FGF-2 inhibits contractile properties of valvular interstitial cell myofibroblasts encapsulated in 3D MMP-degradable hydrogels

Figures

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  FIG. 1.. Morphological changes of encapsulated VICs in response to cytokine treatment. (a) Representative immunostaining images of VICs encapsulated within 3D MMP-degradable hydrogels at day 1 and day 5 of culture in untreated [10% fetal bovine serum (FBS) medium], TGF-β1 (5 ng/ml), and FGF-2 (100 ng/ml) conditions. Minimal spreading (F-actin, red) and diffuse α-SMA (green) are seen at day 1. By day 5, discrete F-actin stress fibers are seen in TGF-β1 and untreated conditions but absent from FGF-2 treated samples. Organized α-SMA fibers are present only in TGF-β1 treated hydrogels. Scale bars = 30 μm. Inset scale bar = 10 μm. (b) Sphericity decreased in all conditions after day 1 with no significant differences between treatments. Values normalized to day 1. (c) Cellular volume increased in untreated and TGF-β1 treated samples. TGF-β1 caused a 1.8-fold increase in cellular volume by day 5. Treatment with FGF-2 resulted in no change in cell volume and a lower cell volume than untreated controls. Values normalized to day 1 (dotted line). (d) Intensity of α-SMA relative to day 1 increased significantly for hydrogels treated with TGF-β1 in comparison to untreated and FGF-2 treated samples.…

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  FIG. 2.. VICs proliferate within 3D MMP-degradable hydrogels in response to cytokine treatment. (a) Representative images for EdU staining of MMP-degradable encapsulated VICs after culturing for 5 days in untreated (10% FBS medium), TGF-β1 (5 ng/ml), or FGF-2 (100 ng/ml) conditions. FGF-2 treatment increased the number of EdU positive cells compared to TGF-β1 and untreated controls. Nuclei (blue) and EdU (green). Scale bars = 50 μm. (b) Quantification of the percent proliferating cells with respect to treatment. FGF-2 treatment increased the number of EdU positive cells to 11.7% ± 4.4% relative to untreated controls. (c) Quantification of the dsDNA content over time for VIC-laden hydrogel with respect to treatment. Treatment with FGF-2 resulted in significantly higher dsDNA after day 5 and day 10 culture, compared to either TGF-β1 or untreated conditions. ****p < 0.0001 and ***p < 0.001.

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  FIG. 3.. mRNA levels of myofibroblast markers and matrix remodeling markers in response to TGF-β1 and FGF-2 treatment. [(a), (b)] Expression of myofibroblast markers in VIC-laden hydrogels relative to L30. Both α-SMA (a) and CTGF (b) were quantified in response to TGF-β1 (5 ng/ml) and FGF-2 (100 ng/ml) treatment and compared to untreated control at days 3, 5, 7, and 10. FGF-2 treatment suppressed the expression of myofibroblast genes, as seen via downregulation of both α-SMA and CTGF with respect to untreated control. Treatment with TGF-β1 resulted in similar mRNA levels of α-SMA at early time points compared to untreated control but was downregulated at later time points. CTGF mRNA expression in TGF-β1 samples was upregulated at day 3 with respect to untreated control but decreased over time. VICs in the untreated condition had an opposite trend with increasing CTGF expression with time, reaching a higher fold expression than TGF-β1 treated VICs at day 10. [(c) and (d)] Expression of matrix associated genes in VICs relative to L30. VIC expression of (c) COL1A1 and (d) MMP1 was measured in response to TGF-β1 (5 ng/ml) and FGF-2 (100 ng/ml) treatment and compared to untreated control at…

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  FIG. 4.. Functional contraction observed in response to treatment with TGF-β1 to VICs cultured in hydrogels versus porcine aortic valve tissue explants. (a) TGF-β1 treatment to VIC-laden MMP-degradable hydrogels resulted in significant contraction (19.5% ± 9.7% decrease in the gel area between day 0 and day 10) as measured via the projected surface area, relative to a 4.4% ± 11.4% decrease in the surface area of untreated control hydrogels. Images demonstrate bulk contraction and opacity due to densification. (b) Valve tissue explants contract with TGF-β1 treatment over 10 days, resulting in a decrease of 36.5% ± 9.1% in the projected surface, relative to 17.9% ± 12.7% reduction in the area for untreated tissue samples. Images demonstrate bulk contraction of tissue explants. Scale bars = 5 mm. ****p < 0.0001 and **p < 0.01. * indicates the significance of TGF-β1 treatment with respect to untreated conditions.

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  FIG. 5.. FGF-2 treatment inhibits VIC contraction and α-SMA protein expression in hydrogels. (a) FGF-2 treatment to VIC-laden MMP-degradable hydrogels resulted in an increase in the projected surface area (3.3% ± 2.6%) over time, which was statistically different from the untreated condition (4.4% ± 11.4% reduction in the area). Images demonstrate the lack of contraction between conditions. (b) Tissue explant contraction in response to FGF-2 treatment (17.2% ± 4.8% reduction in the area) was not statistically different from the untreated controls (17.9% ± 12.7% reduction in the area). Images demonstrate lack of bulk contraction of tissue explants. (c) Percentage α-SMA protein expression relative to untreated conditions (dotted line). TGF-β1 treatment significantly increased protein expression (73.7% ± 31.7%), whereas FGF-2 resulted in a significant decrease (99.6% ± 0.6%). Combinatorial treatment of exogenous cues resulted in significantly lower α-SMA protein expression (96.3% ± 4.3%), similar to that of FGF-2 alone. (d) Representative western blot used for analysis, all lanes from the same blot. Scale bars = 5 mm. ****p < 0.0001, **p < 0.01, and *p < 0.05. * indicates the significance of…

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  FIG. 6.. Hydrogel results validated by similar trends in porcine aortic valve tissue explants. (a) Normalized projected surface area (day 10/day 0) contraction trends with respect to treatment conditions are conserved between MMP-degradable hydrogels and tissue explants. Treatment with TGF-β1 resulted in significant contraction in both culture constructs. (b) Normalized shear storage modulus (day 10/day 1) trends are conserved between hydrogels and tissue explants, where treatment with TGF-β1 resulted in increased stiffness. (c) Hydrogel shear storage modulus measured for samples at day 1 vs. day 10 plus treatment conditions. TGF-β1 treatment resulted in a 1.7-fold increase in G′ between day 1 and day 10. (d) Tissue explant shear storage modulus measured for samples at day 1 vs. day 10 plus treatment conditions. TGF-β1 treatment resulted in a 1.3-fold increase in G′ between day 1 and day 10. ****p < 0.0001, ***p < 0.001, and *p < 0.05.