Myofibroblastic activation of valvular interstitial cells is modulated by spatial variations in matrix elasticity and its organization

Abstract

Valvular interstitial cells (VICs) are key regulators of the heart valve's extracellular matrix (ECM), and upon tissue damage, quiescent VIC fibroblasts become activated to myofibroblasts. As the behavior of VICs during disease progression and wound healing is different compared to healthy tissue, we hypothesized that the organization of the matrix mechanics, which results from depositing of collagen fibers, would affect VIC phenotypic transition. Specifically, we investigated how the subcellular organization of ECM mechanical properties affects subcellular localization of Yes-associated protein (YAP), an early marker of mechanotransduction, and α-smooth muscle actin (α-SMA), a myofibroblast marker, in VICs. Photo-tunable hydrogels were used to generate substrates with different moduli and to create organized and disorganized patterns of varying elastic moduli. When porcine VICs were cultured on these matrices, YAP and α-SMA activation were significantly increased on substrates with higher elastic modulus or a higher percentage of stiff regions. Moreover, VICs cultured on substrates with a spatially disorganized elasticity had smaller focal adhesions, less nuclear localized YAP, less α-SMA organization into stress fibers and higher proliferation compared to those cultured on substrates with a regular mechanical organization. Collectively, these results suggest that disorganized spatial variations in mechanics that appear during wound healing and fibrotic disease progression may influence the maintenance of the VIC fibroblast phenotype, causing more proliferation, ECM remodeling and matrix deposition.

Keywords: Hydrogels; Matrix elasticity; Photopatterning; Valvular interstitial cell (VIC); Yes-associated protein (YAP); α-smooth muscle actin (α-SMA).

Figure 1. VICs culture platform Chemistry with degradation dynamics

A: Copolymerization of PEG400A and PEG with a photodegradable linker creates gels composed of poly(acrylate) kinetic chains (red coils) connected by PEG (blue lines with photodegradable groups (yellow solid sphere). Upon irradiation with UV light, the photodegradable groups were cleaved (yellow open sphere) and decreased the crosslinking density, therefore, a softer gel was created. B: Photodegradable hydrogels can be softened from a storage modulus of 3.37 ± 0.02 kPa to softer moduli of 1.93 ± 0.17 kPa, 1.31 ± 0.02 kPa and 0.69 ± 0.03 kPa by varying the UV irradiation time. C: The viscous modulus was measured to be consistently lower than elastic modulus before and after UV light exposure.

Figure 2. Characterization of YAP and α-SMA activation on uniform substrates

A: Immunostaining of VICs on soft (4 kPa) and stiff (10 kPa) hydrogel surfaces after culturing for 72 hours. On soft hydrogels, VICs have diffuse α-SMA expression in the cytoplasm with 24% α-SMA activation and 40% YAP activation, whereas on stiff substrate, VICs showed α-SMA fiber formation with 79% α-SMA activation and 76% nuclear YAP activation. DAPI (blue), F-actin (orange), α-SMA (green), YAP (red). Scale bars = 50 μm. Inset image is magnification of outlined area, scale bar = 10 μm. B: (i): The percentage of VICs expressing α-SMA fibers and nuclear localized YAP was quantified based on the immunostaining. Both the percentage of α-SMA activated and YAP activated cells increased when the gel modulus of their substrate was increased and there was found to be a high significant difference between 4 and 10 kPa substrates. **** : p<0.0001. **: p<0.01, based on one-way ANOVA analysis. n = 3 with triplicates, more than 200 VICs were analyzed per sample. (ii): Cell morphology was analyzed by quantifying cell area and circularity based on the immunostaining. There was found to be no significant for both cell area and circularity, whereas there is a significant difference for the VICs on 2 kPa substrate. **: compared to 10 kPa, p<0.01.****: compared to 10 kPa, p<0.0001, based on one-way ANOVA analysis. n = 3 with triplicates, more than 200 VICs were analyzed per sample.

Figure 2. Characterization of YAP and α-SMA activation on uniform substrates

A: Immunostaining of VICs on soft (4 kPa) and stiff (10 kPa) hydrogel surfaces after culturing for 72 hours. On soft hydrogels, VICs have diffuse α-SMA expression in the cytoplasm with 24% α-SMA activation and 40% YAP activation, whereas on stiff substrate, VICs showed α-SMA fiber formation with 79% α-SMA activation and 76% nuclear YAP activation. DAPI (blue), F-actin (orange), α-SMA (green), YAP (red). Scale bars = 50 μm. Inset image is magnification of outlined area, scale bar = 10 μm. B: (i): The percentage of VICs expressing α-SMA fibers and nuclear localized YAP was quantified based on the immunostaining. Both the percentage of α-SMA activated and YAP activated cells increased when the gel modulus of their substrate was increased and there was found to be a high significant difference between 4 and 10 kPa substrates. **** : p<0.0001. **: p<0.01, based on one-way ANOVA analysis. n = 3 with triplicates, more than 200 VICs were analyzed per sample. (ii): Cell morphology was analyzed by quantifying cell area and circularity based on the immunostaining. There was found to be no significant for both cell area and circularity, whereas there is a significant difference for the VICs on 2 kPa substrate. **: compared to 10 kPa, p<0.01.****: compared to 10 kPa, p<0.0001, based on one-way ANOVA analysis. n = 3 with triplicates, more than 200 VICs were analyzed per sample.

Figure 3. Characterization of patterned substrates

A: An illustration of VICs seeded on hydrogel surfaces with different organizations of stiff regions (Modified from [15]). The percentages of stiff area is varied together with the organization (regular/random) of these areas. Black indicates areas that are covered with the photomask allowing them to retain the initial modulus of 10 kPa. The white squares indicate areas that are exposed to the UV light, resulting in degradation to 4 kPa. Each small square is 2 μm × 2 μm and each repeat unit is 50 μm × 50 μm. B: AFM elastic moduli maps of (i) 75% stiff (regular pattern), (ii) 11% stiff (regular pattern), and (iii) 75% stiff (random pattern) hydrogels. (iv) Elastic modulus E and surface height h as a function of position x for cross-section AA’ in (ii). Scale bars = 2 mm.

Figure 4. Characterization of a-SMA activation on patterned substrates

A: Immunostaining of VICs on regular and random hydrogel surfaces after culturing in medium with 1% FBS for 72 hours. F-actin fiber formation was seen on 11% and 75% regular and random substrates. α-SMA activation was only seen on the 75% regular substrate. DAPI (blue), F-actin (orange), α-SMA (green). Scale bars = 50 μm. Inset image is magnification of outlined area, scale bar = 10 μm. B: The percentage of VICs expressing α-SMA fibers was quantified based on the immunostaining. For regular patterns α-SMA activation increases correspondingly to stiff percentages. There was found to be a significant increase in α-SMA activation from 25% regular to 75% regular (green line with circles). However on random pattern this increase in α-SMA activation was not profound (orange line with squares), leading to a high significant difference between 75% regular and random substrates.****: p<0.0001, based on one-way ANOVA. n = 3 with triplicates, more than 200 VICs were analyzed per sample.

Figure 5. Characterization of VICs proliferation on patterned substrates

A: The percentage of proliferating cells on 75% regular and random substrates in medium with 1% FBS or 15% FBS was quantified based on EdU staining. For both conditions there was found to be a significant higher percentage of proliferating cells on 75% random substrates than on 75% regular substrates. **: p<0.01, based on one-way ANOVA analysis. n = 3 with triplicates, more than 200 cells were analyzed per sample. (B) The percentage of proliferating cells was quantified within the two subgroups of myofibroblasts and fibroblasts on 75% regular and random substrates. It was seen that the percentage of proliferating cells was higher in the subgroup of fibroblasts compared to the percentage of proliferating cells in the subgroup of myofibroblasts. This was seen for both 75% regular and random substrates. Representative images of both groups are shown, where green is α-SMA, red is EdU positive nucleus and blue is DAPI. Scale bar =50 μm. ****: p<0.0001, based on one-way ANOVA. n = 3 with triplicates, more than 200 cells were analyzed per sample.

Figure 6. Characterization of YAP activation on patterned substrates

A: Immunostaining of VICs on regular and random hydrogel surfaces after culturing for 72 hours. F-actin fiber formation was seen on 11%, 75% regular and random substrates. YAP activation was seen on both the 75% regular and random substrates. DAPI (blue), F-actin (orange), YAP (red). Scale bars = 50 μm. B: The percentage of VICs with intracellular YAP activation was quantified based on the immunostaining. For both regular and random patterns YAP activation increases correspondingly to stiff percentages. There was found to be a significant increase in YAP activation from 11% regular to 75% regular (green line with circles) and 11% random to 75% random (orange line with squares). There was no difference observed between 75% regular and random. **: p<0.01, based on one-way ANOVA. n = 3 with triplicates, more than 200 VICs were analyzed per sample.

Figure 7. Quantification of YAP and α-SMA activation at different time points

The percentages of α-SMA activated cells and YAP activated cells were analyzed at 24, 72 and 120 h. The percentage of α-SMA activated and YAP activated cells increased over time. Furthermore, there was found to be a significant different percentage of YAP activation on 75% regular and random substrates at time point 24 and a significant different percentage of α-SMA activation on 75% regular and random substrates at time point 72. **** p<0.0001 for both 75% regular and random substrates. #: p<0.01 compared between 24 h and 120 h for 75% regular substrates, #: p<0.0001 compared between 24 h and 120 h for 75% random substrates. *: p<0.05. Based on one-way ANOVA analysis. n = 3, more than 200 cells were analyzed per sample.

Figure 8. Characterization of focal adhesion on patterned substrates

A: Focal adhesion formation was seen on 75% regular and 75% random after culturing for 24 hours. It was observed that the focal adhesions from cells on 75% regular are larger in size than the focal adhesions formed on 75% random. DAPI (blue), F-actin (red), Paxillin (green). Scale bars = 30 μm. Inset image is magnification of outlined area, scale bar = 10 μm. B: Quantification of focal adhesion area was based on immunostaining. **: p<0.01, based on non-parametric student t-test. n = 3 with triplicates, at least 20 cells were analyzed per sample.