ABSTRACT: Project #1 system biology studies have highlighted 2 critical lung cytoskeletal effector proteins/genes as central to addressing vascular inflammation, endothelial cell (EC) permeability and the multi-organ failure critical to ARDS mortality and the ARDS vascular endotype. We have convincingly demonstrated the multi-functional non-muscle myosin light chain kinase isoform (MYLK) and its cytoskeletal-binding partner, cortactin (CTTN), are primary regulators of inflammation-induced vascular permeability, leukocyte trafficking, and vascular responses to ventilator-derived mechanical stress. Furthermore, the genes encoding nmMLCK (MYLK) and cortactin (CTTN) harbor genetic variants that confer increased risk of sepsis/trauma-induced ARDS and ARDS mortality in Blacks. In sync with PPG thematic goals, Project #1 is designed to translate mechanistic insights into nmMLCK and cortactin structure and function into novel, effective therapeutic opportunities to reduce ARDS mortality. SA #1 will explore genetic/epigenetic regulation of the non-muscle MYLK and CTTN promoters by: i) ROS–regulated (or sensing) transcription factors (hypoxia-induced factors HIF-1a/HIF-2a, and NRF2), ii) MYLK/CTTN promoter SNPs, and by iii) MYLK/CTTN promoter DNA methylation (Core B). SA #2 will detail EC barrier-responses elicited by S1PR1 and TLR4 receptor activation that are influenced by tyrosine phosphorylation of nmMLCK1, nmMLCK2 (the pro-inflammatory MYLK splice variant) and cortactin; and by MYLK/CTTN coding SNPs (over-represented in Blacks). SA #3 will functionally characterize the involvement of novel nmMLCK-binding proteins (pyruvate kinase M2, kindlin-2) and cortactin-binding proteins (DOCK1/ELMO1) in S1PR1/TLR4-mediated EC cytoskeletal dynamics and barrier regulation. Pyruvate kinase M2 (PKM2), a central regulator of glycolysis and inflammation, selectively binds the nmMLCK1 IgGCAM3 domain to potentially influence EC cytoskeletal-driven barrier restoration. The focal adhesion (FA) regulatory protein, kindlin2, is a Project #3 target gene, and was recently identified as a nmMLCK binding partner likely crucial for linking the cytoskeleton to integrin-mediated cell-ECM focal adhesion and signaling. DOCK1 and ELMO1 are key Rac GTPase and cytoskeletal regulatory proteins and novel cortactin-binding proteins. SA #2 and SA #3 studies utilize Core C/D proteomic and biophysical imaging modalities (super resolution, AFM) to define protein interactions in S1PR1/TLR4-mediated EC spatially-specific cytoskeletal remodeling, gap formation/closure and lamellipodia formation and barrier regulation. SA #4 utilize established Core C rat and porcine ARDS/VILI models to assess a novel barrier-promoting liposome bearing the S1PR1 agonist, Tysiponate, on its outer surface, encargoed with simvastatin (nmMLCK antagonist), bixin (NRF2 agonist, MYLK antagonist), or PIK (nmMLCK peptide inhibitor). Thus, by leveraging the integrated interactions with each PPG Project and Core, Project #1'...