ABSTRACT: The elevated ARDS mortality observed in the COVID-19 pandemic has highlighted the contribution of excessive mechanical stress produced by mechanical ventilation in promoting lethal increases in lung vascular permea- bility. Integrin β4 (ITGB4 gene) and kindlin-2 (FERMT2 gene) are essential adhesion molecules in endothelial cell (EC) focal adhesions (FAs), structures critical for mechano-sensing, for bidirectional signaling between the EC cytoskeleton and the cell-matrix interface, and for EC barrier regulation. The mechanistic basis for dy- namic FA coordination during inflammatory EC barrier dysfunction and subsequent barrier restoration is a fun- damental question that remains unresolved. We speculate that coordinate control of FA structures requires the dynamic interactions of integrin β4 (ITGB4) and kindlin-2 with key PPG cytoskeletal effectors (nmMLCK, cortactin, Dock1, lamellipodin, paxillin) to efficiently assemble functional FAs during EC barrier responses (pe- ripheral cytoskeletal remodeling, lamellipodial formation, gap closure). ITGB4 is a unique mechano-sensing, laminin-binding integrin, and kindlin-2 is a multi-domain mechano-sensing adapter FA protein that recruits struc- tural and signaling molecules to FAs in concert with cytoskeletal rearrangement. We speculate that these EC responses are highly influenced by ITGB4 and kindlin-2 post-translational modifications (PTMs) and coding pol- ymorphisms (SNPs). As reactive oxygen species (ROS) is an important stimulus for FA dynamics and loss of EC barrier integrity, with Core B, SA #1 will characterize the role of three ROS-sensing transcription factors (NRF2, HIF1α/HIF2α), ITGB4/FERMT2 SNPs and DNA methylation in genetic/epigenetic regulation of ITGB4/FERMT2 expression and the influence on generation of the unique ITGB4 alternatively-spliced, barrier- regulatory variant, Integrin β4E (ITGB4E), that we identified as involved in mechano-sensing and EC barrier regulation. With Core D, SA #2 will conduct in depth structure/function studies including 3D live cell imaging of mutant ITGB4 and kindlin-2 fusion proteins (SNPs, PTMs) to characterize ITGB4/kindlin-2 function in spatially- specific EC cytoskeletal rearrangements driving EC barrier-disruption and barrier-restoration. SA #3 will examine the functionality of ITGB4/kindlin-2 interactions within lamellipodia with known and novel FA-binding cytoskeletal partners, including cortactin, Dock1, lamellipodin and highly novel interactions with nmMLCK (Project #1). Finally, utilizing elegant rat and porcine models of LPS/VILI (Core C), SA #4 will assess the therapeutic utility of the SMURF inhibitor A01, or SRI-38832 to augment kindlin-2 expression as cargo in TySIPonate-conjugated liposomes (TySIPosomes, Project #4). Project #3 studies will determine the structure/function and molecular basis for the dynamic FA control by ITGB4 and kindlin-2 and yield important insights into functional relevance of this unique FA signaling axis in restoration of EC barrier function. Our highly translational approaches will also provide actionable EC barrier-regulatory strategies that restore the integrity of the injured pulmonary circulation while yielding insights into ITGB4/FERMT2 variant participation in ARDS health disparities.