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：
10468981
负责人：
Alexander V Ljubimov
金额：
$ 40.5万
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/10468981
关键词：
Address Adult Affect Age Antineoplastic Agents Binding Blindness Cathepsins Cell Proliferation Cells Complications of Diabetes Mellitus Cornea Corneal Diseases DNA DNA Methylation Defect Deoxycytidine Development Diabetes Mellitus Disease Epigenetic Process Epithelial Epithelial Cells Eye Eye diseases Family Future Gene Abnormality Gene Expression Genes Goals Human Image Impaired wound healing Impairment Iris Keratopathy Lead MAP3K7 gene MAPK4 gene MCAM gene Mediating Memory Metabolic Methods MicroRNAs Molecular Mechanisms of Action Nanoconjugate Nature Neuropathy Normal Cell Organ Organ Culture Techniques Pain Pathway interactions Phenotype Phosphorylation Population Process Proteins Public Health Applications Research ROR1 gene Receptor Signaling 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 base bead chip cell motility corneal epithelial stem cells corneal epithelial wound healing corneal epithelium 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 wound healing

Address Adult Affect Age Antineoplastic Agents Binding Blindness Cathepsins Cell Proliferation Cells Complications of Diabetes Mellitus Cornea Corneal Diseases DNA DNA Methylation Defect Deoxycytidine Development Diabetes Mellitus Disease Epigenetic Process Epithelial Epithelial Cells Eye Eye diseases Family Future Gene Abnormality Gene Expression Genes Goals Human Image Impaired wound healing Impairment Iris Keratopathy Lead MAP3K7 gene MAPK4 gene MCAM gene Mediating Memory Metabolic Methods MicroRNAs Molecular Mechanisms of Action Nanoconjugate Nature Neuropathy Normal Cell Organ Organ Culture Techniques Pain Pathway interactions Phenotype Phosphorylation Population Process Proteins Public Health Applications Research ROR1 gene Receptor Signaling 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 base bead chip cell motility corneal epithelial stem cells corneal epithelial wound healing corneal epithelium 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 wound healing

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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广泛的盲目疾病。多达70％的糖尿病患者患者来自神经病和上皮角膜病等角膜问题（伤口愈合受损，溃疡，复发性侵蚀）会损害视力并引起疼痛和不适。糖尿病性角膜病的诊断不足，疗法仍然有症状。我们以前确定在人类糖尿病角膜和通过基因和纳米疗法使它们在人角膜器官培养物中的水平归一化，这也恢复了正常干细胞表型和角膜伤口愈合。表观遗传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中的表观遗传DNA甲基化，并发现了一组基因交替地甲基化与正常角膜。 Wnt5a基因，细胞Wnt调节剂的非规范成员运动，增殖和分化，在糖尿病角膜中被过度甲基化。它的表达减少了糖尿病角膜和富含干细胞的上皮培养物。 WNT5A加入加速伤口愈合糖尿病（但不在正常细胞中），干细胞标记表达增加。抑制糖尿病的抑制 miRNA-203a靶向Wnt5a增加了Wnt5a和糖尿病细胞中伤口愈合。 miRNA-203a抑制剂和 Wnt5a siRNA减少了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Wnt5a，其对糖尿病角膜细胞群体的影响，并在糖尿病角膜细胞中归一化。我们假设糖尿病受损的干细胞表型和上皮伤口的归一化可以通过恢复非规范Wnt5a的正常表达来实现愈合用新型的纳米缀合物抑制microRNA并使用脱甲基剂。具体目的1。鉴定Wnt5a受体介导其对糖尿病上皮细胞和角膜的影响。 Wnt5a 糖尿病长缘细胞和角膜中的受体将通过成像和功能上的siRNA识别，聚焦首先在ROR2上。这将允许调节其在糖尿病细胞中的表达和信号传导，从而增强WNT5A效应。特定目的2。检查Wnt5a对人类上皮伤口愈合和人缘细胞种群的影响器官培养的糖尿病角膜。在这些角膜中，我们将确认使用伤口标准化Wnt5a效应愈合和干细胞标记表达。边缘上皮分化和干细胞种群的变化在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。特定目的3。测试旨在使用培养的糖尿病来增加Wnt5a表达的疗法的效率细胞和有组织的角膜。糖尿病长缘上皮细胞和角膜将用纳米缀合物处理用miR-203a特异性拮抗剂和/或脱甲基化的5-aza-2'-脱氧胞苷（去替替啶）或Zebularine。我们的目标非常适合NEI Vision Research：需求，差距和机遇。这些优先级包括（1）改善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节，以及（3）开发增强伤口愈合过程的方法。