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Circulation Jun 2024Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic...
BACKGROUND
Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.
METHODS
We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 ()-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays.
RESULTS
Transcriptional regulation and trajectory inference from -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor and upregulation of late anticardiac regulators and in knockout cells reflect a necessary role for in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.
CONCLUSIONS
These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.
Topics: Humans; Myocytes, Cardiac; Cell Differentiation; Cell Lineage; Neurofibromin 2; CRISPR-Cas Systems; Human Embryonic Stem Cells
PubMed: 38752370
DOI: 10.1161/CIRCULATIONAHA.122.061335 -
Research Square May 2024Patients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and...
BACKGROUND
Patients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity and in a mouse model of HS/T. This study addresses systemic effects of HS/T on multiorgan EOT in HS/T model.
METHODS
, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (HO) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. , a mouse model of HS/T was used to evaluate the effects of lactated Ringer's (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers.
RESULTS
MSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer , whereas only MSC CM was protective of the Caco-2 epithelial monolayer. , both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice.
CONCLUSIONS
In conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.
PubMed: 38746312
DOI: 10.21203/rs.3.rs-4284907/v1 -
Transplant International : Official... 2024Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more...
Proteomic Analysis of Primary Graft Dysfunction in Porcine Lung Transplantation Reveals Alveolar-Capillary Barrier Changes Underlying the High Particle Flow Rate in Exhaled Breath.
Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more detailed monitoring and understanding of the pathophysiology of PGD. The measurement of particle flow rate (PFR) in exhaled breath is a novel tool to monitor and understand the disease at the proteomic level. In total, 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on postoperative day 3. Exhaled breath particles (EBPs) were evaluated by mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Recipients with PGD had significantly higher PFR [686.4 (449.7-8,824.0) particles per minute (ppm)] compared to recipients without PGD [116.6 (79.7-307.4) ppm, = 0.0005]. Porcine and human EBP proteins recapitulated proteins found in the BAL, demonstrating its utility instead of more invasive techniques. Furthermore, adherens and tight junction proteins were underexpressed in PGD tissue. Histological and proteomic analysis found significant changes to the alveolar-capillary barrier explaining the high PFR in PGD. Exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.
Topics: Animals; Lung Transplantation; Proteomics; Primary Graft Dysfunction; Swine; Humans; Breath Tests; Bronchoalveolar Lavage Fluid; Female; Male; Exhalation
PubMed: 38741700
DOI: 10.3389/ti.2024.12298 -
ACS Applied Bio Materials Jun 2024Cadherin-mediated tension at adherens junctions (AJs) is fundamental for cell-cell adhesion and maintaining epithelial integrity. Despite the importance of manipulating...
Cadherin-mediated tension at adherens junctions (AJs) is fundamental for cell-cell adhesion and maintaining epithelial integrity. Despite the importance of manipulating AJs to dissect cell-cell interactions, existing three-dimensional (3D) multicellular models have not adequately addressed the precise manipulation of these junctions. To fill this gap, we introduce E-cadherin-modified tension gauge tethers (TGTs) at the junctions within spheroids. The system enables both quantification and modulation of junctional tension with specific DNA triggers. Using rupture-induced fluorescence, we successfully measure mechanical forces in 3D spheroids. Furthermore, mechanically strong TGTs can maintain normal E-cadherin-mediated adhesion. Employing toehold-mediated strand displacement allowed us to disrupt E-cadherin-specific cell-cell adhesion, consequently altering intracellular tension within the spheroids. Our methodology offers a robust and precise way to manipulate cell-cell adhesion and intracellular mechanics in spheroid models.
Topics: Cadherins; Spheroids, Cellular; Humans; Cell Adhesion; Biocompatible Materials; Adherens Junctions; Materials Testing; Particle Size
PubMed: 38729097
DOI: 10.1021/acsabm.4c00142 -
Cells May 2024Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several...
Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of β-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-β-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.
Topics: Animals; Actin Cytoskeleton; Actinin; Adherens Junctions; alpha Catenin; beta Catenin; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Integrins; Phosphorylation; Vinculin; Zyxin; Rats
PubMed: 38727316
DOI: 10.3390/cells13090780 -
PloS One 2024Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through...
Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.
Topics: Humans; Adherens Junctions; Antigens, CD; Cadherins; Cell Adhesion; HEK293 Cells; Membrane Proteins; Proteolysis; Ubiquitin-Protein Ligases
PubMed: 38722959
DOI: 10.1371/journal.pone.0290485 -
American Journal of Physiology. Lung... May 2024The house dust mite (HDM) represents a major cause of allergic rhinitis and asthma. We tested whether HDM-induced aeroallergen exposure sensitivity is caused by the...
The house dust mite (HDM) represents a major cause of allergic rhinitis and asthma. We tested whether HDM-induced aeroallergen exposure sensitivity is caused by the innate-immune response in small airway epithelial cells. HDM exposure rapidly activates NFkB/RelA in the Secretoglobin (Scgb1a1+) lineage and upregulates markers of epithelial plasticity. To determine the effect of epithelial NFkB signaling, NFkB was depleted in a tamoxifen (TMX)-inducible -CreER mouse within a CL57B/L6 background. Corn oil or TMX-treated/RelA-depleted (RelA KD) mice were repetitively exposed to airway HDM challenges to induce airway hyperresponsiveness (AHR). Strikingly, we observed that HDM induces hallmarks of epithelial plasticity through upregulation of the mesenchymal core factors SNAI1 and ZEB1 and production of MMP9 that are RelA dependent. Downstream, HDM-induced mucous metaplasia, Th2 polarization, allergen sensitivity and airway hyperreactivity were all reduced in the RelA-depleted mice. Mechanistically, HDM-induced functional and structural barrier disruption was dependent on RelA signaling and associated with active MMP secretion into the bronchoalveolar lavage fluid. To establish the role of MMP2/9 in barrier disruption, we observe that a small-molecule MMP inhibitor (SB-3CT) blocked HDM-induced barrier disruption and activation of plasticity in naïve wild-type mice. Loss of functional barrier was associated with MMP disruption of ZO-1 containing adherens junctions. Overall, this data indicates that host innate signaling in the Scgb1a1+ progenitors is directly linked to epithelial plasticity, MMP9 secretion, and enhanced barrier permeability which allows allergen penetration, sensitization producing allergic asthma (AA) . We propose that maintenance of epithelial integrity may reduce allergic sensitization and AA.
PubMed: 38713619
DOI: 10.1152/ajplung.00066.2024 -
Microbes and Infection Apr 2024African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain...
African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain insights into the host responses to ASFV, we generated recombinant adenovirus Ad5 expressing viral membrane proteins p54, p17, and pB117L individually and infected an alveolar cell line, 3D4/21, with these recombinant viruses. Then, the cell lysates were analyzed using label-free quantification proteomic analysis method. A total of 2158 differentially expressed proteins (DEPs) were identified, of which 817, 466, and 875 proteins were from Ad5-p54-, Ad5-p17-, Ad5-pB117L-infected 3D4/21 cells, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed distinct yet interconnecting patterns of protein interaction networks. Specifically, the Ad5-p54 virus infection enriched the DEPs primarily involved in the metabolic pathways, endocytosis, adherens junction, and SNARE interactions in vesicular transport. The Ad5-p17 virus infection enriched the DEPs in endocytosis, ubiquitin-mediated proteolysis, N-Glycan biosynthesis, and apoptosis, while the Ad5-pB117L virus infection enriched the DEPs in metabolic pathways, endocytosis, oxidative phosphorylation, and focal adhesion. In summary, these results provide a comprehensive proteinomics analysis of the cellular responses to three ASFV membrane proteins, thus facilitating our understanding of ASFV pathogenesis.
PubMed: 38697277
DOI: 10.1016/j.micinf.2024.105348 -
Tissue Barriers May 2024Celiac disease (CD) is characterized by the disruption of the intestinal barrier integrity and alterations in the microbiota composition. This study aimed to evaluate...
Celiac disease (CD) is characterized by the disruption of the intestinal barrier integrity and alterations in the microbiota composition. This study aimed to evaluate the changes in the fecal microbiota profile and mRNA expressions of intracellular junction-related genes in pediatric patients with CD compared to healthy controls (HCs). Thirty treated CD patients, 10 active CD, and 40 HCs were recruited. Peripheral blood (PB) and fecal samples were collected. Microbiota analysis was performed using quantitative real-time PCR (qPCR) test. The mRNA expressions of ZO-1, occludin, β-catenin, E-cadherin, and COX-2 were also evaluated. In active and treated CD patients, the PB expression levels of ZO-1 ( = 0.04 and 0.002, respectively) and β-catenin ( = 0.006 and 0.02, respectively) were lower than in HCs. PB Occludin's level was upregulated in both active and treated CD patients compared to HCs ( = 0.04 and 0.02, respectively). However, PB E-cadherin and COX-2 expression levels and fecal mRNA expressions of ZO-1, occludin, and COX-2 did not differ significantly between cases and HCs (P˃0.05). Active CD patients had a higher relative abundance of the ( = 0.04) and ( = 0.03) phyla compared to treated subjects. The relative abundance of ( = 0.04) and ( = 0.01) genera was lower in active patients in comparison to HCs. Researchers should explore the precise impact of the gut microbiome on the molecules and mechanisms involved in intestinal damage of CD. Special attention should be given to and Enterobacteriaceae, as they have shown a significant correlation with the expression of tight junction-related genes.
PubMed: 38695199
DOI: 10.1080/21688370.2024.2347766 -
Frontiers in Immunology 2024Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We...
BACKGROUND
Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined.
METHODS
Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells.
RESULTS
In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened.
CONCLUSION
Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.
Topics: Animals; Female; Humans; Male; Mice; Caco-2 Cells; Carcinoembryonic Antigen; CD4-Positive T-Lymphocytes; COVID-19; Disease Models, Animal; Galectins; GPI-Linked Proteins; Intestinal Mucosa; SARS-CoV-2; Signal Transduction; Spike Glycoprotein, Coronavirus
PubMed: 38686388
DOI: 10.3389/fimmu.2024.1303356