primary human cardiac fibroblasts  (PromoCell)

Journal: PLOS One

Article Title: The novel mineralocorticoid receptor modulator balcinrenone protects against diet-induced cardiac microvascular dysfunction and plasma potassium elevation in mouse models

doi: 10.1371/journal.pone.0341078

Figure Lengend Snippet: Primary human cardiac fibroblasts were stimulated for 24 hours with or without 10 nM aldosterone, and balcinrenone or eplerenone added to final concentrations of 0.5, 2.5, or 12.5 µM. (A) Collagen 1 and (B) IL-6 concentrations in the supernatant were measured using an enzyme-linked immunosorbent assay. Data are presented as means ± standard deviations. * P < 0.05 versus control; # P < 0.05 versus 10 nM aldosterone. Aldo, aldosterone; Ctrl, control; IL-6, interleukin-6.

Article Snippet: Primary human cardiac fibroblasts (PromoCell, Heidelberg, Germany) were cultured in Fibroblast Growth Medium 3 (PromoCell, Heidelberg, Germany) according to the manufacturer’s instruction and used between passages five and seven.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: Cardiomyocyte-derived TGFB3 attenuates cardiac fibrosis and preserves cardiac function in heart failure

doi: 10.1038/s41598-026-42367-5

Figure Lengend Snippet: TGFB3 upregulation predominantly occurs in cardiomyocytes under pathological conditions. (A) UMAP visualization of single-nucleus RNA sequencing (snRNA-seq) data from mouse hearts (dataset SCP1303, Single Cell Portal). Left: expression pattern of Tgfb3 across all cardiac cell populations, with color intensity representing normalized expression levels. Right: cells colored according to cluster identity. (B) Bar graph showing Tgfb3 expression levels in cardiomyocytes (CM), fibroblasts (FB), and endothelial cells (EC) from sham and TAC groups, derived from a publicly available transcriptomic dataset (GEO accession: GSE180720 ). (C) qPCR analysis of Tgfb3 expression in isolated cardiomyocyte (CM) and non-cardiomyocyte (non-CM) fractions ( n = 4 per group). (D) Immunoblot analysis of TGFB3 protein levels in CM and non-CM fractions. cTnT and αSMA were used as markers of CM and non-CM, respectively; HSP90 served as a loading control. (E) qPCR analysis of Tgfb3 expression in primary cardiomyocytes treated with AngII (5 µM) or vehicle for 24 h ( n = 3 per group). (F) qPCR analysis of Tgfb3 expression in HL-1 cells treated with AngII (5 µM) or vehicle for 24 h ( n = 3 per group). (G) Immunoblot analysis of TGFB3 protein levels in HL-1 cells treated with AngII (5 µM) or vehicle for 48 h; HSP90 served as a loading control. Data are presented as mean ± SEM from three independent experiments. Statistical significance was tested by two-tailed unpaired Student’s t test in ( B, C, E, F ). p-values are indicated above each comparison.

Article Snippet: HEK293T cells (ATCC, CRL-3216) and primary cardiac fibroblasts were maintained in complete medium consisting of high-glucose Dulbecco’s Modified Eagle Medium (DMEM; BasalMedia, L110KJ) supplemented with 10% fetal bovine serum (FBS; Sigma, F8318) and 1% penicillin–streptomycin (BasalMedia, S110JV).

Techniques: RNA Sequencing, Single Cell, Expressing, Derivative Assay, Isolation, Western Blot, Control, Two Tailed Test, Comparison

Journal: Scientific Reports

Article Title: Cardiomyocyte-derived TGFB3 attenuates cardiac fibrosis and preserves cardiac function in heart failure

doi: 10.1038/s41598-026-42367-5

Figure Lengend Snippet: Transcriptomic and mechanistic analysis of fibrosis-related pathways regulated by TGFB3. (A) Volcano plot showing differentially expressed genes in heart tissue from myocardium-specific Tgfb3 knockout (Tgfb3^ΔMyh6) versus control (Tgfb3^fl/fl) mice. (B) Reactome pathway enrichment analysis of upregulated genes from RNA-seq of Tgfb3^ΔMyh6 hearts. (C) Venn diagram and heatmap illustrating five fibrosis-related genes that are highly expressed in the hearts of Tgfb3^ΔMyh6 mice. (D) qPCR analysis of Serpinf1 and Ctgf expression in heart from Tgfb3^fl/fl and Tgfb3^ΔMyh6 mice ( n = 8 per group). (E) Immunoblot analysis of SERPINF1 and CTGF protein expression in heart from Tgfb3^fl/fl and Tgfb3^ΔMyh6 mice. HSP90 was used as a loading control. (F) Representative immunofluorescence images showing the localization of TGFB3 (green), CTGF (cyan), p-SMAD3 (red), and nuclei stained with DAPI (blue) in heart from Tgfb3^fl/fl and Tgfb3^ΔMyh6 mice. Scale bar, 50 μm. (G) qPCR analysis of Acta2, Ctgf and Serpine1 expression in primary cardiac fibroblasts (cFB) treated with vehicle, TGF-β1 (5 ng/mL), and/or TGF-β3 (5 ng/mL) for 24 h ( n = 4 per group). (H) Immunoblot analysis of CTGF and SERPINE1 protein expression in primary cardiac fibroblasts (cFB) treated with Veh, TGF-β1 (5ng/ml) and/or TGF-β3 (5ng/ml) for 48 h. HSP90 serving as a loading control. (I) Immunoblot analysis of p-SMAD3, SMAD3, p-SMAD2 and SMAD2 protein expression in primary cardiac fibroblasts (cFB) treated with Veh, TGF-β1 (5ng/ml) and/or TGF-β3 (5ng/ml) for 15 min. HSP90 serving as a loading control. (J) Luciferase assay results of HEK293T stimulated by Veh, TGF-β1 (5ng/ml) and/or TGF-β3 (5ng/ml) for 48 h. pCAGA12-luc plasmids were used for transfection. (K) Western blot analysis demonstrates co-immunoprecipitation of TGFB1, TGFB3 and TGFBR2 in primary cardiac analysis treated with recombinant TGFB1 and TGFB3. Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t-test ( D, G) and one-way ANOVA by Tukey’s multiple comparisons test ( J ). p-values are indicated above each comparison.

Article Snippet: HEK293T cells (ATCC, CRL-3216) and primary cardiac fibroblasts were maintained in complete medium consisting of high-glucose Dulbecco’s Modified Eagle Medium (DMEM; BasalMedia, L110KJ) supplemented with 10% fetal bovine serum (FBS; Sigma, F8318) and 1% penicillin–streptomycin (BasalMedia, S110JV).

Techniques: Knock-Out, Control, RNA Sequencing, Expressing, Western Blot, Immunofluorescence, Staining, Luciferase, Transfection, Immunoprecipitation, Recombinant, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Decoding the Cardiac Immune Microenvironment and Fibroblast Crosstalk in Radiotherapy Combined with Immunotherapy‐Induced Cardiac Fibrosis Based on Single‐Cell Transcriptomic Analysis

doi: 10.1002/advs.202519216

Figure Lengend Snippet: Cardiac fibroblasts exhibit significant pro‐fibrotic and pro‐inflammatory properties in mouse hearts subjected to ICI combined with CIR. (A) UMAP visualization of scRNA‐seq from fibroblasts in mouse hearts treated with ICI or/and CIR. (B) The proportion of fibroblast subclusters in mice hearts across 12 mouse heart samples from Con, ICI, IR, and iRT groups (left), and after merging by intervention group (right). (C) The number of DEGs in fibroblast subtypes of the mouse hearts for ICl vs Con, IR vs Con, and iRT vs Con. Red blocks represent upregulated genes, while teal blocks represent downregulated genes. (D) Stacked violin plot of the top 3 marker genes for fibroblast subtypes. (E) Heatmap of expression levels of extracellular matrix (ECM) molecules in fibroblasts from Con, ICI, IR, and iRT groups. (F) The box plots showing differential expression levels of pro‐fibrotic and fibrinolysis‐related factors in fibroblasts from cardiac tissues of Con, ICI, IR, and iRT groups. (G) The pro‐fibrotic interaction diagram of fibroblasts acting as receptor cells with other cell types in the iRT group. (H) The chemokine interaction diagram of fibroblasts acting as ligand cells with other cell types in the iRT group. (I) The chord diagram of the cell‐cell interactions networks of CCL, CXCL, and VCAM signaling pathway network in the iRT group. Data are presented as box plots showing the median, interquartile range, and potential outliers. Differences between groups were assessed using the nonparametric Wilcoxon rank‐sum test (F). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary cardiac fibroblasts (#CP‐M074, Procell Life Science & Technology) were isolated from the hearts of 1‐3‐day‐old C57BL/6 mice and authenticated via immunofluorescence staining, which confirmed positive expression of vimentin.

Techniques: Marker, Expressing, Quantitative Proteomics

Journal: Advanced Science

Article Title: Decoding the Cardiac Immune Microenvironment and Fibroblast Crosstalk in Radiotherapy Combined with Immunotherapy‐Induced Cardiac Fibrosis Based on Single‐Cell Transcriptomic Analysis

doi: 10.1002/advs.202519216

Figure Lengend Snippet: The increased infiltrating macrophages demonstrated a notable effect in promoting the activation of fibroblasts in cardiac fibrosis treated with ICI and CIR. (A) UMAP visualization of scRNA‐seq from immune cells in mouse hearts treated with ICI or/and CIR. (B) The proportion of immune cell subtypes in mice hearts across 12 mouse heart samples from Con, ICI, IR, and iRT groups (left), and after merging by intervention group (right). (C) Heatmap of the top 100 DEGs in each subset identified through unsupervised clustering of all the immune cells. Blue indicates lower expression, and red indicates higher expression. The expression scale is shown on the right. (D) Immune cell infiltration was analyzed by immunohistochemistry. CD45 + was used as a marker for leukocytes, F4/80 + for macrophages, and CD4 + and CD8 + were used to identify CD4 + T lymphocytes and CD8 + T lymphocytes, respectively, scale bar = 100 µ m . (E) UMAP visualization of scRNA‐seq from macrophages in mouse hearts treated with ICI and/or CIR. (F) The proportion of macrophage subtypes in mice hearts across 12 mouse heart samples from Con, ICI, IR, and iRT groups (left), and after merging by intervention group (right). (G) The number of DEGs in macrophage subtypes of the mouse hearts for ICl vs Con, IR vs Con, and iRT vs Con. Red blocks represent upregulated genes, while teal blocks represent downregulated genes. (H) The KEGG enrichment analysis of upregulated genes of macrophage subtypes. (I) The chemokine interaction diagram of MPs acting as ligand cells with other cell types in the iRT group. (J) The box plots showing differential expression levels of pro‐fibrotic and fibrinolysis‐related factors in macrophages from cardiac tissues of Con, ICI, IR, and iRT groups. (K) The dot plots showing differential expression levels of pro‐fibrotic and fibrinolysis‐related factors across macrophage subtypes in the iRT group. (L) The box plots showing the expression scores of pro‐fibrotic gene sets in CCR2 + and CCR2 − macrophage clusters. Data are presented as mean ± SD, the one‐way ANOVA (D) were used to compare data. Data are presented as box plots showing the median, interquartile range, and potential outliers, differences between groups were assessed using the nonparametric Wilcoxon rank‐sum test (J, L). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary cardiac fibroblasts (#CP‐M074, Procell Life Science & Technology) were isolated from the hearts of 1‐3‐day‐old C57BL/6 mice and authenticated via immunofluorescence staining, which confirmed positive expression of vimentin.

Techniques: Activation Assay, Expressing, Immunohistochemistry, Marker, Quantitative Proteomics

Journal: Advanced Science

Article Title: Decoding the Cardiac Immune Microenvironment and Fibroblast Crosstalk in Radiotherapy Combined with Immunotherapy‐Induced Cardiac Fibrosis Based on Single‐Cell Transcriptomic Analysis

doi: 10.1002/advs.202519216

Figure Lengend Snippet: Increased IL‐6 expression in the fibroblasts of mice in radioimmunotherapy‐induced cardiac fibrosis. (A) Trend analysis of differential gene expression in fibroblasts from Con, ICI, IR, and iRT groups, categorized into nine distinct expression pattern clusters. (B) Heatmap of the expression levels of the top 20 genes in cluster 1 across the four groups. (C) Distribution and expression of IL‐6 across nine cardiac cell types visualized in feature plot. (D) Distribution and expression of IL‐6 in fibroblasts among Con, ICI, IR, iRT groups visualized in the feature plot. (E) Immunofluorescence analysis of IL‐6 expression in mice cardiac tissues from Con, ICI, IR, and iRT groups at day 28 post‐treatment, scale bar = 50 µ m . (F) Quantification histogram of IL‐6 fluorescence intensity. (G) IL‐6 expression level of cardiac tissues in mice across four groups at day 28 post‐treatment detected by ELISA. (H) qRT‐PCR analysis of IL‐6 transcript levels in cardiac tissues from the four mouse groups at day 28 post‐treatment. (I) The post‐thoracic radiotherapy (post‐RT) and baseline (pre‐RT) of serum IL‐6 expression levels in 29 patients with malignant thoracic tumors were assessed by ELISA. (J) The post‐iRT(post‐iRT) and baseline (pre‐iRT) of serum IL‐6 expression levels in 15 patients with malignant thoracic tumors were assessed by ELISA. (K) Kaplan‐Meier curve showing overall survival in the cohort of 29 thoracic tumor patients. (L) Western blot analysis of Col1, Col3, α‐SMA, TGF‐β, and IL‐6 protein levels in primary mouse fibroblasts stimulated by murine IL‐6 cytokine with or without ruxolitinib for 48 h; quantitative analysis of protein expression levels. (M) qRT‐PCR analysis of IL‐6 transcript levels in primary mouse fibroblasts stimulated by murine IL‐6 cytokine with or without ruxolitinib for 48 h. (N) IF staining of α‐SMA, TGF‐β, Col1, and Col3 in primary mouse fibroblasts stimulated by murine IL‐6 cytokine with or without ruxolitinib for 48 h, scale bar = 50 µ m ; quantitative histograms of corresponding markers. Data are presented as mean ± SD. The Kaplan‒Meier method was used to assess patient survival (K). Differences between groups were assessed using the one‐way ANOVA (F, G, H, L, M, N) and the Wilcoxon signed‐rank test (I,J). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary cardiac fibroblasts (#CP‐M074, Procell Life Science & Technology) were isolated from the hearts of 1‐3‐day‐old C57BL/6 mice and authenticated via immunofluorescence staining, which confirmed positive expression of vimentin.

Techniques: Expressing, Gene Expression, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Staining

Journal: Advanced Science

Article Title: Decoding the Cardiac Immune Microenvironment and Fibroblast Crosstalk in Radiotherapy Combined with Immunotherapy‐Induced Cardiac Fibrosis Based on Single‐Cell Transcriptomic Analysis

doi: 10.1002/advs.202519216

Figure Lengend Snippet: IL‐6 positive fibroblasts involved in promoting cardiac inflammation and fibrosis. (A) UMAP visualization of scRNA‐seq from IL‐6 + and IL‐6 − fibroblasts in mouse hearts treated with ICI or/and CIR. (B) Heatmap of the top 20 DEGs between IL‐6 + and IL‐6 − fibroblasts identified through unsupervised clustering. Blue indicates lower expression, and yellow indicates higher expression. The expression scale is shown on the right. (C) The box plots showing the expression scores of pro‐fibrotic and ECM gene sets in IL‐6 + and IL‐6 − fibroblast clusters. (D) The immunofluorescence co‐localization analysis of inflammatory factor IL‐6 with leukocytes (CD45 + ), macrophages (F4/80 + ), CD8 + T cells, CD4 + T cells, cardiomyocytes (cTnI + ), fibroblasts (Vim + ), and endothelial cells (CD31 + ), scale bar = 50 µ m ; (E) The statistical column plots represent the comparison of colocalization scores among various indicators. (F) Network diagram depicting the interaction frequency between IL‐6 + fibroblasts and other cardiac cell types in Con, ICI, IR, and iRT groups. Line width represents the number of the ligand–receptor pairs. (G) The chemokine interaction diagram of IL‐6 + fibroblasts acting as ligand cells with other cell types in the iRT group. (H) Schematic of the experimental design for radioimmunotherapy in IL‐6 wild‐type (WT) and IL‐6 knockout (KO) mice ( n = 4 per group). (I) qRT‐PCR analysis of IL‐6 transcript levels in cardiac tissues from WT and IL‐6 KO mice. (J) Western blot analysis of Col1, Col3, TGF‐β, α‐SMA, and IL‐6 protein expression in hearts of WT and IL‐6 KO mice treated with radioimmunotherapy; quantitative analysis of protein expression levels ( n = 3 per group). (K) Representative multiplex immunofluorescence images of cardiac tissues from WT and IL‐6 KO mice after combined radioimmunotherapy, scale bar = 50 µ m . (L) Single‐channel immunofluorescence validation and quantitative analysis of the markers in cardiac tissues from WT and IL‐6 KO mice after combined radioimmunotherapy. Staining markers: DAPI (nuclei, blue), Col1 (green), Col3 (yellow), Fibronectin 1 (orange), TGF‐β (red), CD3 (T cells, brown), F4/80 (macrophages, light blue), scale bar = 50 µ m ; the corresponding quantitative histograms of each marker are shown. Data are presented as mean ± SD. The unpaired t‐test (I, J, L) and the one‐way ANOVA (E) was performed to compare data. Data are presented as box plots showing the median, interquartile range, and potential outliers, differences between groups were assessed using the nonparametric Wilcoxon rank‐sum test (C). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary cardiac fibroblasts (#CP‐M074, Procell Life Science & Technology) were isolated from the hearts of 1‐3‐day‐old C57BL/6 mice and authenticated via immunofluorescence staining, which confirmed positive expression of vimentin.

Techniques: Expressing, Immunofluorescence, Comparison, Knock-Out, Quantitative RT-PCR, Western Blot, Multiplex Assay, Biomarker Discovery, Staining, Marker

Journal: Advanced Science

Article Title: Decoding the Cardiac Immune Microenvironment and Fibroblast Crosstalk in Radiotherapy Combined with Immunotherapy‐Induced Cardiac Fibrosis Based on Single‐Cell Transcriptomic Analysis

doi: 10.1002/advs.202519216

Figure Lengend Snippet: Tocilizumab attenuates radioimmunotherapy‐induced cardiac injury and fibrosis via the bulk and single‐cell transcriptomic profiling. (A) Schematic of the of IL‐6 receptor (IL‐6 RA) inhibitor tocilizumab intervention in radioimmunotherapy‐induced cardiac injury mouse model. (B) Survival curves of mice over day 28 in Con, iRT, and Toci groups. Data are presented as Kaplan‐Meier survival curves, statistical analysis was performed using the Log‐rank test ( n = 8 per group). No significant difference in survival rate was observed among the three groups ( p = 0.14). Two cases of acute death occurred in the iRT group, while no mortality was detected in the control and Toci groups. (C) Body weight changes of mice in Con, iRT, and Toci groups at day 28 ( n = 6 per group). (D) HE and Masson's trichrome staining for estimating cardiac injury and fibrosis in Con, iRT, and Toci mice cardiac tissue at day 28 post‐treatment, scale bar = 50 µm. (E) Immunofluorescence analysis of IL‐6 expression levels in mice myocardial tissues from Con, iRT, and Toci groups, scale bar = 50 µ m ; quantification histogram of IL‐6 immunofluorescence intensity. (F) Cardiac tissue IL‐6 expression levels detected by ELISA in mice from Con, iRT, and Toci groups at day 28 ( n = 3 per group). (G) qRT‐PCR analysis of IL‐6 transcript levels in cardiac tissues from Con, iRT, and Toci groups at day 28 post‐treatment ( n = 3 per group). (H) Immunofluorescence staining of α‐SMA, TGF‐β, Col1, and Col3 in cardiac tissues of Con, iRT, and Toci groups at day 28 post‐intervention, scale bar = 50 µ m ; quantitative histograms of corresponding markers. (I) Western blot analysis of Col1, Col3, α‐SMA, and TGF‐β protein levels in myocardial tissues from Con, iRT, and Toci groups at day 28 post‐intervention; quantitative analysis of protein expression levels ( n = 3 per group). (J) Heatmap of the DEGs in cardiac tissues from Toci vs iRT groups based on bulk transcriptome analysis ( n = 3 per group). Blue indicates lower expression, and red indicates higher expression. The expression scale is shown on the right. (K) Expression scores of profibrotic and ECM gene sets from bulk transcriptomic analysis in Con, iRT, and Toci groups at day 28. (L) The dot plot showing IL‐6 expression levels across nine cardiac cell types in Con, iRT, and Toci groups ( n = 3 per group) based on scRNA‐seq. (M) Proportion analysis of IL‐6 + and IL‐6 − fibroblasts in Con, iRT, and Toci groups from scRNA‐seq data. (N) The heatmap displaying expression levels of ECM molecules (left) and profibrotic/fibrinolysis‐related factors (right) in fibroblasts across the three groups based on scRNA‐seq. (O) The dot plot showing IL‐6RA expression across nine cardiac cell types in Con, iRT, and Toci groups based on scRNA‐seq. (P) Proportion analysis of CCR2 + and CCR2 − macrophages in cardiac tissues across the three groups based on scRNA‐seq. (Q) Heatmap of IL‐6 signaling pathway interactions among cardiac cell types in iRT (upper) and Toci (lower) groups based on scRNA‐seq. Data are presented as mean ± SD. Differences between groups were assessed using the one‐way ANOVA (E, F, G, H, I, K), and the nonparametric Wilcoxon rank‐sum test (M, P). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary cardiac fibroblasts (#CP‐M074, Procell Life Science & Technology) were isolated from the hearts of 1‐3‐day‐old C57BL/6 mice and authenticated via immunofluorescence staining, which confirmed positive expression of vimentin.

Techniques: Single Cell, Control, Staining, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot