Epigenetic Reprogramming Via Sodium Butyrate Induces Corneal Myofibroblast Dedifferentiation In Vitro and Inhibits Fibrosis In Vivo.

Abstract

PURPOSE: Abnormal corneal myofibroblast (CMF) differentiation and persistence in stroma after ocular trauma causes corneal fibrosis and impaired vision. This study tested whether epigenetic reprogramming via sodium butyrate (NaB), histone deacetylase inhibitor, could provoke CMF dedifferentiation into precursor/non-opaque corneal fibroblast/keratocyte (CSF) like cells using human in vitro and rabbit in vivo corneal fibrosis models. METHODS: Healthy human cadaver corneas generated CSFs were converted into CMFs by TGF&#x3b2;1 (5ng/ml) and grown in -/+ NaB (5 mM) for 72 hours. Quantitative RT-PCR and immunofluorescence measured changes in profibrotic markers (alpha smooth muscle actin [&#x3b1;SMA], collagen-III [Col-III], fibronectin [FN]) and fibroblast marker (fibroblast-specific protein-1 [FSP1]). Epigenetic reprogramming was measured by quantification of total HDAC activity by commercial-kit and DNA methylation using methylation-specific PCR primers for &#x3b1;SMA, Col-III, and FSP1 genes. Collagen gel contraction assay (CGA) evaluated response of NaB on CMF's contractile function. In vivo clinical utility of NaB in reducing corneal fibrosis was evaluated via stereomicroscopy and Pentacam imaging in live rabbits. RESULTS: NaB treatment to CMFs changed myofibroblast-morphology to fibroblast-like and significantly reduced &#x3b1;SMA (P = 0.0005), Col-III (P = 0.011), FN (P = 0.0004), and increased FSP1 (P = 0.0004) gene expression in human in vitro. Additionally, it significantly reduced total HDAC activity (P = 0.0009) with hypermethylation of &#x3b1;SMA, Col-III, and hypomethylation of FSP1 gene promoters. Also, NaB-treated CMFs showed significantly reduced contractility (P < 0.0001) in CGA. Topical NaB treatment markedly reduced opacity in alkali-injured rabbit corneas in vivo. CONCLUSIONS: Dedifferentiation of CMFs via epigenetic reprogramming by NaB offers an attractive approach to treat corneal fibrosis in vivo. Additional studies are warranted.