Molecular regulation of the capillary barrier in acute critical illness

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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.