Epigenetic mechanism to induce dedifferentiation of corneal myofibroblast to fibroblast

Abstract

Purpose : Excessive formation and residence of myofibroblasts in stroma following ocular trauma/injury leads to loss of corneal transparency and vision impairment. This study sought to determine if corneal myofibroblasts could be de-differentiated into precursor fibroblasts/keratocytes via epigenetic reprogramming using an established in vitro model.

Methods : Primary human corneal stromal fibroblasts (hCSFs) generated from healthy donor corneas were used to generate human corneal myofibroblasts (hCMFs) by exposing cultures to transforming growth factor beta1 (TGFβ1; 5 µg/mL) for 72 h. Sodium Butyrate (NaB), an epigenetic modifier and histone deacetylase inhibitor, was used to induce epigenetic reprogramming by growing hCMFs in +/- NaB for 72 h. Trypan blue exclusion assay and phage-contrast microscopy were used to examine cytotoxicity and morphologic changes. Quantitative reverse transcription PCR (qRT-PCR) and immunofluorescence were used to study the expression of α-smooth muscle actin (α-SMA), collagen type 3 (Col 3), and fibroblast-specific protein 1 (FSP1). The DNA methylation was quantified by measuring the 5-methylcytosine (5-mC) levels with a commercial assay kit.

Results : TGFβ1 treatment converted >95% hCSFs to hCMFs with significant upregulation of α-SMA protein and gene expression. A dose-dependent cytotoxicity assay with NaB (0-100 mM) in CMFs showed IC₅₀ of NaB 20.38 mM. CMFs treated with NaB for 72 h led to the disappearance of typical myofibroblastic morphology and demonstrated a significant reduction in α-SMA, and Col 3, and increase in FSP1 expression compared to control untreated CMFs (p < 0.05). A significantly increased DNA methylation was observed in NaB-treated CMFs compared to the control untreated CMFs (p < 0.05).

Conclusions : Dedifferentiation of corneal myofibroblasts to precursor fibroblasts seems possible via epigenetic reprogramming with NaB. Further studies are warranted.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.