Molecular regulation of the capillary barrier in acute critical illness

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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.