Abstract
Purpose :
Myofibroblasts are associated with pathologic scleral remodeling in myopia and glaucoma development.1,2 We showed reduced myofibroblast alignment with collagen topography in human sclera can be modeled by cyclic strain exposure in vitro.3 Here, we characterize the scleral myofibroblast response to cyclic mechanical strain.
Methods :
Primary human peripapillary scleral (PPS) fibroblasts were cultured on topographically aligned grooves to promote cell alignment, exposed to TGFb (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 fibroblast migration across topographic cues in the absence of strain. 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 significantly reduced after strain (21.7±5.3, p<0.0001) and restored 24 hours after cessation (9.5±2.6°). ERK and TGFbR1 inhibition preserved alignment after strain; however, loss of alignment occurred after ROCK, YAP, or SMAD3 inhibition. TGFb-induced myofibroblast markers were reduced by strain. While TGFb-induced phosphorylation of ERK and SMAD2 was unaffected by strain, SMAD3 phosphorylation was reduced significantly (p=0.0004). Wound healing across grooves was enhanced by ROCK and SMAD3 inhibition but not ERK or TGFbR1 inhibition.
Conclusions :
Strain-induced myofibroblast migration across topographic confinement is ERK dependent and associated with pSMAD3 inhibition. These results inform potential mechanisms of pathologic scleral remodeling.
References
1. Oglesby EN, Tezel G, Cone-Kimball E, et al. Scleral fibroblast response to experimental glaucoma in mice. Mol Vis. 2016;22:82-99.
2. Wu H, Chen W, Zhao F, et al. Scleral hypoxia is a target for myopia control. Proc Natl Acad Sci U S A. Jul 24 2018;115(30):E7091-E7100. doi:10.1073/pnas.1721443115
3. Szeto J, Chow A, McCrea L, et al. Regional Differences and Physiologic Behaviors in Peripapillary Scleral Fibroblasts. Invest Ophthalmol Vis Sci. Jan 2021;62(1):27. doi:10.1167/iovs.62.1.27
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.