Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation

• Published in: Experimental eye research 2018-05, Vol.170, p.101-107
• Main Authors: Thomasy, Sara M., Raghunathan, Vijay Krishna, Miyagi, Hidetaka, Evashenk, Alexander T., Sermeno, Jasmyne C., Tripp, Geneva K., Morgan, Joshua T., Murphy, Christopher J.
• Format: Article
• Language: English
• Subjects: Actins - genetics; Actins - metabolism; Aldehyde Dehydrogenase - genetics; Aldehyde Dehydrogenase - metabolism; Alpha smooth muscle actin; Animals; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Cell Transdifferentiation - drug effects; Cells, Cultured; Cornea - physiology; Cornea - surgery; Corneal Keratocytes - physiology; Elasticity - physiology; Female; Immunohistochemistry; Latrunculin B; Microscopy, Fluorescence; Myofibroblast transformation; Myofibroblasts - physiology; Photorefractive Keratectomy; Proteoglycans - genetics; Proteoglycans - metabolism; Rabbits; Real-Time Polymerase Chain Reaction; RNA, Messenger - genetics; Stromal haze; Substratum stiffness; Thiazolidines - pharmacology; Tomography, Optical Coherence; Transforming Growth Factor beta1 - pharmacology
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2018.02.003

The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P