Spatiotemporal control of early matricellular events in the injured lung

Project Summary/Abstract: Acute lung injury (ALI), clinically known as the acute respiratory distress syndrome (ARDS), is a major cause of ICU morbidity and mortality. ALI/ARDS is an inflammatory lung injury characterized by alveolar barrier disruption and is triggered by both infectious and non-infectious injury. Since no treatment options are directed to the underlying ALI biology, there remains an unmet need to target the mechanisms of ALI. Essential early on for optimal lung repair is the formation of a provisional matrix, which is orchestrated by matricellular proteins that facilitate dynamic interactions between structural matrix proteins and cells directed into the site of injury. Our overarching goal is to better understand early matricellular events in the injured lung, and the mechanisms that are critical for the stabilization of the provisional matrix to identify targetable pathways. Thrombospondin-1 (TSP1) is a matricellular protein that binds to structural matrix proteins and is part of the early provisional matrix. Platelets are a major source of TSP1 and, upon platelet activation, TSP1 is released from α- granules onto the platelet membrane where TSP1 undergoes a conformational change through disulfide interchange mediated by extracellular protein disulfide isomerase (PDI). We posit this conformational change opens a cryptic region of TSP1 and enhances TSP1 anti-protease function. Strikingly, platelet and megakaryocyte-specific (plt/MgK) Thbs1 cKO mice exhibit increased lung permeability with exaggerated neutrophil responses, and enhanced matrix remodeling in the airspaces following lung injury compared to WT mice. The airspace proteome of plt/MgK Thbs1 cKO mice is enriched in proteolytic enzymes that degrade the extracellular matrix (ECM), neutrophil granule contents, increased fibrillar collagen COL1A1, and proteins involved in ECM biosynthesis. Using lung intravital microscopy and 3D scanning microscopy, we show that cKO mice exhibit increased MgK recruitment to sites of collagen in areas of alveolar leak and interstitial space compared to WT mice. These findings lead us to hypothesize that TSP1 is critical for the spatiotemporal control of early matricellular events in the injured lung by stabilizing ECM proteins and regulating cellular activation and inflammation. Utilizing pharmacologic blockade, genetic mouse models, advanced optical imaging, cutting-edge spatial proteomics, untargeted mass spectrometry, and a human ARDS cohort, we propose to (1) determine whether plt/MgK TSP1 instructs neutrophils at the provisional matrix to restrain their activation through PDI; (2) evaluate the mechanism by which plt/MgK TSP1 regulates early matrix remodeling, collagen expression and stabilization; and, (3) investigate the role of TSP1 in the control of MgK and platelet mobilization in the injured lung. Defining the role of TSP1 in the formation of an optimal provisional matrix and barrier repair could prove useful in the rational design of targeted therapeutics.