PROJECT SUMMARY: The COVID-19 pandemic has dramatically highlighted a distinct vascular endotype of ARDS, characterized by profound endothelial cell (EC) permeability, thrombosis and microangiopathy (1; 2), and important unmet needs such as the critical absence of any effective FDA-approved therapies to halt or reverse lethal vascular leak in ARDS/VILI. Loss of lung EC barrier integrity results in vascular leakage, alveolar flooding, and is a critical feature of ARDS multi-organ failure and mortality. Project #4 investigators have demonstrated that sphingosine-1-phos- phate (S1P), a multifunctional lipid mediator, and its analog, Tysiponate (TySIP), effectively reduce vascular leakage and inflammatory lung injury via ligation of S1PR1, a G-protein-coupled receptor highly expressed in lung ECs. S1PR1 ligation rapidly initiates a signaling cascade that reorganizes the EC cytoskeleton via Rac GTPase signaling, enhances junctional integrity, and decreases vascular permeability. In contrast, ligation of the S1PR3 receptor induces Rho GTPase signaling to the cytoskeleton to increase lung permeability. Furthermore, S1PR3 is released into circulating microvesicles in VILI- or LPS-exposed mice serving as a novel ARDS bi- omarker that predicts survival. Both S1PR1 and S1PR3 promoter activity, expression and downstream signaling are influenced by pathophysiologic levels of mechanical stress with selective promoter demethylation, and by promoter SNPs associated with ARDS risk. We identified SELPLG as a novel ARDS susceptibility gene in Blacks, which encodes P-selectin glycoprotein ligand 1 (PSGL1) that via P-selectin (Selp) binding is critically involved in PMN trafficking. Our exciting data suggest interconnected signaling of S1PR/S1PR and PSGL1/SELPLG in regulation of EC vascular leak. Given the major role of ROS in pulmonary vascular leak and PMN trafficking, Specific Aim #1 (SA #1), with strong Core B assistance, will characterize the role of ROS- sensing transcription factors (HIF-1α/2α, NRF2), promoter SNPs (predominant in Blacks) and DNA methylation sites in genetic/epigenetic regulation of S1PR1/S1PR3 and SELPLG/SELP. Leveraging compelling preliminary data and critical Core D support, SA #2 will examine the novel barrier-regulatory roles for AKT nitration, protein deubiquitination (UCHL1), the Rac GTPase activator DOCK1, and the actin-binding FA protein, lamellipodin, in S1PR1 and S1PR3 signaling. SA #3 will characterize the dual influences of S1PR1/S1PR3 on PSGL1 secretion and P-selectin membrane mobilization from EC Weibel-Palade bodies, on signaling to the EC cytoskeleton, and altered EC barrier responses. Leveraging unparalleled Core C expertise in preclinical rat and porcine LPS/VILI models, SA #4 will evaluate the therapeutic effectiveness of the TySIPosome encargoed with either S1PR3 siRNAs or an UCHL1 activator, (CD19H28N205S), and directly assess the efficacy of a competitive inhibitor of P-selectin-PSGL-1 binding, the TSGL-Ig biologic (Tand...