Cytoskeletal Regulation of Lung Endothelial Pathobiology

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DESCRIPTION (Provided by applicant): This revised A1 PPG application remains a highly translational PPG focused on the critical role of the endothelial cell (EC) cytoskeleton in the pathobiology of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). ARDS, a devastating disorder with a mortality of ~30-35%, is defined by increases in lung vascular permeability, a major influence on ARDS mortality. The clinically-relevant PPG studies we have proposed will define the molecular mechanisms by which EC signaling drives cytoskeletal remodeling to either disrupt vascular integrity via production of paracellular gaps (that increases lung vascular permeability) or to restore EC barrier integrity via peripheral cytoskeletal remodeling and formation of cytoskeletal-driven lamellipodia and focal adhesions that promote EC gap closure. The analysis of these biophysical events represent the thematic underpinnings of this PPG and will be conducted by an outstanding translational team of gifted and interactive basic and physician-scientist investigators who will utilize clinically relevant bioactive/biophysical stimuli (VEGF, TNFα, LPS, mechanical stress) both in vitro and in preclinical models of ARDS and VILI. Project #1 will perform sophisticated structure, function and genetic analyses of the multi-functional non-muscle myosin light chain kinase (nmMLCK) isoform in the context of paracellular gap regulation and as a target for lung vascular barrier restoration. As EC barrier-regulatory enhancement is determined by peripheral actin remodeling, Project #2 will provide novel information regarding the interactions between nmMLCK and key actin-binding proteins (cortactin, Ena/VASP-like or EVL, c-Abl) in EC barrierrestorative responses (peripheral cytoskeletal remodeling, lamellipodial dynamics, gap closure). Focal adhesion (FA) proteins are complex participants in both the pathobiology and resolution of ARDS via bidirectional signaling to the cytoskeleton. Project #3 will interrogate the role of the FA proteins, integrin β4 and paxillin, in cytoskeletal linkages to focal adhesion dynamics (assembly/disassembly) and lamellipodialmediated closure of inflammation-induced EC gaps as well as the influence of single nucleotide polymorphisms (SNPs) and post-translational modifications (PTMs) on FA structure /function. Supported by four highly interactive cores, our programmatic approaches are woven into unique PPG features: i) testing of novel ARDS/VILI therapies designed to attenuate the highly druggable lung EC permeability pathway (MLCK inhibitors, S1P, HGF, integrin β4 antibodies) and, ii) the interrogation of ARDS-associated SNPs in key PPG EC barrier-regulatory genes, studies of enormous importance as African descent individuals are a population at high risk for reduced survival in ARDS. By leveraging the outstanding scientific environment at the University of Arizona and long standing collaborations with University of Illinois scientists, this "model translational PPG" is exceptionally suited to provide comprehensive mechanistic understanding of lung vascular barrier regulation, and facilitate the development of therapeutic targets to restore the integrity of the injured pulmonary circulation.