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

• 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
• 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

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 毛细血管渗漏在急性重症患儿中很常见。虽然没有金标准定义存在，但它是临床上 尽管进行了适当的心血管复苏，但仍被认为是新发或恶化的器官衰竭。不幸的是， 尽管几十年来一直在努力研究体积的病理生理过程， 多器官功能障碍综合征的超负荷儿童治疗仅限于强化支持性护理 治疗器官系统衰竭在体外培养的人微血管周围存在渗漏， 内皮细胞（EC）被鉴定为细胞间紧密连接（TJ）的破坏， 单层选择渗透性这种变化可以在EC对细胞因子的反应中建模，包括那些 已知在重症儿童的血浆中升高。然而，靶向特定的细胞因子已经反复地 未能改善患者的预后。我们的首要假设是，虽然大量的泄漏产生 细胞因子可能在急性危重病中升高，只有有限的EC应答， 导致EC之间（帕拉）或通过（反式）EC泄漏的常见信号传导途径具有治疗作用 目标的然而，跨细胞和旁细胞渗漏对渗漏临床表现的相对贡献 (i.e.,器官功能障碍）和导致它们的途径尚不完全清楚。我们将专注于目标 我们在从危重患儿分离的EC中发现了上调的表达（从血管通路插入处收集 设备上，并立即通过单细胞RNA测序进行分析）。这个独特的数据集已经确定了 与细胞旁渗漏相关的候选调节分子和作为新介质的抑瘤素M（OSM） 跨细胞渗漏我们将在我们的TJ形成的培养模型中测试这些靶标对泄漏的贡献。 使用跨内皮细胞技术从健康供体（男性和女性）皮肤和肺中获得人微血管EC 电阻、大分子通量测定、形态学分析、分子工程和免疫- 化学工具目的1将利用肿瘤坏死因子（TNF）模拟细胞旁漏，以验证假设 ArhGEF 15、ArhGAP 21和-26调节RhoB活性，通过下游途径促进连接解体， 直接作用于TJ并扩增新基因转录的激酶。我们还发现福莫特罗， 而不是其他β2-肾上腺素能激动剂，抑制TNF诱导的渗漏，并将研究潜在的机制。目的 2将检验OSM诱导囊泡相关的JAK 3/STAT 3信号传导导致跨细胞免疫应答的假设。 泄漏，这也取决于新的基因表达，并研究福莫特罗如何减少OSM诱导的泄漏。 最后，在目标3中，我们将确定目标1和2中确定的特定途径是否在完整的 使用小鼠中的人皮肤异种移植物在体内和使用机器灌注的人肺离体观察人毛细血管。 拟议的研究将推进我们对毛细血管泄漏如何发生在急性 重症儿童，并评估FDA批准的治疗重新靶向内皮细胞，以防止或逆转 毛细血管渗漏，极大地改善了我们对病情最严重的病人的护理。