High-Magnitude and/or High-Frequency Mechanical Strain Promotes Peripapillary Scleral Myofibroblast Differentiation

Abstract

Purpose: To determine the effects of altered mechanical strain on human peripapillary scleral (ppSc) fibroblast-to-myofibroblast differentiation.

Methods: Eight human ppSc fibroblast cultures were isolated from three paired eyes and two unilateral eyes of five donors using an explant approach. Human ppSc fibroblast isolates were subjected to 1% and 4% cyclic strain at 0.05 to 5 Hz for 24 hours. Levels of α smooth muscle actin (αSMA) mRNA and protein were determined by real-time PCR and immunoblot. Incorporation of αSMA into actin stress fibers was evaluated by confocal immunofluorescent microscopy. Myofibroblast contractility was measured by fibroblast-populated three-dimensional collagen gel contraction assay and phosphorylation of myosin light chain (MLC20).

Results: Human ppSc fibroblasts contained 6% to 47% fully differentiated myofibroblasts before strain application; 4% cyclic strain increased αSMA mRNA and protein expression in ppSc fibroblasts compared with 1% strain applied at 5 Hz, but not at lower frequencies. Seven of eight ppSc fibroblast isolates responded to high-magnitude and high-frequency strain with increased cellular contractility and increased MLC20 phosphorylation. In addition, increasing strain frequency promoted αSMA expression in ppSc fibroblasts under both 1% and 4% strain conditions.

Conclusions: High-magnitude and/or high-frequency mechanical strain promotes differentiation of human ppSc fibroblasts into contractile myofibroblasts, a fibroblast phenotypic change known to be key to tissue injury-repair responses. These findings suggest that the cellular constituent of ppSc may play an important role in the regulation of optic nerve head biomechanics in response to injurious IOP fluctuations.

Figure 1

Isolation of human primary ppSc fibroblasts and identification of endogenous ppSc myofibroblast population. (A) Migration of fibroblasts from a human ppSc tissue explant. (B) Morphology of human ppSc fibroblasts at passage 3. (C) Human ppSc fibroblasts at passage 3 were stained for αSMA (green) and F-actin (red). Nuclei were stained by DAPI (blue). Scale bars: 50 μm.

Figure 2

An increased magnitude of cyclic strain promotes αSMA mRNA and protein expression in human ppSc fibroblasts at 5-Hz strain frequency. Eight human primary ppSc fibroblast isolates were subjected to 1% or 4% cyclic strain at 5 Hz for 24 hours. (A) Levels of αSMA mRNA were determined by real-time PCR; 18S rRNA was used as internal control. The numerical comparative CT value obtained from No. 1837 cells at 1% strain was set to 1. (B) Levels of αSMA protein were determined by immunoblot. Relative levels of αSMA protein were determined by scanning densitometry of the blots and normalized to GAPDH expression. (C) Representative confocal immunofluorescent images showing αSMA expression (green) and incorporation (yellow) into F-actin (red) in ppSc myofibroblasts under 1% and 4% strain conditions. Nuclei were stained with DAPI. Results are the means ± SD of three separate experiments. *P < 0.05 for comparisons as indicated. Scale bars: 50 μm. l.e., long exposure; s.e., short exposure.

Figure 3

High-magnitude cyclic strain does not alter αSMA mRNA and protein expression in human ppSc fibroblasts at lower strain frequencies (0.05 Hz and 0.5 Hz). Eight human primary ppSc fibroblast isolates were subjected to 1% or 4% cyclic strain at 0.5 Hz (A, B) and 0.05 Hz (C, D) for 24 hours. (A, C) Levels of αSMA mRNA were determined by real-time PCR; 18S rRNA was used as internal control. The numerical comparative Ct values obtained from No. 2340 cells at 4% strain (A) and from No. 1837 cells at 1% strain (C) were set to 1, respectively. (B, D) Levels of αSMA protein were determined by immunoblot. Relative levels of αSMA protein were determined by scanning densitometry of the blots and normalized to GAPDH expression. Results are the means ± SD of three separate experiments. *P < 0.05 for comparisons as indicated.

Figure 4

High-magnitude and high-frequency mechanical strain promotes cellular contractility of human ppSc fibroblasts. Eight human primary ppSc fibroblast isolates were subjected to 1% or 4% cyclic strain at 5 Hz for 24 hours. (A) Cells were immediately detached from BioFlex plates by trypsinization and mixed with type I collagen suspension. Fibroblast contractility was assessed by a 3D collagen gel–based assay. (B) The ppSc (myo)fibroblasts were cultured on silicone substrates for 24 hours. Cells were fixed and stained for αSMA (green) and F-actin (red). Nuclei were stained by DAPI (blue). Phase-contrast and confocal immunofluorescent images were taken and overlaid to show the correlation between αSMA expression and wrinkle formation. Arrow indicates a wrinkle-forming myofibroblast. Arrowhead indicates a non–wrinkle-forming fibroblast. Scale bar: 50 μm. (C) Levels of pMLC and total MLC₂₀ (MLC) were determined by immunoblot analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as loading control. Results are the means ± SD of three separate experiments. *P < 0.05 for comparisons as indicated.

Figure 5

Increasing strain frequency is sufficient to promote human ppSc myofibroblast differentiation. Eight human primary ppSc fibroblast isolates were subjected to 1% or 4% cyclic strain at 0.05 Hz, 0.5 Hz, or 5Hz for 24 hours. (A) Levels of αSMA mRNA were determined by real-time PCR; 18S rRNA was used as internal control. The numerical comparative CT values obtained from No. 1837 cells at 4%, 0.05 Hz strain, No. 1861 cells at static condition, No. 1844 cells at 1%, 0.5 Hz stain, and No. 1203 cells at 1%, 0.05 Hz stain were set to 1, respectively. (B) Levels of αSMA, phosphorylated MLC₂₀ and total MLC₂₀ were determined by immunoblot. Glyceraldehyde 3-phosphate dehydrogenase was used as loading control. Results are the means ± SD of three separate experiments. *P < 0.05 for comparisons as indicated. OS and OD indicate paired eyes.