Dual epigenetic regulation of WNT5A in diabetic corneal epithelial wound healing

Abstract

Purpose : Diabetes mellitus (DM) is a leading cause of blindness in working-age adults. It involves various epigenetic modifications such as altered DNA methylation and miRNA changes. In the cornea, DM causes persistent epithelial alterations including impaired wound healing, which may occur due to the dysfunction of limbal epithelial stem cells (LESC). Previously, we reported hypermethylation of WNT5A at the promoter region and up-regulation of Wnt5a regulating miR-203a in diabetic limbal epithelial cells (LEC) enriched in LESC, resulting in decreased Wnt5a expression. Our purpose was to use DNA demethylation and gene therapy approaches to normalize diabetic corneal epithelial wound healing and limbal stem cell expression by increasing Wnt5a expression.

Methods : Diabetic organ-cultured corneas obtained from postmortem human donor eyes were pre-treated with (A) DMSO control or DNA methylation inhibitor, zebularine (20 µM) for 48 hours or (B) biodegradable nanobioconjugates based on polymalic acid (PMLA) scaffold containing a control scrambled antisense oligonucleotide (AON) or AON to miR-203a (supposed to increase Wnt5a) for 4 days. Corneal epithelial 5-mm wounds were created with 1-heptanol, and wound healing was monitored over time. The healed corneas were embedded in OCT and sectioned (5 µm) for immunostaining to determine the limbal stem cell and diabetic marker expression.

Results : In diabetic organ-cultured corneas treated with 20 µM of zebularine, DNMT1 was significantly inhibited and Wnt5a expression was increased, resulting in the acceleration of corneal epithelial wound healing as compared to DMSO controls. Moreover, this treatment also increased the expression of putative stem cell markers, keratins 15 and 17, as well as diabetic markers, integrin α3β1 and nidogen-1, and activated the wound healing mediator, p-Akt at Ser 473. Similarly, treatment with the nanodrug PMLA-AON miR-203a also increased Wnt5a expression and stimulated epithelial wound healing vs. scrambled AON control. It also increased putative LESC (keratins 15 and 17) and diabetic (integrin α3β1 and nidogen-1) marker expression.

Conclusions : Our study documented for the first time the dual regulation of WNT5A in the diabetic cornea through DNA hypermethylation and miRNA action. Inhibition of DNA hypermethylation and miR-203a action can normalize corneal epithelial wound healing and limbal stem cell expression through the upregulation of Wnt5a.

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