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 a smooth muscle actin (aSMA) mRNA and protein were determined by real-time PCR and immunoblot. Incorporation of aSMA 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 (MLC 20 ).RESULTS. Human ppSc fibroblasts contained 6% to 47% fully differentiated myofibroblasts before strain application; 4% cyclic strain increased aSMA 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 MLC 20 phosphorylation. In addition, increasing strain frequency promoted aSMA 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.

The Role of the RhoA/ROCK Signaling Pathway in Mechanical Strain-Induced Scleral Myofibroblast Differentiation

“…The importance of a-SMA expression in myopia sclera was investigated in this study. Although several studies have explored the role of myofibroblasts and biomechanics in the remodeling of the sclera, 6,14,40 none has determined how biomechanical signals are trans- ferred into biochemical signals. Here, using a Flexcell system, it was possible to demonstrate that the RhoA/ROCK pathway regulated the strain-induced differentiation of scleral fibroblast to myofibroblast.…”

Mechanical Strain Induces Distinct Human Scleral Fibroblast Lineages: Differential Roles in Cell Proliferation, Apoptosis, Migration, and Differentiation

“…Primary HSFs were cultured following a collagenase digestion protocol. Briefly, the PPS (2-mm scleral band from the ONH 14 ) and periphery sclera were carefully dissected from the donor eyes and were then digested by collagenase NB4 (Serva, Heidelberg, Germany) overnight at 378C. After filtration (70 lm; Falcon; BD, Franklin Lakes, NJ, USA) and resuspension, HSFs were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) containing 20% FBS and 1% penicillin-streptomycin (Hyclone, South Logan, UT, USA) under the normal condition (378C, 5% CO 2 in a humidified atmosphere).…”

Lamina cribrosa in glaucoma

“…Studies of cells from the LC and sclera indicate that both larger strains and/or higher frequency strain cycling engender cellular changes that are consistent with ECM remodeling [14, 26, 27]. These same effects are seen in other collagenous soft tissues[28], and support the notion that IOP transients play an important role in the connective tissue remodeling signaling cascade.…”

“…Finally, emerging evidence suggests that chronic exposure to elevated IOP may also spur remodeling-based stiffening of the sclera [8] and LC [9], presumably driven by cellular responses to elevated strain [10]. Connective tissue remodeling cascades in scleral fibroblasts and LC cells/astrocytes are preferentially activated in response to cyclic strain [11–14], analogous to IOP transients, which have been measured in both humans and animals. In summary, the ONH reacts to IOP and its transients as a structural system, and the resulting strains are wholly dependent on the structural characteristics of the individual eye.…”