Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells
Creators
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Shah, Ruchi1
- Spektor, Tanya M.1, 2
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Weisenberger, Daniel J.3
- Ding, Hui1
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Patil, Rameshwar1, 4
- Amador, Cynthia1
- Song, Xue-Ying1
- Chun, Steven T.1, 5
- Inzalaco, Jake1, 5
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Turjman, Sue1, 6
- Ghiam, Sean1, 7
- Jeong-Kim, Jiho1, 5
- Tolstoff, Sasha1, 8
- Yampolsky, Sabina V.1, 9
- Sawant, Onkar B.
- Rabinowitz, Yaron S.1
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Maguen, Ezra
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Hamrah, Pedram10
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Svendsen, Clive N.1, 5
- Saghizadeh, Mehrnoosh1, 5
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Ljubimova, Julia Y.1, 11
- Kramerov, Andrei A.1
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Ljubimov, Alexander V.1, 5
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1.
Cedars-Sinai Medical Center
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2.
Kura Oncology (United States)
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3.
University of Southern California
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4.
Loma Linda University Medical Center
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5.
University of California, Los Angeles
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6.
Mount St. Mary's College
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7.
Tel Aviv University
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8.
California Institute of Technology
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9.
Duke University
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10.
Tufts Medical Center
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11.
Terasaki Foundation
Abstract
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 Cβ; 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.
Copyright and License
© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Acknowledgement
We thank S. Svendsen (Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, USA) for a thorough editing of the manuscript and V. Punj (Keck School of Medicine, University of Southern California, USA) for bioinformatics analysis. Parts of this work were presented at Gordon Research Conferences on Wnt signalling (2019), Cornea and Ocular Surface Biology and Pathology (2020), International Society for Stem Cell Research (2019, 2022), Cell Symposia (2018, 2022), and The Association for Research in Vision and Ophthalmology (2019, 2021, 2022).
Funding
Open access funding provided by SCELC, Statewide California Electronic Library Consortium. This work was supported by grants from the National Institutes of Health (R01EY013431 to AVL; R01EY031377 to AVL; R01EY025377 to MS; R01CA206220 to JYL) and funding from the Board of Governors Regenerative Medicine Institute (to AVL).
Contributions
RS contributed to conceptualisation, design and methodology, supervision, investigation and analyses, and writing, reviewing, editing, and approving the manuscript. TMS, DJW, HD, RP, CA, XYS, STC, JI, STu, SG, JJK, STo, SVY and PH contributed to design and methodology, investigation, reviewing, editing and approving the manuscript. OBS, YSR, and EM contributed to design and methodology, data collection, reviewing, editing, and approving the manuscript. CNS contributed to design and methodology, funding acquisition, reviewing, editing, and approving the manuscript. MS contributed to conceptualisation, design and methodology, supervision, funding acquisition, project administration, investigation, reviewing, editing, and approving the manuscript. JYL contributed to conceptualisation, design and methodology, funding acquisition, reviewing, editing and approving the manuscript. AAK contributed to conceptualisation, design and methodology, supervision, investigation and analyses, reviewing, editing, and approving the manuscript. AVL contributed to conceptualisation, design and methodology, supervision, investigation and analyses, funding acquisition, project administration, and writing, reviewing, editing, and approving the manuscript. AVL is the guarantor of this work.
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).
Conflict of Interest
The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.
Files
125_2023_5960_MOESM1_ESM.pdf
Additional details
Identifiers
- ISSN
- 1432-0428
- PMCID
- PMC10474199
Funding
- Statewide California Electronic Library Consortium
- National Institutes of Health
- R01EY013431
- National Institutes of Health
- R01EY031377
- National Institutes of Health
- R01EY025377
- National Institutes of Health
- R01CA206220
- Cedars-Sinai Medical Center
- Board of Governors Regenerative Medicine Institute