The Cedars-Sinai research is the first to discover disease-related changes in the cornea and proposes potential therapeutic approaches to correct diabetic wound healing.
Summary Objectives/hypotheses Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and wound healing problems due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to discover epigenetic alterations in LESC-enriched diabetic versus non-diabetic human limbal epithelial cells (LEC) and identify novel diabetic markers that can be targeted for therapy to normalize corneal epithelial wound healing and expression. of stem cells. Methods Human limbal epithelial cells (LECs) were isolated or organ-cultured corneas obtained from autopsy eyes of 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 phosphor array analysis. Wound healing was studied using a scrape assay on LEC or 1-heptanol wounds on organ cultured corneas. Cultured corneas from organs and LECs were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or 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) for miR-203a or a scrambled AON control (15–20 µmol/l). Results There was significant differential DNA methylation between diabetic and non-diabetic LECs. The WNT5A promoter was hypermethylated in diabetic LEC accompanied by a markedly decreased Wnt-5a protein. Treatment of diabetic human limbal epithelial cells (LECs) 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 the expression of LESC and diabetic marker. Wnt-5a treatment in diabetic human limbal epithelial cells (LECs) increased phosphorylation of members of the non-canonical Ca2+-dependent pathway (phospholipase Cγ1 and protein kinase Cβ; by 1.15-fold [p<0.05] and 1.36 times [p<0.05], respectively). In diabetic LECs, zebularine treatment increased Wnt-5a levels by 1.37-fold (p<0.01) and stimulated wound healing in a dose-dependent manner with a 1.6-fold increase (p< 0.01) at 24 h. Furthermore, zebularine also improved wound healing by 30% (p<0.01) in corneas cultured from diabetic organs and increased the expression of LESC and diabetic markers. Transfection of these cells with WNT5A siRNA abolished zebularin’s stimulation of wound healing, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of organ-cultured corneal and diabetic human limbal epithelial cells (LECs) with NBC improved wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively. with higher 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 novel wound healing stimulator of corneal epithelium that can be used to improve wound healing and stem cells in the diabetic cornea. |
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Cedars-Sinai researchers have provided new insight into how diabetes slows wound healing in the eye, identifying for the first time two changes in the cornea linked to the disease.
The findings, published in the peer-reviewed journal Diabetologia , also identified three therapeutic pathways that reversed these changes and partially restored wound-healing function to the cornea, a discovery that could ultimately inform new treatments for diabetes.
"We found that diabetes induces more cellular changes than we previously knew," said Alexander Ljubimov, PhD, director of the Eye Program at the Cedars-Sinai Board of Governors Institute for Regenerative Medicine and senior author of the paper. “The discovery does not affect the gene sequence, but involves specific DNA modifications that alter gene expression, known as epigenetic alterations.”
More than 37 million people in the United States, 11% of the population, have diabetes, a systemic disorder that can lead to kidney disease, heart disease, amputations, strokes and nerve damage. Most diabetes medications are designed to increase glucose tolerance or supply depleted insulin, but do not address the molecular and cellular changes and their associated complications.
The new research also identifies for the first time an important role for Wnt-5a, a secreted signaling protein that the researchers found responsible for wound healing in the cornea and the function of stem cells, cells capable of differentiating into many types of cells. cells.
“Current treatments only address symptoms, so there is an urgent need to understand the molecular mechanisms of diabetes-related wound healing problems,” said Ruchi Shah, PhD, a scientist in Ljubimov’s laboratory and first author of the study. . “Understanding this novel epigenetically regulated wound healing mechanism could lead to therapeutic treatments that could help patients avoid further long-term eye health problems.”
Although much of the focus on diabetic eye disease is on the retina, up to 70% of patients with diabetes suffer from corneal problems .
In advanced diabetes, corneal stem cells become dysfunctional and the cornea heals more slowly and less completely after injury or procedures such as cataract surgery and laser treatment for diabetic retinopathy.
To identify the epigenetic changes discovered in this study, changes that are not integrated into the genome from birth but were introduced later, Ljubimov and his team compared cells from the corneas of six diabetic patients with those of five healthy donors. They found that in diabetic corneas, the protein product of the WNT5A gene was repressed. Furthermore, in diabetic samples they found an increase in the microRNA that inhibits WNT5A.
The team of scientists then induced injury to cultured corneal cells and corneal organ cultures, and tested three interventions designed to normalize Wnt-5a protein expression. They added the Wnt-5a protein directly; introduced a DNA methylation inhibitor, originally approved to treat cancer; and targeted microRNA levels with a novel gene therapy approach using a nanoscale compound. The team developed the compound, which uses synthetic molecules to block microRNA, as a substitute for a viral gene therapy that they found toxic to stem cells.
The three therapeutic methods, in the diabetic samples, stimulated the production of stem cell markers and improved tissue regeneration, accelerating wound healing.
“New therapies to reverse epigenetic effects could improve corneal function and may also be significant in other diabetic complications,” said Clive Svendsen, PhD, director of the Board of Governors of the Institute for Regenerative Medicine and co-author of the study. “This work certainly helps advance the field.”
The researchers will continue to analyze their data to better understand the mechanisms of WNT5A and other genes related to wound healing. They are also studying a combination therapy to target both microRNA and DNA methylation in the hopes that it will more completely normalize wound healing by increasing the Wnt-5a protein.
“Our goal is to develop sustained-release topical medications for corneal wound healing,” Ljubimov said. “Drugs that are approved by the FDA [Food and Drug Administration] and could be easily applied may be one of the most promising approaches for future effective therapies.”
Funding: This work was supported by National Institutes of Health grant numbers R01EY013431482, R01EY031377, R01EY025377, and R01CA206220; and the Cedars-Sinai Board of Governors Institute for Regenerative Medicine.
