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 < 0.001) without TGFβ1. Administration of topical
Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial
wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to
DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses,
Lat-B modulates
mRNA and
protein expression of α-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human
glaucoma patients,
Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied
Lat-B to determine if this compound can reduce stromal
fibrosis.