FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

Abstract

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes

“…In the corneal stroma, the differentiation of corneal keratocytes into myofibroblasts has been shown to depend on ECM stiffness ( Dreier et al, 2013 ; Petroll and Lakshman, 2015 ; Maruri et al, 2020 ; Petroll et al, 2020 ), but it is not known how keratocytes sense changes in the mechanical properties of the ECM and whether signaling downstream of FAs is involved. Inhibition of FAK can disrupt keratocyte differentiation on rigid substrata (e.g., tissue-culture plastic) ( Yeung et al, 2021 ), but it is unclear whether differences in FAK activity are associated with stiffness-dependent differences in keratocyte behavior.…”

Extracellular Vesicles Secreted by Corneal Myofibroblasts Promote Corneal Epithelial Cell Migration

“…Protein isolation and western blot analyses were performed as previously described [ 7 , 8 , 12 ]. In brief, proteins from isolated EVs were extracted with RIPA buffer (10 mM Tris, 150 nM NaCl, 1% deoxycholic acid, 1% Triton X, 0.1% SDS, and 1 mM EDTA) plus protease inhibitors (aprotinin, PMSF, and sodium orthovanadate).…”

“…Moreover, some HCFs can further differentiate to human corneal myofibroblasts (HCMs) under the synergistic action of serum and transforming growth factor β1 (TGF-β1). HCMs are characterized by increased cell size and alpha smooth muscle actin (αSMA) expression [ 10 , 11 , 12 ]. HCMs are responsible for the increased secretion of matrix metalloproteinases (MMPs), collagenases, and chemokines that govern ECM remodeling and collectively mediate wound closure [ 13 ].…”

Extracellular Vesicles in Corneal Fibrosis/Scarring

“…The resulting fibroblasts has a spindle-shaped morphology, possess multiple nucleoli, and lack cytoplasmic granules [ 39 ]. Fibroblasts are a proliferative and migratory cell type which secrete an array of repair-type ECM components, collagenases, and proteinases, that function in reconstructing the damaged stroma [ 63 ]. Not only do corneal fibroblasts serve to protect the stroma, but they act as sentinel cells to sense injury or infection by recognizing changes to the surrounding ECM, damage-associated molecular patterns (DAMPs) derived from damaged cells, pathogen-associated molecular patterns (PAMPs) derived from infectious microbes, and host cytokines through various receptors [ 64 ].…”

“…As the corneal healing process advances into the last stages, corneal fibroblasts can further differentiate into myofibroblasts under the synergistic action of serum and TGFβ1 [ 63 ]. Myofibroblasts are not prevalent in the uninjured cornea and are activated upon injury.…”