试这些目标对TJ形成培养模型中泄漏的贡献来自健康供体（男性和女性）皮肤和肺的人类微血管EC使用跨内皮电阻，大分子通量测定，形态分析，分子工程和免疫 - 化学工具。 AIM 1将利用肿瘤坏死因子（TNF）模拟细胞细胞泄漏以检验假设那个arhgef15，arhgap21和-26调节RHOB活动，通过下游促进连接式拆卸激酶直接作用于TJ并扩增新的基因转录。我们还发现福特罗洛尔，但没有其他β2-肾上腺素能激动剂抑制TNF诱导的泄漏，并将研究潜在的机制。目的 2将检验以下假设：OSM诱导囊泡相关的JAK3/STAT3信号传导，导致跨细胞泄漏也取决于新的基因表达，并研究了配方器如何减少OSM诱导的泄漏。最后，在AIM 3中，我们将确定目标1和2中确定的特定途径是否完整地概括人体毛细血管在体内使用人体皮肤Xenographographics在小鼠中，并在体内使用机器化的人肺。拟议的研究将促进我们对毛细管泄漏如何急性泄漏的基本理解重症儿童病患者并评估FDA批准的疗法重新定位到内皮，以防止或反向毛细管泄漏，大大改善了我们最恶心的患者的护理。