Wnt5a, a New Diabetic Corneal Marker Related to Wound Healing

Wnt5a，一种与伤口愈合相关的新糖尿病角膜标记物

基本信息

批准号：
10682429
负责人：
Alexander V Ljubimov
金额：
$ 41.75万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10682429
关键词：
Acceleration Address Adult Affect Age Antineoplastic Agents Binding Blindness Cathepsins Cell Proliferation Cells Combined Modality Therapy Complications of Diabetes Mellitus Cornea Corneal Diseases DNA DNA Methylation Decitabine Defect Deoxycytidine Development Diabetes Mellitus Diagnosis Disease Epigenetic Process Epithelial Cells Epithelium Eye Eye diseases Family Future Gene Abnormality Gene Expression Genes Goals Growth Human Hypermethylation Image Impaired wound healing Impairment Iris Keratopathy MAP3K7 gene MAPK4 gene MCAM gene Mediating Memory Metabolic Methods Methylation MicroRNAs Molecular Mechanisms of Action Nanoconjugate Nature Neuropathy Normal Cell Operative Surgical Procedures Organ Culture Techniques Pain Pathway interactions Phenotype Phosphorylation Population Prediction of Response to Therapy Process Proliferating Proteins Public Health Applications Research ROR1 gene Recurrence Research Project Grants Retina Signal Transduction Signaling Molecule Small Interfering RNA Source Surface System Testing Therapeutic Tissues Transplantation Treatment Efficacy Ulcer Viral Vision research Visual impairment WNT5A gene WNT5A protein Zebularine antagonist bead chip biomarker identification cell motility corneal epithelial stem cells corneal epithelial wound healing corneal epithelium demethylation diabetic diabetic patient effective therapy efficacy testing epigenetic regulation epithelial stem cell epithelial wound gene therapy improved inhibitor innovation lens limbal member nano nanodrug nanopolymer nanotherapy novel protein expression receptor restoration single-cell RNA sequencing stem cell biomarkers stem cell population stem cells stromelysin 2 wound healing

项目摘要

Diabetes is the most widespread blinding disease in working-age adults. Up to 70% of diabetic patients suffer from corneal problems including neuropathy and epithelial keratopathy (impaired wound healing, ulcers, recurrent erosions) that impair vision and cause pain and discomfort. Diabetic keratopathy is underdiagnosed, and therapy remains symptomatic. We previously identified markers altered in human diabetic corneas and normalized their levels in human corneal organ cultures by gene and nano therapy, which also restored normal stem cell phenotype and corneal wound healing. Epigenetic changes may contribute to diabetic complications. Therefore, we examined epigenetic DNA methylation in human diabetic corneas and found a set of genes abnormally methylated vs. normal corneas. WNT5A gene, a noncanonical member of Wnt regulators of cell motility, proliferation and differentiation, was hypermethylated in diabetic corneas. Its expression was reduced in diabetic corneas and stem cell-enriched epithelial cultures. Wnt5a addition accelerated wound healing in diabetic (but not in normal cells), with stem cell marker expression increase. Inhibition of diabetes-increased miRNA-203a targeting WNT5A increased Wnt5a and wound healing in diabetic cells. miRNA-203a inhibitor and WNT5A siRNA reduced wound healing in normal cells. We will identify mechanisms of action of new diabetic marker Wnt5a, its effects on diabetic corneal cell populations, and normalize its levels in diabetic corneal cells. We hypothesize that normalization of diabetes-impaired stem cell phenotype and epithelial wound healing may be achieved by restoring normal expression of noncanonical Wnt5a by blocking its inhibiting microRNA with a novel nanoconjugate and using demethylating agents. Specific Aim 1. To identify Wnt5a receptor(s) mediating its effects on diabetic epithelial cells and corneas. Wnt5a receptors in diabetic limbal cells and corneas will be identified by imaging and functionally, by siRNAs, focusing first on ROR2. This will allow modulating their expression and signaling in diabetic cells to boost Wnt5a effects. Specific Aim 2. To examine Wnt5a effects on epithelial wound healing and limbal cell populations in human organ-cultured diabetic corneas. In these corneas, we will confirm normalizing Wnt5a effects using wound healing and stem cell marker expression. Changes in limbal epithelial differentiated and stem cell populations upon Wnt5a (or inhibitors) treatment will be examined by single cell RNA-seq vs. control normal cells. Specific Aim 3. To test the efficacy of therapies aimed at increasing Wnt5a expression using cultured diabetic cells and organ-cultured corneas. Diabetic limbal epithelial cells and corneas will be treated with nanoconjugate with miR-203a specific antagonist and/or with demethylating 5-Aza-2′-deoxycytidine (Decitabine) or Zebularine. Our aims fit well the priorities set in the NEI Vision Research: Needs, Gaps, and Opportunities. These priorities include (1) improving the transplantation of cultured corneal epithelial cells, (2) understanding of the epigenetic regulation of wound healing, and (3) developing methods to enhance the wound healing process.

糖尿病是工作年龄成年人中最常见的致盲疾病，高达 70% 的糖尿病患者患有糖尿病。角膜问题，包括神经病变和上皮性角膜病变（伤口愈合受损、溃疡、复发性糜烂）会损害视力并引起疼痛和不适，但糖尿病性角膜病的诊断不足。我们之前在人类糖尿病角膜中发现了标记物，并且治疗仍然有效。通过基因和纳米疗法使它们在人类角膜器官培养物中的水平正常化，这也恢复了正常干细胞表型和角膜伤口愈合的变化可能导致糖尿病并发症。因此，我们检查了人类糖尿病角膜的表观遗传DNA甲基化，发现了一组基因异常甲基化与正常角膜的 WNT5A 基因，细胞 Wnt 调节因子的非规范成员。运动、增殖和分化，在糖尿病角膜中高度甲基化，其表达在糖尿病角膜中降低。糖尿病角膜和富含干细胞的上皮培养物的添加可加速伤口愈合。糖尿病（但正常细胞中不存在），随着干细胞标志物表达的增加而抑制糖尿病。靶向 WNT5A 的 miRNA-203a 可以增加糖尿病细胞中的 Wnt5a 和伤口愈合。 WNT5A siRNA 减少正常细胞的伤口愈合我们将确定新糖尿病的作用机制。标记物 Wnt5a，其对糖尿病角膜细胞群的影响，并使其在糖尿病角膜细胞中的水平正常化。我们追求糖尿病受损干细胞表型和上皮伤口的正常化通过阻断非经典 Wnt5a 的正常表达，可以实现治愈使用新型纳米缀合物和去甲基化剂抑制 microRNA。具体目标 1. 鉴定 Wnt5a 受体介导其对糖尿病上皮细胞和角膜的影响。糖尿病角膜缘细胞和角膜中的受体将通过成像和功能性的 siRNA 进行识别，重点关注首先是 ROR2，这将允许调节它们在糖尿病细胞中的表达和信号传导，以增强 Wnt5a 的作用。具体目标 2. 检查 Wnt5a 对人类上皮伤口愈合和角膜缘细胞群的影响在这些角膜中，我们将使用伤口来确认 Wnt5a 的正常化效果。角膜缘上皮分化和干细胞群的愈合和干细胞标志物表达的变化。 Wnt5a（或抑制剂）治疗后，将通过单细胞 RNA-seq 与对照正常细胞进行检查。具体目标 3. 使用培养的糖尿病患者来测试旨在增加 Wnt5a 表达的疗法的功效细胞和器官培养的角膜将用纳米缀合物进行处理。与 miR-203a 特异性拮抗剂和/或去甲基化 5-Aza-2'-脱氧胞苷（地西他滨）或 Zebularine 一起使用。我们的目标非常符合 NEI 愿景研究中设定的优先事项：需求、差距和机遇。优先事项包括（1）改进培养的角膜上皮细胞的移植，（2）了解伤口愈合的表观遗传调控，以及（3）开发增强伤口愈合过程的方法。