Deane Mosher

Fibronectin (FN) is a key extracellular matrix glycoprotein indispensable for the development and function of major vertebrate organ systems. Autosomal dominant FN mutations cause corner fracture-type spondylometaphyseal dysplasia (SMDCF). However, the molecular pathomechanisms underlying this pathology are not known. Here, we have employed patient-derived induced pluripotent stem cells (iPSCs) as an SMDCF cell culture model to investigate the consequences of FN mutations in mesenchymal stem cells (MSCs) and differentiated cartilage-producing chondrocytes. The FN mutations impaired protein secretion from MSCs leading to significant accumulation of FN within the cells and strong reduction of FN in the extracellular matrix. Analyses of plasma samples from SMDCF patients showed a similar reduction in circulating FN. In mutant MSCs, FN accumulated in ribosome-covered intracellular vesicles …

Cartilage acidic protein‐1 (CRTAC1) is produced by several cell types, including Type 2 alveolar epithelial (T2AE) cells that are targeted by SARS‐CoV2. Plasma CRTAC1 is known based on proteomic surveys to be low in patients with severe COVID‐19. Using an ELISA, we found that patients treated for COVID‐19 in an ICU almost uniformly had plasma concentrations of CRTAC1 below those of healthy controls. Magnitude of decrease in CRTAC1 distinguished COVID‐19 from other causes of acute respiratory decompensation and correlated with established metrics of COVID‐19 severity. CRTAC1 concentrations below those of controls were found in some patients a year after hospitalization with COVID‐19, long COVID after less severe COVID‐19, or chronic obstructive pulmonary disease. Decreases in CRTAC1 in severe COVID‐19 correlated (r = 0.37, p = 0.0001) with decreases in CFP (properdin), which …

BackgroundThe pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches.Methods and resultsHere we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN …

IL5 and IL33 are major activating cytokines that cause circulating eosinophils to polarize, adhere, and release their granule contents. We correlated microscopic features of purified human blood eosinophils stimulated for 10 min with IL5 or IL33 with phosphoproteomic changes determined by multiplexed isobaric labeling. IL5 caused phosphorylation of sites implicated in JAK/STAT signaling and localization of pYSTAT3 to nuclear speckles whereas IL33 caused phosphorylation of sites implicated in NFκB signaling and localization of RELA to nuclear speckles. Phosphosites commonly impacted by IL5 and IL33 were involved in networks associated with cytoskeletal organization and eosinophil adhesion and migration. Many differentially regulated phosphosites were in a diverse set of large proteins—RAB44, a “large RAB” associated with crystalloid granules; NHSL2 and VIM that change localization along with the nucleus during polarization; TNFAIP3 vital for control of NFκB signaling, and SRRM2 and PML that localize, respectively, to nuclear speckles and PML bodies. Gene expression analysis demonstrated differential effects of IL5 and IL33 on IL18, CCL5, CSF1, and TNFSF14. Thus, common effects of IL5 and IL33 on the eosinophil phosphoproteome are important for positioning in tissues, degranulation, and initiation of new protein synthesis whereas specific effects on protein synthesis contribute to phenotypic heterogeneity.KEY POINTSIL33 and IL5 impact common pathways of eosinophil cytoskeletal reorganization, adhesion and migration.Each lobe of the human eosinophil nucleus has a specific anatomy poised for new onset of cytokine …

Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury+ mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation.

A method to inhibit neutrophil recruitment to damaged tissue, thereby inhibiting inflammation in a subject. The method includes administering to an anti-inflammatory amount of a myeloid-derived growth factor (“MYDGF”). Also disclosed are corresponding pharmaceutical compositions of matter containing the MYDGF.

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Eosinophils deposit their damaging products in airway tissue, likely by degranulation and cytolysis. We previously showed that priming blood eosinophils with IL3 strongly increased their cytolysis on aggregated IgG. Conversely, IL5 priming did not result in significant eosinophil cytolysis in the same condition. Therefore, to identify critical events protecting eosinophils from cell cytolysis, we examined the differential intracellular events between IL5- and IL3-primed eosinophils interacting with IgG. We showed that both IL3 and IL5 priming increased the eosinophil adhesion to IgG, phosphorylation of p38, and production of reactive oxygen species (ROS), and decreased the phosphorylation of cofilin. However, autophagic flux as measured by the quantification of SQSTM1-p62 and lipidated-MAP1L3CB over time on IgG, with or without bafilomycin-A1, was higher in IL5-primed compared to IL3-primed eosinophils. In addition, treatment with bafilomycin-A1, an inhibitor of granule acidification and autophagolysosome formation, enhanced eosinophil cytolysis and DNA trap formation in IL5-primed eosinophils. Therefore, this study suggests that increased autophagy in eosinophils protects from cytolysis and the release of DNA, and thus limits the discharge of damaging intracellular eosinophilic contents.

Introduction: Fibronectin (FN) has key roles in cell adhesion, differentiation, migration, alignment and immune modulation in the developing heart, but is of low abundance in the postnatal heart. Replenishing injured myocardium with insoluble FN could be a novel therapeutic approach; however, methods to produce ECM enriched with insoluble FN have remained elusive. Our team recently developed a method to produce decellularized fibronectin-rich (dFN) sheets assembled by dense human cardiac myofibroblast (CMF) cultures. We discovered that the transmembrane protein, Sushi Containing Domain 2 (SUSD2), is broadly expressed on cultured CMFs. Hypothesis: We hypothesized that SUSD2 abundance predicts whether CMFs produce intact dFN sheets. Methods: Primary CMFs were isolated from healthy left ventricles of human cadaveric donors. CMFs were sorted by median fluorescence intensity (MFI) into …