Substratum compliance modulates corneal fibroblast to myofibroblast transformation

Abstract

Purpose: The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor β1 (TGF-β1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-β1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas.

Methods: Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (> 1 gigapascal [GPa]). Cells were cultured in media containing TGF-β1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot.

Results: Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-β1.

Conclusions: These data indicate that substrate compliance modulates TGF-β1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.

Keywords: biophysical cues; corneal wound healing; myofibroblast transformation; α-smooth muscle actin.

Figure 1

Transforming growth factor β1 induces the expression of α-SMA in HCFs. Shown is a representative graph from one of at least three experiments demonstrating the relative mRNA expression of α-SMA in HCFs cultured on TCP for 72 hours with various concentrations of TGF-β1 and 10% serum added to the medium. Maximal expression of α-SMA was achieved at 1 ng/mL of TGF-β. Data are mean ± SD. Asterisks indicate statistically significant differences compared with the expression in cells cultured without added TGF-β1 (***P < 0.001 and **P < 0.01, one-way ANOVA, followed by Holm-Sidak pairwise comparison test).

Figure 2

Substratum compliance restricts TGF-β1–induced expression of α-SMA in HCFs. Shown is a representative graph from one of at least three independent experiments demonstrating the average amounts of relative α-SMA mRNA expression in HCFs grown on gels and TCP cultured with 0, 1, or 10 ng/mL of TGF-β1 as determined by qPCR. The addition of TGF-β1 induced an increased α-SMA expression on all substrates. Following treatment with 1 or 10 ng/mL of TGF-β1, HCFs grown on very stiff substrate (TCP) had markedly more α-SMA expression compared with cells grown on more compliant substrates. In the presence of 1 or 10 ng/mL of TGF-β1, the lowest α-SMA expression was measured in HCFs grown on 4-kPa gels. Treatment with 1 or 10 ng/mL of TGF-β1 significantly increased α-SMA mRNA expression (P < 0.001) on TCP compared with culture without TGF-β1. The expression of α-SMA in HCFs on TCP treated with 10 ng/mL of TGF-β1 was arbitrarily set as 1.0. Data are mean ± SD. Indicated are statistically significant differences in α-SMA expression between cells grown on compliant substrates and TCP for each TGF-β1 concentration (***P < 0.001 and **P < 0.01) and between cells grown on 4-kPa gels and less compliant gels (###P < 0.001) (two-way ANOVA, followed by Holm-Sidak pairwise comparison test).

Figure 3

Substratum compliance modulates α-SMA mRNA expression in RCFs. Treatment with 1 or 10 ng/mL of TGF-β1 significantly increased α-SMA mRNA expression (P < 0.001) on TCP compared with culture without TGF-β1. Following treatment with TGF-β1, cells on the most compliant substrate (4 kPa), mimicking the normal rabbit stroma, expressed significantly reduced amounts of α-SMA mRNA compared with any other substrate. Shown is a representative graph from one of three independent experiments. The expression of α-SMA in RCFs on TCP treated with 10 ng/mL of TGF-β1 was arbitrarily set as 1.0. Data are mean ± SD. Indicated are statistically significant differences in the α-SMA expression between cells grown on compliant substrates and TCP for each TGF-β1 concentration (***P < 0.001) and between cells grown on 4-kPa gels and less compliant gels (###P < 0.001, ##P < 0.01, and #P < 0.05) (two-way ANOVA, followed by Holm-Sidak pairwise comparison test).

Figure 4

Substratum compliance limits the transformation to the myofibroblast phenotype. Representative images show the fluorescent staining of α-SMA (green), F-actin (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in RCFs cultured in the absence or presence of 10 ng/mL of TGF-β1 on substrates with varying compliance. The majority of cells on TCP displayed strong staining for α-SMA when exposed to TGF-β1, thus confirming the myofibroblast phenotype in these cells. In contrast, few cells (on 28-kPa gels) or no cells (on 4-kPa gels) stained positive for α-SMA, even in the presence of 10 ng/mL of TGF-β1. All cells show clear staining of F-actin, although the incidence of stress fibers was greatest in cells on TCP. Scale bar: 100 μm; ×10 objective.

Figure 5

Substratum compliance modulates α-SMA protein expression in RCFs. A representative Western blot from one experiment demonstrates the reduced expression of α-SMA protein in RCFs cultured for 72 hours with 10 ng/mL of TGF-β1 on more compliant substrates compared with TCP. (A) As a loading control, GAPDH was detected as a 36-kDa band, while α-SMA was detected as a 42-kDa band. (B) A densitometry analysis was performed to account for differences in the loaded protein amounts. The graph shows mean ± SEM values obtained from five independent experiments. Asterisks indicate statistically significant differences in the α-SMA protein expression between cells on compliant substrates and TCP (***P < 0.001 and **P < 0.01, one-way ANOVA, followed by Holm-Sidak pairwise comparison test). The differences in the α-SMA expression in cells on the different gels were not statistically different from each other.