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: 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
• 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

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 既往健康儿童的毛细血管渗漏发生在休克的初期，与 心脏骤停。毛细血管渗漏会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。尽管具有如此重要的临床意义，但目前还没有已知的治疗方法或逆转方法。 毛细管渗漏是因为其潜在的机制未知。重要器官中的毛细血管内皮细胞 通过细胞间紧密连接(TJ)的形成形成连续的、选择性的渗透屏障，TJ控制 细胞旁的流量和精确地调节跨细胞作用。毛细血管泄漏是由于其中一种或两种情况的破坏造成的 流程。然而，即使是跨细胞和细胞旁渗漏的相对贡献也没有确定。 我们的主要假设是，虽然临床侮辱产生失代偿性休克可能是可变的和 它们是冗余的，它们汇聚在一起，以激活ECs中的最终公共机制，这些机制可以针对这些机制进行预防或逆转 毛细血管渗漏。在休克和心脏骤停的初始状态中信号的这种冗余是缺乏的原因 以个体调解人为目标的临床益处。我们的假设是通过比较 正常儿童单个内皮细胞与早期内皮细胞的转录图谱比较 与心脏骤停相关的休克，确定培养的微血管内皮细胞中的候选分子 用于产生跨细胞或旁细胞泄漏的特定结构变化。我们将测试这些组件的功能 人微血管内皮细胞来源的正常供体(男女)培养模型中的分子 皮肤和肺形成TJ，使用跨内皮细胞电阻和大分子流量分析， 形态分析、分子工程和免疫化学工具。在目标1中，我们将确定 从早期休克儿童分离的内皮细胞中增加的小GTP酶及其调节因子 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 健康供体真皮毛细血管内皮细胞TJs断裂的细胞旁渗漏。我们还将调查 福莫特罗抑制肿瘤坏死因子诱导的渗漏的机制。在目标2中，我们研究了肿瘤抑素-m(OSM)如何， 在我们的转录分析中，受体和下游信号分子也上调了， 在我们的模型中诱导跨细胞渗漏而不干扰TJ。具体地说，我们将测试OSM假设 激活JAK/STAT/p38-MAPK信号，导致AP-1依赖基因表达增加，并 囊泡转运增加。我们还将探索OSM的作用如何在药物上受到抑制 福莫特罗。最后，在目标3中，我们将确定目标1和2的发现是否是完整的血管 利用体外灌流的人体器官和体内移植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促进我们对 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 防止或逆转泄漏及其病理后果。