rat cardiac fibroblasts  (Cell Applications Inc)

Journal: Cell Systems

Article Title: Cold and hot fibrosis define clinically distinct cardiac pathologies

doi: 10.1016/j.cels.2025.101198

Figure Lengend Snippet: Acute myocardial infarction results in cold fibrosis (A) Mathematical model of the myofibroblast-macrophage cell circuit predicts three possible outcomes: hot fibrosis, cold fibrosis, and healing based on the abundance of these two populations (axes). The basin of attraction for the healing state is bounded by a separatrix. The scheme also denotes the growth factor signaling as either ON (black lines) or OFF (gray lines) at each specific fixed point. (B) Single-cell RNA sequencing (scRNA-seq) dynamic analysis of total macrophage and myofibroblast populations (% of total interstitial cells) in an adult mouse heart following-MI data obtained from Forte et al. Data are represented as cell composition fold change (FC) to day 0. These data are further described in Figures S1 A and S1B. UMAP of clusters highlighting macrophages (green, full line) and myofibroblasts (red, straited line). Cell-type clusters also include smooth muscle cells, fibroblasts, endothelial cells, granulocytes, NK/T cells, and B cells. (C) Breeding strategy used to create the macrophage-myofibroblast double-reporter (MAMY) mice. First, we crossed Postn MCM/MCM mice with Rosa26 tdTom a to/tdTom a to mice to obtain an established tamoxifen-inducible cardiac myofibroblasts lineage-tracer , . We then crossed these mice with a global monocyte/macrophage reporter-Cx3cr1 GFP/GFP . Triple-heterozygote mice were used as experimental animals (Postn MCM/+ Rosa26 TdTomto/+ Cx3cr1 GFP/+ ). (D) Experimental scheme whereby MAMY mice (Postn MCM/+ Rosa26 TdTomto/+ Cx3cr1 GFP/+ ) underwent sham or MI by permanent left anterior descending artery (LAD) ligation. To label myofibroblasts with tdTomato, mice were given daily tamoxifen injections following MI (red line). Cardiac function was tracked using 2D echocardiography at multiple time points following MI: 7 days prior to MI (baseline), and 7, 14, and 28 days post-MI (black arrows). Hearts were collected for histological fibrosis and immunofluorescence (IF) analysis on days: 2, 4, 7, 14, and 28 post-MI/sham (red arrows). (E) Ejection fraction (EF; %) analysis was performed on MI/Sham operated MAMY mice (Sham, n = 4; MI, n = 7). To extract the direct % change in EF between days 28 post-MI and baseline, we calculated the ΔEF value per animal . Red, MI; blue, sham. Results are represented as mean ± SEM. Statistical analysis performed using two-way ANOVA with Sidak’s adjusted p values (left) or two-tailed unpaired t test (right). Individual points represent individual biological replicates. (F) Representative histology images of MAMY hearts stained with sirius red on days 2, 4, 7, 14, and 28 post-MI/sham. Scale bar, 1 mm. Collagen, red; healthy myocardium, yellow. (G) MAMY mice fibrosis area/left ventricle (LV) in % was calculated per timepoint: Sham ( n = 12), day 2 ( n = 7), day 4 ( n = 4), day 7 ( n = 9), day 14 ( n = 4), and 28 ( n = 13) post-MI. Mix-max violin plot represent the median (middle full line) and quartiles (striated lines). Statistical analysis performed using one way ANOVA with Tukey’s multiple comparisons test. (H–K) Quantification of spatiotemporal distribution of cardiac-troponin-T cells (cardiomyocytes, cTnT+; purple), the lineage-traced (tdTomato+; red) myofibroblasts, and macrophages (GFP+, green) in Sham hearts and infarct zone of MI operated hearts. cTnT+, tdTomato+, and GFP+ cells were also measured at the suture area of 28 days post-MI hearts ( , Figure S1 C). (H) Representative immunofluorescence (IF) images of Sham ( n = 5; 1,487 ± 118.5 SEM cells per replicate) and MI operated hearts on days 2 ( n = 4; 1,088 ± 341.8 SEM cells per replicate), 4 ( n = 4; 1,027 ± 352.6 SEM cells per replicate), 7 ( n = 4; 1,792 ± 153.3 SEM cells per replicate), 14 ( n = 3; an average of 1,471 ± 275.2 SEM cells per replicate), and 28 ( n = 5; 1,627 ± 248.7 and 1,049 ± 315.9 SEM cells per replicate for infarct zone and suture, respectively) post-MI. Images are represented as either single channel for GFP, tdTomato, and cTnT, as a merged IF image and as a cell-type map that illustrates the identified cells as representative points. White scale bar, 100 μm. cTnT+ cells (I), GFP+ cells (J), and tdTomato+ cells (K) were quantified as count per 100 μm neighborhood or as % of cells per 100 μm neighborhood ( , Figure S1 C). Results are represented as mean ± SEM. In (I)–(K), infarct zone is denoted as a full circle, day 28 suture is denoted as an upside-down triangle. Statistical analysis performed using the Mann-Whitney test with Bonferroni adjusted p values (I–K). (L) Experimental scheme whereby adult Hsd:ICR (CD1) mice hearts underwent MI by permanent LAD ligation and further processed by flow cytometry . LV samples below the suture of MI or sham operated mice were collected at 6 different time points following injury at days: 0/Sham ( n = 11), 2 ( n = 5), 4 ( n = 5), 7 ( n = 7), 14 ( n = 5), and 65 ( n = 5). (M–Q) (M) Representative flow cytometry plots of the gating scheme used to identify (N) total cardiac macrophages (out of total immune cells- CD45 + ), (O) infiltrating cells (monocytes and neutrophils out of total immune cells-CD45 + ), (P) cardiac activated macrophages, measured by CD11b mean fluorescence intensity (MFI), and (Q) Resident macrophages (TIM4 + macrophages out of total macrophages. Data presented as mean ± SEM. Statistical analysis performed using one way ANOVA with Dunnett’s multiple comparisons procedure.

Article Snippet: For primary cardiac myofibroblast enriched cultures, cells were first isolated from adult (10-12 weeks) female ICR mice hearts using a neonatal dissociation kit (Miltenyi Biotec,130-098-373) and gentleMACS homogenizer (Miltenyi Biotec).

Techniques: RNA Sequencing, Ligation, Immunofluorescence, Two Tailed Test, Staining, MANN-WHITNEY, Flow Cytometry, Fluorescence

Journal: Cell Systems

Article Title: Cold and hot fibrosis define clinically distinct cardiac pathologies

doi: 10.1016/j.cels.2025.101198

Figure Lengend Snippet: Chronic ventricular pressure overload results in hot fibrosis (A) Experimental scheme whereby MAMY mice (Postn MCM/+ Rosa26 TdTomto/+ Cx3cr1 GFP/+ ) underwent sham or transverse aortic constriction (TAC) to induce chronic pressure overload. To label myofibroblasts with tdTomato, mice were given 7 daily tamoxifen injections immediately following TAC, and on days 13–14 and 27–28 post-TAC (red line). Cardiac function was tracked using 2D echocardiography at baseline (−7) and on multiple time points following TAC: 7, 14, and 28 days (black arrows). Hearts were collected for histological fibrosis and immunofluorescence (IF) analysis 28 post-MI/sham (red arrows). Heart weight (HW) to body weight (BW) ratios were further analyzed 28 days following TAC (green arrow). (B–E) Temporal echocardiography measurements of MAMY mice following TAC/sham, including (B) aortic pressure gradient (mmHg), (C) left ventricle anterior wall diameter in diastole (LVAW;d, in mm), (D) left ventricle posterior wall diameter in diastole (LVPW;d, in mm), and (E) ejection fraction (EF; %). Results are represented as mean ± SEM. Statistical analysis performed using two-way ANOVA with Sidak’s adjusted p values. (F) HW/BW ratios (mg/gr) of MAMY mice sham ( n = 4) and TAC ( n = 9) hearts 28 days post-TAC. Mix-max violin plot represent the median (middle full line) and quartiles (striated lines). Statistical analysis performed using two-tailed unpaired t test. (G) Left: representative images of MAMY hearts sirius red stained sections, 28 days post-TAC/sham. Scale bar, 1 mm. Right: fibrosis in TAC hearts was quantified separately based on its histological presentation: interstitial, replacement and perivascular fibrosis. Scale bar, 100 μm. Collagen, red; healthy myocardium, yellow. (H) MAMY mice TAC interstitial ( n = 9) and replacement ( n = 8) fibrosis (fib) field of views (FOVs) were compared with healthy sham ( n = 4) myocardium (data presented as average %/FOV). Mix-max violin plot represent the median (middle full line) and quartiles (striated lines). Statistical analysis performed using one way ANOVA with Tukey’s multiple comparisons test. (I) MAMY mice TAC perivascular fibrosis ( n = 9) was compared with sham perivascular regions ( n = 4). Average fibrosis area/FOV (%) was calculated. Mix-max violin plot represent the median (middle full line) and quartiles (straited lines). Statistical analysis performed using two-tailed unpaired t test. (J–M) MAMY mice neighborhood quantification of cardiac-troponin-T cells (cardiomyocytes, cTnT+; purple), the lineage-traced (tdTomato+, red) myofibroblasts, and macrophages (GFP+, green) in TAC/Sham hearts ( , similar analysis demonstrated for MI hearts in Figure S1 C). (J) Representative IF images of interstitial and perivascular sham ( n = 4; 1,833 ± 117.4 and 946.5 ± 71.68 SEM cells per replicate, respectively), and interstitial ( n = 6; 1,422 ± 156.7 SEM cells per replicate), replacement ( n = 6; 1,474 ± 286.5 SEM cells per replicate) and perivascular ( n = 6; 963.5 ± 68.77 SEM cells per replicate) fibrosis (fib) in hearts 28 days post-TAC. Images are represented as either single channel for: GFP (green), tdTomato (red), and cTnT (purple), as a merged IF image and as a cell-type map illustrated the identified cells as representative points. White scale bar, 100 μm. cTnT+ cells (K), GFP+ cells (L), and tdTomato+ cells (M) were quantified as count per 100 μm neighborhood or as % of cells per 100 μm neighborhood ( , Figure S1 C). Results are represented as mean ± SEM. Each dot represents a single biological replicate. Statistical analysis performed using the Mann-Whitney test with Bonferroni-adjusted p values when appropriate.

Article Snippet: For primary cardiac myofibroblast enriched cultures, cells were first isolated from adult (10-12 weeks) female ICR mice hearts using a neonatal dissociation kit (Miltenyi Biotec,130-098-373) and gentleMACS homogenizer (Miltenyi Biotec).

Techniques: Immunofluorescence, Two Tailed Test, Staining, MANN-WHITNEY

Journal: Cell Systems

Article Title: Cold and hot fibrosis define clinically distinct cardiac pathologies

doi: 10.1016/j.cels.2025.101198

Figure Lengend Snippet: Cold fibrosis after MI is conserved in humans and in a clinically relevant porcine model (A and B) (A) Representative human left ventricle spatial transcriptomics slides (Visium) of patients following acute-myocardial infarction (MI) and non-transplanted donor hearts. Samples were divided based on time following MI as either: uninjured ( n = 10), early (days 0–15 post-MI; n = 6), and late (30 days+; n = 6). (B) Abundance of fibroblasts and myeloid cells was quantified based on deconvolution scores of cell types per spot . Myofibroblast were calculated as enrichment of the mean myofibroblast state score within spots with a minimal 10% value of cell-type abundance . Statistical analysis used Wilcoxon tests with Benjamini-Hochberg adjusted p values. (C) Experimental design of pig MI experiment: adult (3 months old) pigs underwent MI by temporarily occluding their LAD using a balloon catheter . Following reperfusion (balloon deflation), pigs were immediately treated with recombinant human Agrin (rhAgrin) or Saline control, in an antegrade trajectory. Injured pig hearts were collected at either day 3 ( n = 4 for rhAgrin; n = 3 for Saline) or day 28 ( n = 4 for rhAgrin; n = 3 for saline) and dissected to distinct tissue areas (infarct and remote zones). Remote and infarcted samples were subjected to bulk-mRNA sequencing and histology for fibrosis assessment. (D and E) Representative sirius red staining images are shown from (D) day 3 and (E) day 28 post-MI. Fibrosis was quantified as the average % fold change (FC) between infarct/remote zone sections for each pig individually. Fibrosis (day 3 or day 28) for Saline and rhAgrin hearts was measured using two-tailed unpaired t test. Scale bars: 1 mm. striated line denotes FC = 1. n.s, non-significant difference. (F and G) Heatmaps based on log 2 transformed normalized counts for all (upregulated and downregulated) differentially expressed genes (defined by |log 2 fold change| ≥ 1, p -adjusted value < 0.05 and max raw counts > 30) between remote and infarct zones for Saline and rhAgrin-treated hearts at day 3 (5,961 genes) (F) and 28 (1,079 genes) (G) post-MI. Rows represent genes and columns represent each biological sample and its spatial distribution according to infarct or remote zone. Data are represented as mean ± SD. (H) Hierarchical clustering per condition (day [3 or 28] +treatment [rhAgrin or Saline]), based on the 1,000 most variable genes. Triangles and circles represent remote and infarct zones, respectively. (I) Deconvolution of bulk-mRNA sequencing of pig hearts following MI of either rhAgrin (blue) or Saline-treated (red) samples. Macrophage and myofibroblast abundances were assessed by gene signatures as FC, between infarct and remote zones ( , C, and Table S5 ). (J) Macrophage and myofibroblast abundances, based on deconvolution of bulk-mRNA sequencing (as in I). Treated samples were compared per time point (day 3 or 28), separately by two-tailed unpaired Student t test. Results are represented as mean ± SEM.

Article Snippet: For primary cardiac myofibroblast enriched cultures, cells were first isolated from adult (10-12 weeks) female ICR mice hearts using a neonatal dissociation kit (Miltenyi Biotec,130-098-373) and gentleMACS homogenizer (Miltenyi Biotec).

Techniques: Recombinant, Saline, Control, Sequencing, Staining, Two Tailed Test, Transformation Assay

Journal: Cell Systems

Article Title: Cold and hot fibrosis define clinically distinct cardiac pathologies

doi: 10.1016/j.cels.2025.101198

Figure Lengend Snippet: In cold fibrosis, macrophages return to homeostatic functions whereas fibroblasts acquire a persistent fibrotic cell state (A) Pareto analysis was used to characterize the continuum of gene expression within each cell type as distributed between specialist and generalist cells. Pareto analysis was done using ParTI. Fibroblasts (including myofibroblasts) from day 0 (black) and day 3 (green) following MI fill in a 5-vertex polyhedron in gene expression space ( p value < 0.02). The cells are projected on the first three principal components . (B) Gene expression of the top enriched genes for the day 0 + 3 post-MI fibroblast archetypes. Upper: cells within the polyhedron are colored by the normalized mRNA expression level of each gene (on a scale of 0–1). Lower: mRNA expression level plotted as a function of the euclidean distance from each archetype (in gene expression space) where cells were binned into 10 bins and the mean expression across the bins was computed. (C) Fold change (FC) of the proportion of fibroblasts from day 3 (green) and day 0 (black) that are closest to each archetype. (D) Macrophages from day 0 (black) and day 3 (green) following MI fill in a 5-vertex polyhedron in gene expression space ( p value < 0.001). The cells are projected on the first three principal components . (E) Gene expression of the top enriched genes for the day 0 + 3 post-MI macrophage archetypes. Upper: cells within the polyhedron are colored by the normalized mRNA expression level of each gene (on a scale of 0–1). Lower: mRNA expression level plotted as a function of the euclidean distance from each archetype (as in B). (F) FC of proportion of macrophages from day 3 (green) and day 0 (black) that are closest to each archetype. (G) Fibroblasts (including myofibroblasts) from day 0 (black) and day 28 (red) following MI fill in a 4-vertex polyhedron in gene expression space ( p value < 0.0001). The cells are projected on the first three principal components . (H) Gene expression of the top enriched genes for the day 0 + 28 post-MI fibroblast archetypes. Upper: cells within the polyhedron are colored by the normalized mRNA expression level of each gene (on a scale of 0–1). Lower: mRNA expression level plotted as a function of the euclidean distance from each archetype (as in B). (I) FC of proportion of fibroblasts from day 28 (red) and day 0 (black) that are closest to each archetype. (J) Macrophages from day 0 (black) and day 28 (red) following MI fill in a 4-vertex polyhedron in gene expression space ( p value < 0.02). The cells are projected on the first three principal components . (K) Gene expression of the top enriched genes for the day 0 + 28 post-MI macrophage archetypes. Upper: cells within the polyhedron are colored by the normalized mRNA expression level of each gene (on a scale of 0–1). Lower: mRNA expression level plotted as a function of the euclidean distance from each archetype (as in B). (L) FC of proportion of macrophages from day 28 (red) and day 0 (black) that are closest to each archetype. Archetypes for macrophages and fibroblasts are denoted based on color (1, red; 2, green; 3, blue; 4, yellow; 5, purple).

Article Snippet: For primary cardiac myofibroblast enriched cultures, cells were first isolated from adult (10-12 weeks) female ICR mice hearts using a neonatal dissociation kit (Miltenyi Biotec,130-098-373) and gentleMACS homogenizer (Miltenyi Biotec).

Techniques: Gene Expression, Expressing

Journal: Cell Systems

Article Title: Cold and hot fibrosis define clinically distinct cardiac pathologies

doi: 10.1016/j.cels.2025.101198

Figure Lengend Snippet: Targeting the autocrine loop of myofibroblasts by inhibiting TIMP1 reduces fibrosis after acute MI in adult mice (A) Phase plot where the axes represent the number of cells. The separatrix (dashed lines) separates the basins of attraction of healing (gray area) and cold fibrosis (white). The separatrix is calculated with reference (WT) parameters (black), and with a 30% decrease in the production rates of the macrophage paracrine growth factor (green), myofibroblast paracrine growth factor (blue), and myofibroblast autocrine growth factor (red). (B) Simulations of the cell-circuit response (red lines) to acute injury that leads to cold fibrosis with reference (WT) parameters (left). Healing is seen with a 42% or larger decrease in the myofibroblast autocrine growth factor production rate. (C) Schematic representation of NicheNet analysis to identify ligand-receptor interactions between fibroblasts (Fs), macrophages (Ms), and myofibroblasts (mFs) at days 3 and 28 following MI. NicheNet performed on the scRNA-seq data of Figures 1 B, A, and S1B. (D) Heatmap of potential ligands based on F, M, and mF differentially expressed (DE) genes at days 3 (right) and 28 (left) post-MI. Ligand scores (Pearson correlation) and average ligand expression per cell type are presented as mean ± SD (by either white-orange scale or blue-red scale, respectively). (E and F) Weighted interactions between F, M, and mF following MI (days 3 and 28) . In (E), line color represents weighted interaction strength; circle size represents the fold change (FC) shift in cluster size compared with day 0. In (F), individual weighted interactions/pair of communicating cells is plotted as a bar graph for both days 3 and 28. Green arrow, increase in weighted interaction strength; red arrow, decrease in weighted interaction strength. Inset graph showing the individual weighted interactions in log 10 scale. (G) Representative immunofluorescence images of TIMP1 (green), LRP1 (red), alpha smooth muscle actin (ɑSMA, gray), and DAPI (blue) in primary adult cardiac myofibroblasts cultures. ( n = 5 biological replicates, 2,510 ± 515 SEM cells per replicate). Scale bars, 50 μm, white frame represents the inset on the right. (H) Quantification of images based on their TIMP1, LRP1, and ɑSMA protein expression (as % of total cells) presented as mean ± SEM. Statistical analysis performed using one way ANOVA with Tukey’s multiple comparisons test. (I) Top: schematic representation of cardiac myofibroblasts proliferation assay in vitro . Primary cardiac myofibroblasts were treated in cell culture for 48 h with either recombinant mouse TIMP1 (rmTIMP1) or anti-TIMP1 neutralizing antibodies (ɑTIMP1) and the appropriate control (either PBS or IgG, respectively). Cells were further incubated with 5-ethynyl-2′-deoxyuridine (EdU) to directly measure cell cycle using flow cytometry. Bottom: cardiac myofibroblast proliferation was assessed in cultures by means of delta EdU + (%) cells of control (either PBS or IgG) and 48-h-treated cells (either rmTIMP1 or ɑTIMP1, respectively). In rmTIMP1 and ɑTIMP1 experiments, n = 4 and 5 biological replicates, respectively. Data are represented as mean ± SEM. Avg, average. Statistical analysis performed using two-tailed paired t test. (J) Top: schematic representation of non-human-primate (NHP) cardiac fibroblasts (cFs) proliferation assay in vitro . Primary cardiac fibroblasts were treated in cell culture for 48 h with either full recombinant human TIMP1 (rhWT-TIMP1; n = 3 replicates) or with the human N terminus fragment of TIMP1 (rhN-TIMP1; n = 3 replicates) anti-TIMP1 and control (untreated cells; n = 3 replicates). Cells were further incubated with 5-ethynyl-2′-deoxyuridine (EdU) to directly measure cell cycle using flow cytometry. Bottom: NHP-cFs proliferation was assessed in cultures by FC of EdU + (%) cells relative to control, respectively. Data are represented as mean ± SEM. Statistical analysis performed using one way ANOVA with Tukey’s multiple comparisons test. (K) Schematic experimental plan by which adult MAMY mice underwent MI and treated with either TIMP1 neutralizing antibodies (ɑTIMP1, n = 5) or IgG control ( n = 4). To activate the MerCreMer system and obtain tdTomato expression, tamoxifen was injected daily following MI . To label proliferating cells, mice were induced with a 24 h EdU pulse on day 3, and hearts were collected on day 4 post-MI for histological and immunofluorescence analysis. (L) Representative immunofluorescence images of MAMY hearts treated with either ɑTIMP1 or IgG control 4 days post-MI and 24 h EdU pulse. Scale bar, 100 μm. GFP-green, tdTomato-red, EdU-white, and DAPI-blue. Following segmentation and annotation cells were further analyzed based on their proliferative state, EdU±: myofibroblasts (tdTomato+), macrophage (GFP+), other (tdTomato−GFP−). Images are represented either by their immunofluorescence channels or as a cell-type proliferation map illustrates the identified cells and their proliferative state as representative points. (M) Quantification of proliferative myofibroblasts (tdTomato+EdU+) abundance (% of cells) per cellular neighborhood (100 μm in diameter) in the infarct zone of ɑTIMP1 or IgG-control-treated MAMY hearts ( , Figure S9 A). Left: distribution of data per tissue slice per biological replicate. Each color represents a biological replicate. Right: average tdTomato+EdU+/neighborhood per biological replicate. Results are represented as mean ± SEM. Blue circles: IgG control; red triangle: ɑTIMP1. Statistical analysis performed using the Mann-Whitney test. (N) Schematic experimental plan by which adult mice underwent MI and treated with either ɑTIMP1 ( n = 12 biological replicates) or IgG control ( n = 11 biological replicates) immediately after injury onset and 3 days after injury. At 35 days left ventricular (LV) fibrosis was assessed by sirius red staining. (O) Representative sequential sirius red staining images along the base to apex axis of ɑTIMP1/IgG-control-treated hearts. Scale bars: 2 mm. (P) Fibrosis parameters presented as % of total sections that are either transmural, non-transmural, or sections with no scar , and scar area out of the LV quantification. Statistical analysis performed using two-tailed unpaired t test.

Article Snippet: For primary cardiac myofibroblast enriched cultures, cells were first isolated from adult (10-12 weeks) female ICR mice hearts using a neonatal dissociation kit (Miltenyi Biotec,130-098-373) and gentleMACS homogenizer (Miltenyi Biotec).

Techniques: Expressing, Immunofluorescence, Proliferation Assay, In Vitro, Cell Culture, Recombinant, Control, Incubation, Flow Cytometry, Two Tailed Test, Injection, MANN-WHITNEY, Staining

Journal: Journal of Thoracic Disease

Article Title: TIMP1 regulation of cardiac fibroblast proliferation via the TGF-β/Smad pathway in an in vitro model of atrial fibrillation

doi: 10.21037/jtd-2025-1088

Figure Lengend Snippet: Effect of ang II on the expression of TIMP1 , TGF-β/Smad pathway-related proteins, and α-SMA. (A) qRT-PCR was performed to assess the mRNA expression levels of TIMP1 in rat cardiac fibroblasts treated with different concentrations of ang II (1 nM, 10 nM, 100 nM, and 1 μM). (B) qRT-PCR was also performed to assess the mRNA expression levels of TIMP1 in rat atrial fibroblasts subjected to different durations of 1 μM of ang II treatment (12, 24, and 48 hours). (C) WB was used to detect the expression of TGF-β1, p-Smad2, p-Smad3, Smad2, Smad3 and α-SMA after 48 hours of treatment with 1 μM of ang II. (D) The results of WB analysis for the same proteins are shown. Each experiment was performed with at least three biological replicates. *, P<0.05. TIMP1 , tissue inhibitor of metalloproteinases-1; ang II, angiotensin II; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting.

Article Snippet: Rat primary cardiac fibroblasts were purchased from Stemcell Biotechnology Co., Ltd. (cat. no. STM-CE-3303; Shanghai, China; https://www.stemrecell.com/primary-cell-rat-fibroblast/cardiacmuscle.html ).

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Thoracic Disease

Article Title: TIMP1 regulation of cardiac fibroblast proliferation via the TGF-β/Smad pathway in an in vitro model of atrial fibrillation

doi: 10.21037/jtd-2025-1088

Figure Lengend Snippet: TIMP1 modulation altered the ang II-mediated effects on rat atrial fibroblast viability and proliferation. (A,B) qRT-PCR and WB were used to detect the TIMP1 knockdown efficiency in rat cardiac fibroblasts treated with 1 μM of ang I for 48 hours. (C,D) Evaluation of TIMP1 overexpression efficiency in rat atrial fibroblasts after 48 hours of 1-μM ang II treatment. (E) qRT-PCR was used to detect the expression of TIMP1 in cardiac fibroblasts treated with 1 μM of ang II for 48 hours following knockdown or overexpression of TIMP1 . (F) Cell viability of rat cardiac fibroblasts was assessed by CCK-8 assay after treatment with 1 μM of ang II, either alone or in combination with negative control (NC), TIMP1 knockdown (si- TIMP1 -1), or TIMP1 overexpression (over- TIMP1 ). (G) Colony formation assay results of rat cardiac fibroblasts treated with control, 1 μM of ang II alone, ang II combined with NC, si- TIMP1 -1, or over- TIMP1 . Observed using a stereomicroscope and photographed with a scanner. Each experiment was performed with at least three biological replicates. *, P<0.05. TIMP1 , tissue inhibitor of metalloproteinases-1; ang II, angiotensin II; CCK-8, Cell Counting Kit-8; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting.

Article Snippet: Rat primary cardiac fibroblasts were purchased from Stemcell Biotechnology Co., Ltd. (cat. no. STM-CE-3303; Shanghai, China; https://www.stemrecell.com/primary-cell-rat-fibroblast/cardiacmuscle.html ).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Expressing, CCK-8 Assay, Negative Control, Colony Assay, Control, Cell Counting, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Thoracic Disease

Article Title: TIMP1 regulation of cardiac fibroblast proliferation via the TGF-β/Smad pathway in an in vitro model of atrial fibrillation

doi: 10.21037/jtd-2025-1088

Figure Lengend Snippet: Effects of TIMP1 regulation on fibrosis marker expression and TGF-β/Smad signaling in ang II-induced rat cardiac fibroblasts. (A-C) The mRNA expression levels of fibrotic markers, collagen I , collagen III , and α-SMA in rat cardiac fibroblasts were analyzed via qRT-PCR and WB assay. The experimental groups of rat cardiac fibroblasts were as follows: control, 1 μM of ang II, 1 μM of ang II + NC, 1 μM of ang II + si- TIMP1- 1, and 1 μM of ang II + over- TIMP1 . (D,E) WB analysis of the protein expression levels of the TGF-β/Smad3 signaling pathway components in rat cardiac fibroblasts, including TGF-β1, phosphorylated Smad2 (p-Smad2), phosphorylated Smad3 (p-Smad3), Smad2, and Smad3. The experimental groups of rat cardiac fibroblasts were as follows: control, 1 μM of ang II, 1 μM of ang II + NC, 1 μM of ang II + si- TIMP1- 1, and 1 μM of ang II + over- TIMP1 . Each experiment was performed with at least three biological replicates. *, P<0.05. TIMP1 , tissue inhibitor of metalloproteinases-1; ang II, angiotensin II; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting.

Article Snippet: Rat primary cardiac fibroblasts were purchased from Stemcell Biotechnology Co., Ltd. (cat. no. STM-CE-3303; Shanghai, China; https://www.stemrecell.com/primary-cell-rat-fibroblast/cardiacmuscle.html ).

Techniques: Marker, Expressing, Quantitative RT-PCR, Control, Negative Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biomaterials Research

Article Title: Myofibroblast-Targeting Extracellular Vesicles: A Promising Platform for Cardiac Fibrosis Drug Delivery

doi: 10.34133/bmr.0179

Figure Lengend Snippet: FAP expression is up-regulated in the myocardium of isoproterenol (ISO) mice and in cardiac fibroblasts upon TGF-β1 stimulation. (A) Quantitative polymerase chain reaction (qPCR) analysis showing messenger RNA (mRNA) expression levels of Col1α1 , Acta2 , and FAP in heart tissue from ISO-treated mice. n = 6. Data are presented as mean ± SEM. ** P < 0.01; **** P < 0.0001. (B) Western blotting analysis of Col1α1, α-smooth muscle actin (α-SMA), and FAP protein expressions in the hearts of mice treated with ISO or saline. (C) Sirius Red staining and immunohistochemical staining for FAP in hearts from ISO-treated mice. (D) mRNA expression levels of Col1α1 , Acta2 , and FAP in neonatal rat cardiac fibroblasts (NRCFs) treated with TGF-β1 (10 ng/ml) for 24 h, assessed by qPCR. (E) Western blotting analysis of FAP protein expression in NRCFs after TGF-β1 treatment. (F) Flow cytometry analysis of FAP protein expression in NRCFs following TGF-β1 treatment. (G) Immunofluorescence (IF) analysis of FAP protein expression in NRCFs post-TGF-β1 treatment. IHC, immunohistochemical; Col I, type I collagen; PBS, phosphate-buffered saline; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Primary cultures of neonatal rat cardiomyocytes (CMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from 1- to 3-d-old Sprague Dawley rats.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Saline, Staining, Immunohistochemical staining, Flow Cytometry, Immunofluorescence, Control

Journal: Biomaterials Research

Article Title: Myofibroblast-Targeting Extracellular Vesicles: A Promising Platform for Cardiac Fibrosis Drug Delivery

doi: 10.34133/bmr.0179

Figure Lengend Snippet: Targeted delivery of αFAP-EVs into activated myofibroblasts in vitro. (A to C) Confocal fluorescence and flow cytometry analysis of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) uptake in neonatal rat cardiomyocytes (NRCMs) and neonatal rat cardiac fibroblasts (NRCFs) treated with 100 μg/ml DiD-labeled αFAP-EVs at the indicated time points ( n = 3). Quantification of mean DiD fluorescence intensity using the ImageJ software (A). Flow cytometry analysis was performed to assess the extent of DiD internalization by cells (B and C). (D) NRCFs were treated with TGF-β1 for 24 h, followed by treatment with 50 μg/ml of red fluorescent DiD-labeled con-EVs or αFAP-EVs. Activation of NRCFs was assessed by α-SMA staining. Scale bar = 20 μm. (E) Quantification of EV uptake by NRCFs using mean intracellular fluorescence intensity, analyzed with ImageJ software. (F to H) In vivo distribution of 100 μg of DiD-labeled con-EVs or αFAP-EVs injected via the tail vein into ISO-treated mice, detected by noninvasive bioluminescence imaging 24 h postinjection. Fluorescence images were captured (F), and the fluorescence intensity in the ISO model group and control group was measured (G and H). CMs, cardiomyocytes; CFs, cardiac fibroblasts.

Article Snippet: Primary cultures of neonatal rat cardiomyocytes (CMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from 1- to 3-d-old Sprague Dawley rats.

Techniques: In Vitro, Fluorescence, Flow Cytometry, Labeling, Software, Activation Assay, Staining, In Vivo, Injection, Imaging, Control

Journal: Biomaterials Research

Article Title: Myofibroblast-Targeting Extracellular Vesicles: A Promising Platform for Cardiac Fibrosis Drug Delivery

doi: 10.34133/bmr.0179

Figure Lengend Snippet: αFAP-EL@CLD enhances accumulation in the fibrotic regions of ISO-treated mice. (A) Confocal analysis of NRCFs treated with TGF-β1 for 24 h, followed by the addition of 50 μg/ml red fluorescent DiD-labeled Con-EL@CLD or αFAP-EL@CLD. Activation of NRCFs was evaluated by α-SMA staining. Scale bar = 20 μm. (B and C) In vivo fluorescence distribution of 100 μg of DiD-labeled Con-EL@CLD or αFAP-EL@CLD injected into the tail vein of ISO-treated mice, detected by noninvasive bioluminescence imaging 24 h postinjection (B). Quantification of fluorescence intensity in the hearts and livers of each group (C). (D) Confocal immunofluorescence analysis of heart tissues from ISO-treated mice, showing FAP antibody staining (green), DiD (red), and DAPI (blue) for nuclei.

Article Snippet: Primary cultures of neonatal rat cardiomyocytes (CMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from 1- to 3-d-old Sprague Dawley rats.

Techniques: Labeling, Activation Assay, Staining, In Vivo, Fluorescence, Injection, Imaging, Immunofluorescence

Journal: Biomaterials Research

Article Title: Myofibroblast-Targeting Extracellular Vesicles: A Promising Platform for Cardiac Fibrosis Drug Delivery

doi: 10.34133/bmr.0179

Figure Lengend Snippet: αFAP-EL@CLD loaded with Agomir-29b inhibits myoFb activation in vitro. (A) Schematic illustration of the procedure used to produce αFAP-EL@CLD loaded with miR-29b (αFAP-EL@CLD: miR-29b). (B) TEM images showing the shape and size of Con-EL@CLD: miR-29b and αFAP-EL@CLD: miR-29b. (C) NTA indicating the size and size distribution of Con-EL@CLD: miR-29b and αFAP-EL@CLD: miR-29b. (D and E) NRCFs were treated with 10 ng/ml TGF-β1 for 48 h, followed by treatment with 50 μg/ml αFAP-EL@CLD: miR-29b at 24 h. The activation level of NRCFs was evaluated by detecting the fluorescence intensity of α-SMA at 48 h. (E) Histogram of α-SMA fluorescence-positive area statistics. (F) Quantitative PCR analysis of the mRNA levels of Acta2 and Col1α1 in each treatment group.

Article Snippet: Primary cultures of neonatal rat cardiomyocytes (CMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from 1- to 3-d-old Sprague Dawley rats.

Techniques: Activation Assay, In Vitro, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Biomaterials Research

Article Title: Myofibroblast-Targeting Extracellular Vesicles: A Promising Platform for Cardiac Fibrosis Drug Delivery

doi: 10.34133/bmr.0179

Figure Lengend Snippet: αFAP-EL@CLD loaded with GW788388 protects against cardiac fibrosis. (A) Schematic illustration of the procedure used to produce αFAP-EL@CLD loaded with GW788388 (αFAP-EL@CLD: GW788388). (B) TEM images showing the shape and size of Con-EL@CLD: GW788388 and αFAP-EL@CLD: GW788388. (C) NTA indicating the size and size distribution of Con-EL@CLD: GW788388 and αFAP-EL@CLD: GW788388. (D) The cumulative release curves of GW788388 in Con-EL@CLD and αFAP-EL@CLD in FBS ( n = 3). (E to G) NRCFs were treated with 10 ng/ml TGF-β1 for 48 h, followed by treatment with 50 μg/ml αFAP-EL@CLD: GW788388 at 24 h. (E) The activation level of NRCFs was evaluated by detecting the fluorescence intensity of α-SMA at 48 h. (F) Quantitative PCR analysis of the mRNA levels of Acta2 and Col1α1 in each treatment group. (G) The phosphorylation of SMAD family member 3 (Smad3) was detected by Western blotting. (H) Histological analysis of mouse hearts after 15 d of treatment, using H&E staining, Masson’s trichrome staining, and Sirius Red staining to evaluate the degree of cardiac fibrosis.

Article Snippet: Primary cultures of neonatal rat cardiomyocytes (CMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from 1- to 3-d-old Sprague Dawley rats.

Techniques: Activation Assay, Fluorescence, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Staining