Fibroblast growth factor 2 inhibits myofibroblastic activation of valvular interstitial cells

Abstract

Heart valve disease is a growing problem worldwide. Though very common in older adults, the mechanisms behind the development of the disease aren't well understood, and at present the only therapeutic option is valve replacement. Valvular interstitial cells (VICs) may hold the answer. These cells can undergo pathological differentiation into contractile myofibroblasts or osteoblasts, leading to thickening and calcification of the valve tissue. Our study aimed to characterise the effect of fibroblast growth factor 2 (FGF-2) on the differentiation potential of VICs. We isolated VICs from diseased human valves and treated these cells with FGF-2 and TGF-β to elucidate effect of these growth factors on several myofibroblastic outcomes, in particular immunocytochemistry and gene expression. We used TGF-β as a positive control for myofibroblastic differentiation. We found that FGF-2 promotes a 'quiescent-type' morphology and inhibits the formation of α-smooth muscle actin positive myofibroblasts. FGF-2 reduced the calcification potential of VICs, with a marked reduction in the number of calcific nodules. FGF-2 interrupted the 'canonical' TGF-β signalling pathway, reducing the nuclear translocation of the SMAD2/3 complex. The panel of genes assayed revealed that FGF-2 promoted a quiescent-type pattern of gene expression, with significant downregulations in typical myofibroblast markers α smooth muscle actin, extracellular matrix proteins, and scleraxis. We did not see evidence of osteoblast differentiation: neither matrix-type calcification nor changes in osteoblast associated gene expression were observed. Our findings show that FGF-2 can reverse the myofibroblastic phenotype of VICs isolated from diseased valves and inhibit the calcification potential of these 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.