Fig 1: Proposed model of EndMT induction in MV by post-MI plasma.sFRP3-deficient post-MI plasma releases the brake on Wnt signaling, increases FOXM1 transcriptional activity, nuclear localization of Slug, which initiates EndMT within days after MI. EndMT indicates endothelial-to-mesenchymal transition; FOXM1, forkhead box M1; MI, myocardial infarction; MV, mitral valve; NS, not significant; sFRP3, secreted frizzled-related protein 3; TGFß, transforming growth factor beta; VE, vascular endothelial; and Wnt, wingless-related integration site.
Fig 2: sFRP3 is reduced in post-MI plasma. A, Representative images of ovine cytokine arrays exposed to ovine plasma. sFRP3 in sham (n=3) and MI (n=6) plasma were measured. Values from signal intensity in arrays using ImageJ software, normalized to the positive controls of each individual array (B) and ELISA (C) were quantified. Fold changes (FC) were calculated by normalizing values against naïve plasma. Mean±SD were graphed. P value was calculated using Mann-Whitney test and revealed a significant difference in the mean of the 2 groups. Two-sample Wilcoxon test revealed that there was no significant difference in the mean quantified sFRP3 when the observations were categorized based on the sex of the animals (P=0.4762). D, sFRP3 values MI (n=6) plasma were normalized to naïve (n=4), measured by cytokine array, and plotted against infarct size (normalized for heart size as infarct relative to total LV endocardial surface area) and (E) LVEF. The fitted regression lines in D and E showed a significant slope parameter with P=0.0132 and P=0.0185, respectively. F, Mitral VECs were treated with 3 individual ovine baseline (before MI), after MI, and sFRP3 supplemented post-MI plasma for 10 minutes and lysates were subjected to western blot analysis. G, Band densities from 3 independent assays were quantified and subjected to 1-way ANOVA with Tukey’s multiple comparisons test. a-SMA indicates a-smooth muscle actin; EndMT, endothelial-to-mesenchymal transition; FC, fold changes; LV, left ventricular; LVEF, left ventricular ejection fraction; MI, myocardial infarction; MMP, matrix metalloproteinase; MNCs, mononuclear cells; qPCR, quantitative polymerase chain reaction; RBCs, red blood cells; sFRP3, secreted frizzled-related protein 3; TGFß, transforming growth factor beta; VE, vascular endothelial; and VEC, valve endothelial cell.
Fig 3: FOXM1 contributes to EndMT and can be suppressed by sFRP3. A, Mitral VECs were treated with sham, post-MI plasma, and post-MI plasma with Siomycin A (1 µmol/L) for 24 hours before immunofluorescence staining using FOXM1 antibody (green; scale bar: 20 µm). B, Percentage of the cells positive for nuclear FOXM1 per total nuclei from 4 independent immunofluorescence assays were graphed. Statistical analysis was conducted using 1-way ANOVA with Tukey’s multiple comparisons test. C, Mitral VECs were treated with MI plasma (n=6) with and without Siomycin A (1 µmol/L) for 24 hours before qPCR analysis. Mean±SD from 3 independent assays were graphed. P values were calculated using the Wilcoxon signed-rank test and were significant (P<0.05) for all tested genes. D, Mitral VECs were treated with MI plasma (n=6) supplemented with sFRP3 (250 ng/mL) for 24 hours before qPCR analysis. Mean±SD from 3 independent assays were graphed. P value was calculated using nonparametric Mann-Whitney test. E, FOXM1 staining in mitral VECs following exposure to post-MI plasma and post-MI plasma supplemented with 250 ng/mL of sFRP3 for 24 hours. DAPI was used to stain nuclei (scale bar: 50 µm). F, Mean±SD of % cells positive for nuclear FOXM1 per total nuclei from 5 independent immunofluorescence assays with 2 individual post-MI plasma were graphed. P values were calculated using Mann-Whitney test. EndMT indicates endothelial-to-mesenchymal transition; FC, fold changes; FOXM1, forkhead box M1; MI, myocardial infarction; NS, not significant; qPCR, quantitative polymerase chain reaction; sFRP3, secreted frizzled-related protein 3; SioA, Siomycin A; and VEC, valve endothelial cell.
Fig 4: sFRP3 inhibits EndMT. A and B, Mitral VECs were treated with MI plasma (n=6) supplemented with 0, 250, or 1250 ng/mL sFRP3 for 24 hours before qPCR. Mean±SD from three independent assays were graphed. P values were calculated using nonparametric Mann-Whitney test. C, Mitral VECs were treated with post-MI plasma±sFRP3 (250 ng/mL) for 24 hours before immunofluorescent staining using anti-Slug (red). Naïve and sham plasma served as control. DAPI was used to stain nuclei (blue). Both black-and-white and colored merged images are shown to ease visualization (scale bar: 20 µm). D, Number of nuclei positive for Slug divided by total nuclei from 4 wells—duplicates incubated with 2 individual plasmas for each group of animals—were graphed. P values were calculated using 1-way ANOVA with Sidak’s multiple comparisons test. EndMT indicates endothelial-to-mesenchymal transition; FC, fold changes; LVEF, left ventricular ejection fraction; MI, myocardial infarction; qPCR, quantitative polymerase chain reaction; sFRP3, secreted frizzled-related protein 3; and VEC, valve endothelial cell.
Fig 5: FOXM1 is increased in mitral VECs after MI. A, Hierarchical clustering and heat map of normalized read counts of 206 significantly regulated mRNA transcripts in mitral VECs treated with naïve (n=3) and post-MI plasma (n=3). Red color represents higher expression and green color indicates the lower expression. One minus Pearson correlation metric was used for hierarchical clustering. B, Hierarchical clustering and heat map of log2 fold change of FOXM1-related genes. Red color represents the higher expression and green color indicates the lower expression for each mRNA. One minus Pearson correlation metric was used for hierarchical clustering. C, Ovine mitral VECs were exposed to naïve (n=3), sham (n=3), and MI (n=6) plasma for 24 hours before qPCR analysis. All values were normalized to average naïve and graphed. Data represent mean±SD of 3 independent assays. P values were calculated using nonparametric Mann-Whitney test. D, Equal numbers of ovine mitral VECs were seeded and treated with sham (n=4) or MI (n=5) plasma for 24 hours before cell count. Fold change was calculated using average number of cells in triplicates for all sham controls. Mean±SD from 2 independent assays were graphed. P values were calculated using nonparametric Mann-Whitney test. E through G, Mitral valve tissue from animals with naïve (E), sham (F), and inferior MI (G) were stained for FOXM1 expression using immunohistochemistry (scale bar: 500 µm). H, Number of cells expressing FOXM1 divided by total nuclei from 10 fields per each individual section was graphed. P values were calculated using 1-way ANOVA with Tukey’s multiple comparisons test. I, Post-MI MV was costained with anti-FOXM1 (red) and anti-CD31 (green) using immunofluorescence staining and DAPI staining to visualize nuclei. Arrows indicate FOXM1+ nuclei. scale bar: 50 µm. a-SMA indicates a-smooth muscle actin; CCNB1, Cyclin B1; FC, fold changes; FOXM1, forkhead box M1; MI, myocardial infarction; MV, mitral valve; qPCR, quantitative polymerase chain reaction; sFRP3, secreted frizzled-related protein 3; TGFß, transforming growth factor beta; VE, vascular endothelial; and VEC, valve endothelial cell.
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