Abstract The protective intestinal mucosal barrier is a specialized domain responding to and interacting with different luminal noxious substances and the microbiome. Acute gut barrier dysfunction occurs commonly in patients with critical surgical disorders such as trauma, thermal injury, sepsis, shock, massive surgical operations, and ischemic postconditioning. Gut barrier dysfunction leads to the translocation of luminal toxic substances and bacteria to the blood stream and, in some instances, results in multiple organ dysfunction syndrome (MODS) and death. Effective therapies to preserve gut barrier integrity are limited, because of poorly understood mechanisms of acute gut barrier dysfunction in various critical surgical conditions. Recently, intercellular crosstalk directed by the secretion of extracellular vesicles (EVs) has been gaining increasing attention. EVs released from many cell types, including intestinal epithelial cells (IECs), can transfer a variety of bioactive molecules to neighboring or distant tissues and hence play previously unrecognized functional roles. The goal of this competitive renewal application is to determine the role and mechanism of EV noncoding RNAs (ncRNAs) in the control of intestinal barrier function under critical surgical conditions by carrying out a series of multi-disciplinary studies. Our preliminary data indicate that the small noncoding vault RNASs (vtRNAs) are critical transcripts present in EVs released from IECs, and that the levels of EV vtRNAs increase in patients with sepsis and mice exposed to septic stress. Ectopically expressed vtRNAs decrease the levels of tight junctions and disrupt the intestinal barrier function. Mice bearing a maternal deletion of exon 1 in the H19 gene (H19-/-) exhibit induced autophagy and decreased release of vtRNA-enriched EVs after septic stress. Building on these exciting observations, we now propose the paradigm-shifting hypothesis that vtRNAs in EVs secreted by IECs in response to critical surgical stress play an important role in the pathogenesis of acute gut barrier dysfunction, and that EV release from IECs is tightly regulated by long ncRNA H19 and RNA-binding protein HuR via autophagy. Three specific aims are proposed to test the hypothesis: 1) to define the roles of the vtRNA cargo of EVs generated from human enterocytes in the regulation of intestinal barrier function during critical surgical stress; 2) to identify novel targets of EV vtRNAs that play an important role in triggering intestinal epithelial barrier dysfunction in response to critical surgical stress; and 3) to test if H19 and HuR regulate the formation of vtRNA-rich EVs in IECs by altering autophagy. Completion of these specific aims will uncover novel mechanisms underlying the pathogenesis of acute gut barrier dysfunction in patients with critical surgical disorders. It will also shed information needed to develop new biomarkers for deteriorating acute gut barrier dysfunction and/or MODS an...