Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

Systemic diseases and the cornea.

There is a number of systemic diseases affecting the cornea. These include endocrine disorders (diabetes, Graves' disease, Addison's disease, hyperparathyroidism), infections with viruses (SARS-CoV-2, herpes simplex, varicella zoster, HTLV-1, Epstein-Barr virus) and bacteria (tuberculosis, syphilis and Pseudomonas aeruginosa), autoimmune and inflammatory diseases (rheumatoid arthritis, Sjögren's syndrome, lupus erythematosus, gout, atopic and vernal keratoconjunctivitis, multiple sclerosis, granulomatosis with polyangiitis, sarcoidosis, Cogan's syndrome, immunobullous diseases), corneal deposit disorders (Wilson's disease, cystinosis, Fabry disease, Meretoja's syndrome, mucopolysaccharidosis, hyperlipoproteinemia), and genetic disorders (aniridia, Ehlers-Danlos syndromes, Marfan syndrome). Corneal manifestations often provide an insight to underlying systemic diseases and can act as the first indicator of an undiagnosed systemic condition. Routine eye exams can bring attention to potentially life-threatening illnesses. In this review, we provide a fairly detailed overview of the pathologic changes in the cornea described in various systemic diseases and also discuss underlying molecular mechanisms, as well as current and emerging treatments.

Non-canonical Wnt signaling in the eye.

Wnt signaling comprises a group of complex signal transduction pathways that play critical roles in cell proliferation, differentiation, and apoptosis during development, as well as in stem cell maintenance and adult tissue homeostasis. Wnt pathways are classified into two major groups, canonical (ß-catenin-dependent) or non-canonical (ß-catenin-independent). Most previous studies in the eye have focused on canonical Wnt signaling, and the role of non-canonical signaling remains poorly understood. Additionally, the crosstalk between canonical and non-canonical Wnt signaling in the eye has hardly been explored. In this review, we present an overview of available data on ocular non-canonical Wnt signaling, including developmental and functional aspects in different eye compartments. We also discuss important changes of this signaling in various ocular conditions, such as keratoconus, aniridia-related keratopathy, diabetes, age-related macular degeneration, optic nerve damage, pathological angiogenesis, and abnormalities in the trabecular meshwork and conjunctival cells, and limbal stem cell deficiency.

MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

Gene Therapy in the Anterior Eye Segment.

This review provides comprehensive information about the advances in gene therapy in the anterior segment of the eye, including cornea, conjunctiva, lacrimal gland, and trabecular meshwork. We discuss gene delivery systems, including viral and non-viral vectors as well as gene editing techniques, mainly CRISPR-Cas9, and epigenetic treatments, including antisense and siRNA therapeutics. We also provide a detailed analysis of various anterior segment diseases where gene therapy has been tested with corresponding outcomes. Disease conditions include corneal and conjunctival fibrosis and scarring, corneal epithelial wound healing, corneal graft survival, corneal neovascularization, genetic corneal dystrophies, herpetic keratitis, glaucoma, dry eye disease, and other ocular surface diseases. Although most of the analyzed results on the use and validity of gene therapy at the ocular surface have been obtained in vitro or using animal models, we also discuss the available human studies. Gene therapy approaches are currently considered very promising as emerging future treatments of various diseases, and this field is rapidly expanding.