Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Protein-protein interaction network analysis: (A) Protein-protein interaction network of cells treated with TAK-242 with TGFβ compared to TGFβ alone. (B) Top 20 hub genes identified using cytohubba in Cytoscape. (C) qPCR showing the fold increase in IL6 expression with TGFβ treatment in cardiac fibroblasts in vitro,n = 6,p < 0.05.

Article Snippet: Primary human cardiac fibroblasts (Promo Cell, Germany) were used for bulk RNA sequencing, and immortalized human cardiac fibroblasts (ABM, Canada) were utilized for all other functional assays and confirmatory in vitro experiments.

Techniques: Expressing, In Vitro

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: The expression of TLRs in adult cardiac fibroblasts: (A) FACS analysis for TLR4 in human cardiac fibroblasts with unstained control and cardiac fibroblasts stained with anti-TLR4 APC antibody. (B) Western blot and quantitative analysis showing the expression of TLR4 in CF in growth medium with 10 % serum, 2 % serum and with LPS stimulation.(C) Dot plot representing the expression of different TLRs in human cardiac fibroblast clusters. (D) Dot plot representing the average expression and percentage cells expressing different Tlr genes in healthy mouse adult Col1-GFP labelled cardiac fibroblasts by single-cell RNA seq data. (E) Temporal changes in gene expression of Tlr4 following cardiac injury: UMAP demonstrating the expression of Tlr4 in Col1-GFP single-cell RNA seq data of healthy and at different time points at 7 days, 14 days, and 30 days following injury. (F) Dot plot demonstrating the expression of Tlr4, Myd88, Tril, Traf6, Tab2, Mapk14, Nfkb1, and Jun in healthy control and injured Col1-GFP labelled cardiac fibroblasts at 7 days, 14 days and 30 days post-injury by single-cell sequencing (n = 3).

Article Snippet: Primary human cardiac fibroblasts (Promo Cell, Germany) were used for bulk RNA sequencing, and immortalized human cardiac fibroblasts (ABM, Canada) were utilized for all other functional assays and confirmatory in vitro experiments.

Techniques: Expressing, Control, Staining, Western Blot, RNA Sequencing Assay, Sequencing

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Expression of genes encoding cytokines: (A) Expression of cytokines and related genes in mouse Col1-GFP cardiac fibroblasts in healthy control and on different days following injury. (B) Expression of cytokines and related genes in human cardiac fibroblast clusters of healthy and heart failure patients' sample. (C) Expression of cytokines in human cardiac fibroblasts in basal conditions in vitro.

Article Snippet: Primary human cardiac fibroblasts (Promo Cell, Germany) were used for bulk RNA sequencing, and immortalized human cardiac fibroblasts (ABM, Canada) were utilized for all other functional assays and confirmatory in vitro experiments.

Techniques: Expressing, Control, In Vitro

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Inhibiting TLR4 signaling reduces differentiation and migration of myofibroblasts: (A) qPCR showing the expression of fibrotic genes, Collagen1A1 and α-Sma in cardiac fibroblasts treated with TGF-β, n = 8. (B) qPCR showing the expression of fibrotic markers Collagen1A1 and α-Sma in TGF-β plus TAK-242 treated cells compared to TGF-β with vehicle alone control,n = 5.(C) Scratch wound assay to determine the migration of cardiac fibroblasts in the presence of TGF−β plus TAK-242, TGF-β alone and controls cells in medium with 10 % FBS and 2 % FBS. (D) Percentage area of cardiac fibroblast migration in TGF-β condition versus TGF-β plus TAK-242 treatment and controls,n = 4. (E) Cardiac fibroblasts seeded on contractable collagen gel matrices were assayed for gel contraction after treatment with TGF-β and TGF-β plus TAK-242 for 24 h, n = 4. Data are represented as the mean ± SE, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, ns = p > 0.05.

Article Snippet: Primary human cardiac fibroblasts (Promo Cell, Germany) were used for bulk RNA sequencing, and immortalized human cardiac fibroblasts (ABM, Canada) were utilized for all other functional assays and confirmatory in vitro experiments.

Techniques: Migration, Expressing, Control, Scratch Wound Assay Assay

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Inhibiting TLR4 signaling reduces the expression of extracellular matrix genes: (A) GO enrichment analysis to determine the biological processess that are significantly altered with TGF-β treatment (for 48 h) in cardiac fibroblasts compared to control cells without TGF-β. (B) GO enrichment analysis to determine the biological processess significantly inhibited by TAK-242 in TGF-β treated cardiac fibroblasts. (C) Heatmap showing the scaled expression of extracellular matrix genes with TAK-242 treatment. (D) Expression of genes encoding profibrotic factors and extracellular matrix by qPCR in TGF-β+TAK-242 compared to TGF-β only, n = 4. Data are represented as the mean ± SE, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001.

Article Snippet: Primary human cardiac fibroblasts (Promo Cell, Germany) were used for bulk RNA sequencing, and immortalized human cardiac fibroblasts (ABM, Canada) were utilized for all other functional assays and confirmatory in vitro experiments.

Techniques: Expressing, Control

Journal: Cells

Article Title: Primary Human Cardiomyocytes and Cardiofibroblasts Treated with Sera from Myocarditis Patients Exhibit an Increased Iron Demand and Complex Changes in the Gene Expression

doi: 10.3390/cells10040818

Figure Lengend Snippet: Venn diagram ( A ) of overlapped up- and downregulated genes detected between two cell types (hCF and hCM). Significantly changed genes in hCM ( B ) and hCF ( C ) treated with sera from acute MCD patients vs. healthy controls. Student’ t -test, p FDR < 0.1, n = 3 per group. Abbreviations: hCM, human cardiomyocytes; hCF, human cardiofibroblasts.

Article Snippet: Primary human cardiofibroblasts (hCF, PromoCell) were precultured in Fibroblast Growth Medium supplemented with the recommended Supplemented Mix (PromoCell) and passaged 5 times before the experiment.

Techniques:

Journal: Cells

Article Title: Primary Human Cardiomyocytes and Cardiofibroblasts Treated with Sera from Myocarditis Patients Exhibit an Increased Iron Demand and Complex Changes in the Gene Expression

doi: 10.3390/cells10040818

Figure Lengend Snippet: Selected hints of the significantly dysregulated canonical pathways in hCM ( A ) and hCF ( B ) treated with sera from acute MCD patients vs. healthy controls. Selected hints of the significantly dysregulated cardiotoxicity functions in hCM ( C ) and hCF ( D ) treated with sera from acute MCD patients vs. healthy controls. Identified with Ingenuity Pathway Analysis (IPA) using respectively 1138 and 1050 protein-coding transcripts that changed significantly. Fisher’s exact test, p < 0.05. Abbreviations: hCM, human cardiomyocytes; hCF, human cardiofibroblasts; ERK5, extracellular-signal-regulated kinase 5; iNOS, inducible nitric oxide synthase; PAK, p21-activated kinases; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-7, interleukin 7; STAT3, signal transducer and activator of transcription 3; IL-6, interleukin 6.

Article Snippet: Primary human cardiofibroblasts (hCF, PromoCell) were precultured in Fibroblast Growth Medium supplemented with the recommended Supplemented Mix (PromoCell) and passaged 5 times before the experiment.

Techniques:

Journal: Cells

Article Title: Primary Human Cardiomyocytes and Cardiofibroblasts Treated with Sera from Myocarditis Patients Exhibit an Increased Iron Demand and Complex Changes in the Gene Expression

doi: 10.3390/cells10040818

Figure Lengend Snippet: The expression of TFR1 in human cardiomyocytes ( A – C ) and in human cardiofibroblasts ( D – F ) treated with patients’ sera at the mRNA and protein level, together with representative immunoblots ( C , F ). ANNOVA test followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test followed by Dunn’s multiple comparisons test (depending on data normality). * p < 0.05; ** p < 0.01; *** p < 0.001. The association between the protein level of TFR1 in human cardiac myocytes treated with patient sera and markers of iron status in patients’ peripheral blood i.e., transferrin saturation ( G ), serum iron ( H ), and serum ferritin ( I ). Gene and protein expressions were normalized to mean value in control group. Spearman’s rank correlation. * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: AU, arbitrary units; C, healthy controls, H, hospitalization (acute phase); 6wk, after 6 weeks of clinical recovery; TFR1, transferrin receptor 1; TSAT, transferrin saturation.

Article Snippet: Primary human cardiofibroblasts (hCF, PromoCell) were precultured in Fibroblast Growth Medium supplemented with the recommended Supplemented Mix (PromoCell) and passaged 5 times before the experiment.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: Cardiomyocyte-derived exosomes regulate cardiac fibroblast to myofibroblast transformation. ( A ) Schematic diagram for studying the effects of human cardiomyocyte (HCM)-derived exosome uptake by human cardiac fibroblasts (HCF) using early endosome antigen 1(EEA1) staining. ( B ) Immunocytochemistry and confocal microscopy identified the time-dependent intake and regulation of HCM-derived exosomes by EEA1 in HCFs. Nuclear DNA, exosomes, and EEA1 were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue), Dil dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine, red), and GFP fluorescence dye (green), respectively. Data are representative of three independent experiments ( n = 3). ( C ) HCFs were treated with or without HCM-derived exosomes, HCM+MCT-derived exosomes, or rTHBS2 (THBS2 recombinant protein, 1 μg/mL) for 48 h to assess cardiomyocyte-derived exosomal THBS2-mediated fibroblast to myofibroblast transformation. Expression of the myofibroblast marker proteins, α-SMA, β-catenin, fibronectin, and vimentin was examined by immunocytochemistry and confocal microscopy with Imaris software for confocal image 3D reconstruction. ( D , E ) Myofibroblast marker protein expression in myocardial tissues of MCT-induced PAH mice was examined by western blotting and immunohistochemistry. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05).

Article Snippet: Primary human cardiac fibroblasts (HCF; C-12375; PromoCell, Heidelberg, Germany) and primary human cardiac myocytes (HCM; ScienCell Research Laboratories, Carlsbad, CA, USA) were isolated from the ventricles of an adult heart and cultured in a specific culture media, namely, HCF growth medium (C-23010; PromoCell) and HCM medium (ScienCell Research Laboratories) supplemented with 1% penicillin/streptomycin solution (ScienCell Research Laboratories, Carlsbad, CA, USA) and 5% fetal bovine serum.

Techniques: Derivative Assay, Transformation Assay, Staining, Immunocytochemistry, Confocal Microscopy, Labeling, Fluorescence, Recombinant, Expressing, Marker, Software, Western Blot, Immunohistochemistry, MANN-WHITNEY