Wnt5a, a New Diabetic Corneal Marker Related to Wound Healing

糖尿病是劳动年龄成年人中最普遍的致盲疾病。高达70%的糖尿病患者 角膜问题，包括神经病变和上皮性角膜病变(伤口愈合受阻、溃疡、 反复侵蚀)损害视力并引起疼痛和不适。糖尿病角膜病变被低估了， 治疗仍然是有症状的。我们之前发现了人类糖尿病角膜中改变的标志物 通过基因和纳米疗法使它们在人类角膜器官培养中的水平正常化，这也恢复了正常 干细胞表型与角膜伤口愈合。表观遗传改变可能导致糖尿病并发症。 因此，我们检测了人类糖尿病角膜的表观遗传dna甲基化，发现了一组基因。 异常甲基化的角膜与正常的角膜。Wnt5A基因--细胞Wnt调节因子的非规范成员 糖尿病角膜的运动性、增殖性和分化均发生了高甲基化。它的表达在 糖尿病角膜和富含干细胞的上皮细胞培养。WNT5A添加剂加速伤口愈合 糖尿病(但不在正常细胞中)，随干细胞标记物表达增加。加强对糖尿病的抑制 靶向Wnt5a的miRNA-203a增加了Wnt5a，促进了糖尿病细胞的伤口愈合。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种群的影响，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 修复可以通过阻断非规范WNT5a的正常表达来实现 使用新型纳米偶联物和去甲基化试剂抑制microRNA。 具体目的1.鉴定WNT5a受体(S)介导其对糖尿病上皮细胞和角膜的作用。WNT5A 糖尿病角膜缘细胞和角膜中的受体将通过成像和功能上的siRNAs、聚焦来识别 在ROR2上排名第一。这将允许在糖尿病细胞中调节它们的表达和信号，以增强Wnt5a的作用。 具体目的2.检测Wnt5a对人角膜上皮损伤愈合和角膜缘细胞数量的影响 器官培养的糖尿病角膜。在这些角膜中，我们将确认使用创伤使WNT5a效果正常化 愈合和干细胞标记物的表达。角膜缘上皮分化和干细胞群体的变化 在Wnt5a(或抑制剂)处理后，将通过单细胞RNA-seq与对照正常细胞进行比较。 具体目标3.测试旨在通过培养的糖尿病患者增加Wnt5a表达的治疗方法的有效性 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结合物治疗 用miR-203a特异性拮抗剂和/或去甲基5-氮杂-2‘-脱氧胞苷(地西他滨)或ZeBularine。 我们的目标非常符合NEI远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 伤口愈合的表观遗传调控，以及(3)发展促进伤口愈合过程的方法。

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.