Fibroblast growth factor 2 inhibits myofibroblastic activation of valvular interstitial cells

Fig 1. vWF immunostaining. hVICs and hVECs stained for vWF (green), phalloidin (red), and DAPI (blue). Scale bars represent 100μm.

Fig 2. FIB media is pro-proliferative. (A) Fluorescence data from alamarBlue proliferation assay (n = 4), normalised to day 0. The two treatment groups were compared to control at each time point using an ordinary 2-way ANOVA and (B) the gradients of the line of best fit derived from a simple linear regression model of 4 repeats, normalized to control. Both treatments were compared to control using an ordinary one-way ANOVA. * p<0.05, #p<0.01.

Fig 3. FGF-2 and TGF-β differentially influence cell morphology. (A) depicts representative images of hVICs on day 2 and 4, immunostained for α-SMA (red), vimentin (green), and DAPI (blue). Scale bars represent 100μm. The following morphological characteristics are derived from pooling days 2 and 4, as the 2-way ANOVA showed that the ‘time’ factor did not significantly affect any parameter. Comparisons are ordinary one-way ANOVAs between control and the experimental groups. For image analysis, 6 biological replicates were used (n = 6); 6 representative images were taken from each biological replicate per treatment per timepoint, representing >75 cells per experimental group. These are: (B) cell area in μm2, (C) aspect ratio, the long axis divided by the short axis of each cell, (D) the ‘circularity’ of each cell. This measure is defined by 4π(area/perimeter2), and E) the length of the major axis. * p<0.05, #p<0.01, error bars represent 95% CI.

Fig 4. FGF-2 downregulates ACTA2 expression. RT-qPCR results of ACTA2 (α-SMA) and VIM (vimentin) expression. In a mixed effects ANOVA, experimental groups were compared to control at each time point. * p<0.05, #p<0.01, dots represent biological replicates (n = 7 on day 2, n = 5 on day 4), error bars represent 95% CI.

Fig 5. FGF-2 reduces SMAD immunostaining, no effect on expression of SMAD genes. (A) shows hVICs on day 2 stained for activated SMAD2/3 complex (green), counterstained with phalloidin (red), scale bars represent 100μm. Representative images of day 4 did not appreciably differ from day 2, and are available in the supplementary materials (S3). (B) shows RT-qPCR results of expression of SMAD2 and SMAD3 respectively at day 2 and 4. * p<0.05, #p<0.01, dots represent biological replicates (n = 7 on day 2, n = 5 on day 4), error bars represent 95% CI.

Fig 6. FGF-2 inhibits focal adhesion formation. (A) shows paxillin staining of hVICs on day 2, scale bars represent 100μm. Representative images of day 4 did not appreciably differ from day 2, and are available in the supplementary materials (S3). (B) is the number of mature focal adhesions per cell. n = 4 biological replicates were used, 10 images per replicate per group. Experimental groups were compared to control using a 2-way ANOVA. * p<0.05, #p<0.01, error bars represent 95% CI.

Fig 7. FGF-2 inhibits nodular calcification. (A) shows alizarin red stained hVICs on day 4. Scale bars represent 100μm. (B) shows the number of calcific nodules per well on day 2 and day 4, counted manually from n = 4 biological repeats, with 2 wells per group. A 2-way ANOVA compared the experimental groups with control at each time point. * p<0.05, #p<0.01, error bars represent 95% CI.

Fig 8. FGF-2 has no effect on expression of osteoblast-associated genes. Results of RT-qPCR on a panel of 7 osteoblast associated genes. Note that expression of BGLAP and SP7 was below the detectable level. * p<0.05, #p<0.01, dots represent biological replicates (n = 7 on day 2, n = 5 on day 4), error bars represent 95% CI.

Fig 9. FGF-2 promotes matrix breakdown expression pattern. RT-qPCR results on a panel of matrix associated genes. * p<0.05, #p<0.01, dots represent biological replicates (n = 7 on day 2, n = 5 on day 4), error bars represent 95% CI.

Fig 10. FGF-2 upregulates BMP2 expression. RT-qPCR results of BMPs 2, 4, and 6. In a mixed effects model, experimental groups were compared to control at each time point. * p<0.05, #p<0.01, dots represent biological replicates (n = 7 on day 2, n = 5 on day 4), error bars represent 95% CI.

Fig 11. Scleraxis, potential myofibroblast marker, is upregulated by TGF-β. (A) shows representative images of hVICs stained for scleraxis (green) and phalloidin (red) on day 4. Representative images of day 2 did not appreciably differ from day 4, and are available in the supplementary materials (S3). Scale bars represent 100μm. (B) shows RT-qPCR results of SCX (scleraxis) gene expression. In a mixed effects model, experimental groups were compared to control at each time point. * p<0.05, #p<0.01, dots represent biological replicates (n = 7 on day 2, n = 5 on day 4), error bars represent 95% CI.

Fig 12. Summary of findings.