Diabetes mellitus (DM) is a major cause of vision loss worldwide. Diabetic corneas have epithelial alterations such as impaired wound healing, possibly due to dysfunctional limbal epithelial stem cells (LESC). We previously used gene therapy and DNA demethylation to modulate DM-altered proteins cathepsin F (increased in DM), c-Met (decreased in DM), and Wnt-5a (decreased in DM) to stimulate wound healing and LESC marker expression. Here, we used combined therapy with a DNA methylation inhibitor and a nanobioconjugate (NBC) targeting cathepsin F and c-Met. Human donor diabetic organ-cultured corneas (6 pairs) were treated with 20 µM of biodegradable NBC based on polymalic acid scaffold containing antisense oligonucleotides (AON) to cathepsin F and miR-409 (c-Met inhibitor) or control scrambled AON twice for 2 days, with 20 µM of DNA demethylator zebularine or DMSO control for 2 days. One cornea received combined treatment, and the fellow one, combined controls. Healing of 5-mm epithelial wounds made with 1-heptanol was monitored, and marker expression was examined by immunostaining or western blot. Organ-cultured DM corneas treated with combined zebularine and NBC suppressed DNA methyltransferase 1 and cathepsin F, and increased Wnt-5a and c-Met. This resulted in significant decrease (by 38.4%, p<0.02) of corneal epithelial wound healing time vs. DMSO and control NBC. Zebularine alone stimulated healing by 29.4%, and NBC alone, by 20.4%. Combination treatment increased the expression of putative LESC markers, keratins 15 and 17. DM markers integrin α3β1 and nidogen-1, and wound healing mediators p-Akt and p-p38 also had increased staining. There was no change in activated caspase-3 staining, indicating lack of treatment toxicity.Combination therapy with DNA methylation inhibitor and NBC targeting diabetes-altered protein markers may thus have a potential for the treatment of impaired diabetic corneal epithelial wound healing.
A.V. Ljubimov: None.
National Institutes of Health (EY013431; EY031377; EYEY025377)
