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TGFβ treatment alters the scleral fibroblast migratory response to cyclic strain in an ERK-dependent manner

Purpose Myofibroblasts are associated with scleral remodeling in myopia and glaucoma. We showed reduced myofibroblast alignment with topographic cues in sclera can be modeled by cyclic strain exposure of aligned myofibroblasts in vitro. Here, we characterize the scleral myofibroblast response to cyclic mechanical strain. Methods Human peripapillary scleral (PPS) fibroblasts were cultured on topographically aligned grooves to promote cell alignment, exposed to TGFβ (2 ng/ml) in the presence of vehicle or kinase inhibitors, and exposed to uniaxial strain (1 Hz, 5%, 12-24 hours). Alignment with grooves was determined at baseline, immediately following strain, and 24 hours after strain cessation with 0° being completely aligned and 90° being perpendicular to grooves. A wound healing assay was developed to investigate further fibroblast migration across topographic cues. Transcriptional profiling of myofibroblasts with or without strain was performed by RT-PCR and pERK, pSMAD2, and pSMAD3 levels were measured by immunoblot. Results Pre-strain alignment (6.2±1.5°) was reduced after strain (21.7±5.3°, p<0.0001) and restored 24 hours after cessation (9.5±2.6°). ERK, FAK, and ALK5 inhibition preserved alignment following strain; however, alignment reduction was not inhibited by ROCK, YAP, or SMAD3 inhibition. TGFβ-induced myofibroblast markers were reduced by strain. While TGFβ-induced phosphorylation of ERK and SMAD2 was unaffected by strain, SMAD3 phosphorylation was reduced (p=0.0004). Wound healing across grooves was enhanced by ROCK and SMAD3 inhibition but not ERK or TGFβR1 inhibition. Conclusions Strain-induced myofibroblast migration across topographic confinement is ERK dependent and associated with pSMAD3 inhibition. These results provide insight into potential mechanisms of pathologic scleral remodeling. Precis Glaucomatous scleral remodeling is driven by cellular activity. Here we find that scleral myofibroblasts have an exaggerated response to mechanical strain that is ERK dependent, associated with pSMAD3 inhibition and mitigated TGFβ signaling.