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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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87\u00b120.89 years) and diabetic (71.93\u00b19.29 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\n                      <jats:italic>WNT5A<\/jats:italic>\n                      siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng\/ml), DNA methylation inhibitor zebularine (1\u201320 \u00b5mol\/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15\u201320 \u00b5mol\/l).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      There was significant differential DNA methylation between diabetic and non-diabetic LEC.\n                      <jats:italic>WNT5A<\/jats:italic>\n                      promoter was hypermethylated in diabetic LEC accompanied with markedly\u00a0decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.05) and 37% (\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca\n                      <jats:sup>2+<\/jats:sup>\n                      -dependent non-canonical pathway (phospholipase C\u03b31 and protein kinase C\u03b2; by 1.15-fold [\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.05] and 1.36-fold [\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p&lt;0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with\n                      <jats:italic>WNT5A<\/jats:italic>\n                      siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of\n                      <jats:italic>WNT5A<\/jats:italic>\n                      hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.01) and 23.3% (\n                      <jats:italic>p<\/jats:italic>\n                      &lt;0.05), respectively, with increased expression of LESC and diabetic markers.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions\/interpretation<\/jats:title>\n                    <jats:p>\n                      We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of\n                      <jats:italic>WNT5A<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Data availability<\/jats:title>\n                    <jats:p>\n                      The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 (\n                      <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE229328\">https:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE229328<\/jats:ext-link>\n                      ).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical Abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1007\/s00125-023-05960-1","type":"journal-article","created":{"date-parts":[[2023,7,17]],"date-time":"2023-07-17T19:02:35Z","timestamp":1689620555000},"page":"1943-1958","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells"],"prefix":"10.1007","volume":"66","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9533-1747","authenticated-orcid":false,"given":"Ruchi","family":"Shah","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanya M.","family":"Spektor","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel J.","family":"Weisenberger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Ding","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rameshwar","family":"Patil","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia","family":"Amador","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue-Ying","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven T.","family":"Chun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jake","family":"Inzalaco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sue","family":"Turjman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean","family":"Ghiam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiho","family":"Jeong-Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sasha","family":"Tolstoff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabina V.","family":"Yampolsky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Onkar B.","family":"Sawant","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaron S.","family":"Rabinowitz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ezra","family":"Maguen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pedram","family":"Hamrah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clive N.","family":"Svendsen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mehrnoosh","family":"Saghizadeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia Y.","family":"Ljubimova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrei A.","family":"Kramerov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2398-5319","authenticated-orcid":false,"given":"Alexander V.","family":"Ljubimov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2023,7,18]]},"reference":[{"issue":"1","key":"5960_CR1","doi-asserted-by":"publisher","first-page":"69","DOI":"10.4103\/0301-4738.178150","volume":"64","author":"R Raman","year":"2016","unstructured":"Raman R, Gella L, Srinivasan S, Sharma T (2016) Diabetic retinopathy: an epidemic at home and around the world. 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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).","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Acknowledgements"}},{"value":"The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 (\n                      \n                      ).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Data availability"}},{"value":"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).","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Funding"}},{"value":"The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Authors\u2019 relationships and activities"}},{"value":"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.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Contribution statement"}}]}}