Metformin mitigates aortic valve degeneration in an ex vivo three-dimensional tissue model

Figures

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  Fig. 1. Metformin inhibits degeneration of valvular interstitial cells (VICs) and aortic valve (AV) leaflets. A Calcium content in VIC cultures determined by colorimetric assay and normalized to protein content in pd and pd + metformin treatment (n = 5). B Representative microscopic images (left) and quantification of alizarin red S calcium staining of treated VICs (right). (n = 28) Scale bar = 400 µm. C Representative macroscopic images of treated AV leaflets. Scale bar = 1 cm. D Quantification of opaque area of treated AV leaflets presented as percentage of total AV leaflet area. (n = 11). E Representative microscopic image of alizarin red S calcium staining of treated AV leaflets. Scale bar = 500 µm. F Quantification of alizarin red S calcium stained areas as percentage of total AV area (n = 7). p-values were calculated by using either nonparametric Kruskal-Wallis-test with Dunn’s post hoc correction (A) or nonparametric Mann-Whitney-U test (B, D, F). Data are presented as mean ± SEM. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001. AV – aortic valve, µM – micrometer, pd – pro-degenerative, VIC – valvular interstitial cell.

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  Fig. 2. Metformin decreases alkaline phosphatase (ALP) activity in both VICs and AV leaflets. A Representative microscopic images of ALP activity staining in VICs treated with pd and pd + metformin (left). Scale bar = 100 µm. Quantification of ALP staining (n = 3 – 4, right). B ALP activity in VIC culture supernatants at day 2, 5, and 7. C ALP activity in VIC cultures normalized to total protein content at day 7 (n = 16). D Representative microscopic images of AV leaflets stained for ALP activity. Scale bar = 500 µm. E Quantification of ALP-stained areas shown as percentage of total leaflet area (n = 7 – 10). F ALP activity in AV leaflet culture supernatants at day 3, 9, 15, 21, and 28 (n = 7 – 13). G ALP activity of AV leaflets normalized to total protein content at day 28 (n = 4). p-values were calculated by using either nonparametric Mann-Whitney-U test (A, C, E, G) or two-way analysis of variance (ANOVA) with Sidak’s post-hoc test (B, F). Data are presented as mean ± SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. ALP – alkaline phosphatase, AV – aortic valve, µM – micrometer, pd – pro-degenerative, VIC – valvular interstitial cell.

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  Fig. 3. Metformin modulates the expression of genes involved in osteogenic differentiation. Gene expression analysis of osteogenic markers of VIC cultures (A) and AV leaflets (B). Relative x-fold gene expression of SPP1, BGLAP, BMP2, and RUNX2 (n = 6). p-values were calculated by using nonparametric Wilcoxon-signed-rank test. Data are presented as mean ± SEM. * = p < 0.05. AV – aortic valve, BMP2 – bone morphogenic protein 2, BGLAP – osteocalcin, SPP1 – osteopontin, pd – pro-degenerative, RUNX2 – runt-related transcription factor 2, VIC – valvular interstitial cell.

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  Fig. 4. Metformin reduces proliferation, lactate dehydrogenase activity, and alleviates myofibroblastic differentiation in VICs and AV leaflets. A Assessment of cell proliferation in VIC cultures using BrdU incorporation for VICs treated with pd and pd + metformin at 24, 48, and 72 hours (h). (n = 11–12). B LDH content in VIC culture supernatants (n = 5). C Relative x-fold ACTA2 and VIM gene expression of treated VICs (n = 6). D Representative microscopic images of fluorescent DAPI staining of AV leaflets (left). Scale bar = 50 μm. Quantification of cell nuclei per µm2 of DAPI-stained AV leaflets (right) (n = 5). E LDH content in AV leaflet culture supernatants (n = 7). F Relative x-fold ACTA2 and VIM gene expression of AV leaflets (n = 5–6). p-values were calculated by using either two-way analysis of variance (ANOVA) with Sidak’s post-hoc test (A), nonparametric Mann-Whitney-U test (B, D, E)or nonparametric Wilcoxon-signed-rank test (C, F). Data are presented as mean ± SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. ACTA2 – alpha smooth muscle actin, AV – aortic valve, BrdU – 5-bromo-2’-deoxyuridine, DAPI - fluorescent 4′,6-diamidino-2-phenylindole, LDH – lactate…

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  Fig. 5. Metformin preserves extracellular matrix (ECM) architecture in AV leaflets and modulates the expression of genes involved in ECM remodeling. Gene expression analysis of ECM markers of VIC cultures (A) and AV leaflets (B). Relative x-fold gene expression of COL1A1, COL3A1, COL5A1, TGFB1, MMP2, and MMP9, is shown (n = 5–6). p-values were calculated by using nonparametric Wilcoxon-signed-rank test. Data are presented as mean ± SEM. * = p < 0.05. Representative microscopic images of Movat’s pentachrome staining of AV leaflets treated with pd and pd + metformin at culture day 28 (C) and 56 (D). Scale bar = 500 μm. AV – aortic valve, COL1A1 – alpha- type I collagen, COL3A1 – alpha- type III collagen, COL5A1 – alpha- type V collagen, ECM – Extracellular matrix, MMP2 – matrix metalloproteinase 2, MMP9 – matrix metalloproteinase 9, µm – micrometer, TGFB1 – transforming growth factor beta 1.

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  Fig. 6. Metformin treatment induces AMP-activated protein kinase (AMPK) phosphorylation in VICs and AV leaflets. A Representative cropped Western blot images of VIC cultures (left) for AMPK, phosphorylated AMPK (pAMPK), and β-actin at 1, 3, and 6 h under control conditions (10% culture medium) and metformin treatment. Density analysis (right) for quantification of relative AMPK phosphorylation, normalized to β-actin (n = 6). Uncropped Western blot membrane images are displayed in Supplementary Figure S6. B Representative cropped Western blot images of VIC cultures (left) for AMPK, pAMPK, and GAPDH after 7 days of cultivation under pd and pd + metformin. Density analysis (right) for quantification of relative AMPK phosphorylation, normalized to GAPDH (n = 11). Uncropped Western blot membrane images are displayed in Supplementary Figure S7. C Representative cropped Western blot images of AV leaflets (left) after 28 days of cultivation under pd and pd + metformin for AMPK, pAMPK, and GAPDH. Density analysis (right) for quantification of relative AMPK phosphorylation, normalized to GAPDH (n = 15). Uncropped Western blot membrane images are displayed in Supplementary Figure S8. p-values were…