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

• 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
• 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

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普遍的致盲疾病。高达70%的糖尿病患者 包括神经病变和上皮角膜病变（伤口愈合受损，溃疡， 复发性糜烂），损害视力并引起疼痛和不适。糖尿病性角膜病变诊断不足， 治疗仍然是对症治疗我们先前确定了人类糖尿病角膜中改变的标记物， 通过基因和纳米治疗使它们在人类角膜器官培养物中的水平正常化，这也恢复了正常的 干细胞表型与角膜创伤愈合表观遗传变化可能导致糖尿病并发症。 因此，我们检测了人类糖尿病角膜中的表观遗传DNA甲基化，发现了一组基因， 异常甲基化与正常角膜。WNT 5A基因是细胞Wnt调节子中的一个非经典成员 运动、增殖和分化，在糖尿病角膜中是高甲基化的。其表达减少， 糖尿病角膜和富含干细胞的上皮培养物。Wnt 5a的加入加速了创伤愈合， 糖尿病（但不是在正常细胞中），干细胞标志物表达增加。抑制糖尿病-增加 靶向WNT 5A的miRNA-203 a增加了糖尿病细胞中的Wnt 5a和伤口愈合。miRNA-203 a抑制剂和 WNT 5A siRNA减少正常细胞中的伤口愈合。我们将确定新的糖尿病的作用机制， 标记物Wnt 5a，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。 我们假设糖尿病受损的干细胞表型和上皮损伤的正常化 通过阻断非经典Wnt 5a的正常表达，可以实现治愈。 用新的纳米缀合物和使用脱甲基化剂抑制微小RNA。 具体目标1。鉴定介导其对糖尿病上皮细胞和角膜作用的Wnt 5a受体。Wnt5a 糖尿病角膜缘细胞和角膜中的受体将通过成像和功能，通过siRNA，聚焦 首先是ROR 2。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Wnt 5a的作用。 具体目标2。研究Wnt 5a对人类上皮伤口愈合和角膜缘细胞群的影响 器官培养的糖尿病角膜在这些角膜中，我们将使用伤口愈合来证实正常化Wnt 5a效应。 愈合和干细胞标志物表达。角膜缘上皮分化和干细胞群的变化 在Wnt 5a（或抑制剂）处理后，将通过单细胞RNA-seq相对于对照正常细胞来检查。 具体目标3。为了测试旨在使用培养的糖尿病细胞增加Wnt 5a表达的疗法的功效， 细胞和器官培养的角膜。糖尿病角膜缘上皮细胞和角膜将用纳米缀合物治疗 与miR-203 a特异性拮抗剂和/或与去甲基化5-氮杂-2 ′-脱氧胞苷（地西他滨）或Zebularine。 我们的目标非常符合NEI愿景研究中设定的优先事项：需求，差距和机会。这些 优先事项包括（1）改进培养的角膜上皮细胞的移植，（2）了解 创伤愈合的表观遗传调节，和（3）开发增强创伤愈合过程的方法。