June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Wnt5a promotes diabetic corneal epithelial wound healing and limbal stem cell expression
Ruchi Shah; Tanya M Spektor; Vasu Punj; Sue Turjman; Sean Ghiam; Jiho Kim; Sasha Tolstoff; Cynthia Amador; Steven Takeshi Chun; Daniel J Weisenberger; Mehrnoosh Saghizadeh; Andrei A Kramerov; Alexander V Ljubimov
Author Affiliations & Notes
  • Ruchi Shah
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Tanya M Spektor
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Vasu Punj
    University of Southern California Keck School of Medicine, Los Angeles, California, United States
  • Sue Turjman
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Sean Ghiam
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Jiho Kim
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Sasha Tolstoff
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Cynthia Amador
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Steven Takeshi Chun
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Daniel J Weisenberger
    University of Southern California Keck School of Medicine, Los Angeles, California, United States
  • Mehrnoosh Saghizadeh
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
    University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Andrei A Kramerov
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
  • Alexander V Ljubimov
    Eye Program, Cedars-Sinai Medical Center Board of Governors Regenerative Medicine Institute, Los Angeles, California, United States
    University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Ruchi Shah, None; Tanya Spektor, None; Vasu Punj, None; Sue Turjman, None; Sean Ghiam, None; Jiho Kim, None; Sasha Tolstoff, None; Cynthia Amador, None; Steven Chun, None; Daniel Weisenberger, None; Mehrnoosh Saghizadeh, None; Andrei Kramerov, None; Alexander Ljubimov, None
  • Footnotes
    Support  NIH R01 EY031377 EY013431
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 847. doi:
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      Ruchi Shah, Tanya M Spektor, Vasu Punj, Sue Turjman, Sean Ghiam, Jiho Kim, Sasha Tolstoff, Cynthia Amador, Steven Takeshi Chun, Daniel J Weisenberger, Mehrnoosh Saghizadeh, Andrei A Kramerov, Alexander V Ljubimov; Wnt5a promotes diabetic corneal epithelial wound healing and limbal stem cell expression. Invest. Ophthalmol. Vis. Sci. 2021;62(8):847.

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Abstract

Purpose : Diabetes mellitus appears to be an epigenetic disease with DNA methylation and histone acetylation changes. It causes persistent corneal epithelial alterations including impaired wound healing, which may occur due to the dysfunction of limbal epithelial stem cells (LESC). Previously, DNA methylation analysis showed WNT5A gene to be hypermethylated in diabetic primary cultured human limbal epithelial cells (LEC) enriched in LESC. Our purpose was to examine the effect of Wnt5a on diabetic corneal epithelial wound healing and limbal stem cell expression.

Methods : LEC were isolated from age-matched autopsy non-diabetic and diabetic limbal rims and eyes with IRB approval. Scratch wound healing was carried out in LEC treated with recombinant Wnt5a (200 ng/mL) or DNA methylation inhibitor, zebularine (1, 5, 10 µM), or transfected with siRNA to WNT5A or with miR-203a mimic or inhibitor. Healing of epithelial wounds created with 1-heptanol was monitored in organ-cultured diabetic corneas with or without the addition of exogenous Wnt5a. Protein expression was determined in these LEC and corneas using Western blot and immunostaining.

Results : Diabetic LEC and corneal epithelium showed significantly decreased expression of Wnt5a protein apparently due to hypermethylation of WNT5A gene. Wnt5a treatment significantly stimulated wound healing in cultured cells and organ-cultured corneas and increased the expression of putative stem cell markers, K15 and K17. In contrast, siRNA knockdown of WNT5A in non-diabetic LEC decreased wound healing vs. control. Zebularine significantly decreased the expression of DNA methyltransferase DNMT1 and 5-methylcytosine and increased Wnt5a expression with an acceleration of wound healing in diabetic LEC. siRNA experiments suggested that demethylating effect was directly related to Wnt5a level changes. Transfection of LEC with Wnt5a-suppressing miR-203a significantly retarded wound healing in non-diabetic LEC, whereas miR-203a inhibitor increased Wnt5a expression and promoted wound healing in diabetic LEC.

Conclusions : In summary, this study confirms the epigenetic changes in diabetic limbal epithelium with reproducible differential methylation in non-diabetic and diabetic primary LEC. Wnt5a is a new diabetic marker in the cornea that appears to be under dual inhibition in diabetic corneas by DNA hypermethylation and miRNA action.

This is a 2021 ARVO Annual Meeting abstract.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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Copyright © 2024 Association for Research in Vision and Ophthalmology.
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