MyoD gene silencing promotes corneal fibroblast de-differentiation and reverses fibrosis

Abstract

Purpose : Myofibroblasts proliferate, de-differentiate, and transform to precursor resident fibroblasts in various non-ocular tissues. Our ongoing studiesfound that corneal myofibroblasts (CMFs) are not terminally differentiated albeit undergo proliferation and can acquire differing phenotype based on the stimulants (cytokines, heparin and genes). This study tested a hypothesis thatde-differentiation of CMFs into precursor corneal stromal fibroblasts (CSFs) by silencing of MyoD (basic helix-loop-helix) gene to inhibit alpha smooth muscle actin (αSMA) transcriptional machinery is an innovative approach to cure corneal fibrosis using an in vitromodel.

Methods : Donor human corneas obtained from Eye Banks were used to generate human corneal stromal fibroblasts (CSFs). CMFs were produced by growing CSFs in 5ng/ml TGFβ1 for 72h under serum-free conditions.MyoD-RNAi and control scrambled-RNAi plasmids were obtained from Dr. VictorThannickal, University of Alabama, Birmingham. The DNA sequences of each plasmid were confirmed using DNA core lab.Non-transfected and MyoD/scrambled-RNAi transfected hCMFs were grown in +/- 5ng/ml TGFβ1 for 72h under serum-free conditions. Lipofectamine-3000 and PEI2-GNPs nanoparticles were used for gene transfer. Immunofluorescence, western blotting, qPCR, confocal microscopy, and slot blots quantified mRNA, protein, and delivered-gene levels.

Results : We found that tissue sections from donor fibrotic human cornea showed significantly high expression of MyoD compared to the normal human cornea (41-63%; p<0.05). Likewise, CMFs showed markedly increased MyoD expression compared to the CSFs in vitro (p<0.01). The MyoD-RNAi or scrambled-RNAi delivery into CSFs and CMFsdid not alter their appearance, viability, or proliferationin normal culture conditions. MyoD-RNAi transfected CMFs grown with TGFβ1 in serum-free condition acquired phenotype similar to CSFs in a time-dependent manner (12-24% in 24h, 43-53% in 48h, and 79-87%-72h). Furthermore, MyoD-RNAi silencing in CMFs demonstrated significantly deceased αSMAmRNA and protein levels in a time dependent manner (71-83%; p<0.001). This pilot data and ongoing mechanistic studies suggest that MyoD gene silencing promotes de-differentiation of corneal myofibroblasts and their reversal to precursor CSFs.

Conclusions : Human corneal myofibroblasts are not terminally differentiated and undergo proliferation and de-differentiation to fibroblasts in vitro.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.