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Purpose: MiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing., Methods: Our previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs., Results: Overexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1β, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a., Conclusion: Our study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions., (Copyright © 2022. Published by Elsevier Inc.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

PurposeMiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing.MethodsOur previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs.ResultsOverexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1β, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a.ConclusionOur study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.

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.

Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells.

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 (β-catenin-dependent) or non-canonical (β-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. [ABSTRACT FROM AUTHOR]