Shah Ruchi, Amador Cynthia, Poe Adam J, Spektor Tanya M, Bhandary Priyanka, Wang Yizhou, Wang Zhiping Paul, Weisenberger Daniel J, Borges Vanessa F, Sawant Onkar B, Maguen Ezra, Hamrah Pedram, Kramerov Andrei A, Saghizadeh Mehrnoosh, Ljubimov Alexander V
Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United Sates.
Board of Governors Regenerative Medicine Institute Eye Program, Cedars-Sinai Medical Center, Los Angeles, California, United Sates.
Invest Ophthalmol Vis Sci. 2025 Feb 3;66(2):64. doi: 10.1167/iovs.66.2.64.
Persistent epithelial alterations such as delayed wound healing are a key feature of diabetic corneal disease. Previously, we reported that epigenetic changes in the diabetic cornea led to the suppression of Wnt-5a, and that addition of Wnt-5a accelerated wound healing. In this study, we set to determine which Wnt receptor(s) mediated Wnt-5a induced stimulation of diabetic corneal epithelial wound healing.
Human limbal epithelial cells (LECs) were isolated from postmortem diabetic and non-diabetic donor eyes for single-cell RNA sequencing (scRNA-seq) and DNA methylation analysis. These analyses were validated by qRT-PCR, western blot, or immunostaining of corneal tissue sections. Cultured primary LECs were transfected with small interfering RNA (siRNA) to specific Wnt receptors to evaluate their role in scratch wound healing in the presence or absence of 200 ng/mL Wnt-5a.
Single-cell RNA sequencing analysis revealed differential gene expression of Wnt receptors, ROR2, MCAM, FZD5, FZD6, and FZD7. DNA methylation arrays showed hypomethylation of ROR2 gene promoter in diabetic versus non-diabetic LECs by 41.3% (**P < 0.01) resulting in increased ROR2 protein expression. Non-diabetic cells transfected with siRNA to knockdown ROR2 but not FZD5, FZD6, FZD7, MCAM, and RYK showed significantly decreased wound healing by approximately 50% (*P < 0.05) versus control siRNA. In diabetic LECs, knockdown of ROR2 significantly inhibited wound healing by 40% (*P < 0.05) and of FZD5 partially blocked wound healing that could not be restored by the addition of Wnt-5a.
Wnt-5a seems to mediate wound healing in diabetic LECs mainly through receptor tyrosine kinase like orphan receptor 2 with Frizzled-5 serving as a possible co-receptor with a smaller effect.
持续性上皮改变，如伤口愈合延迟，是糖尿病角膜病的一个关键特征。此前，我们报道糖尿病角膜中的表观遗传变化导致Wnt-5a表达受抑制，并且添加Wnt-5a可加速伤口愈合。在本研究中，我们旨在确定哪种Wnt受体介导Wnt-5a诱导的糖尿病角膜上皮伤口愈合刺激作用。
从死后的糖尿病和非糖尿病供体眼中分离人角膜缘上皮细胞（LEC），用于单细胞RNA测序（scRNA-seq）和DNA甲基化分析。这些分析通过qRT-PCR、蛋白质印迹或角膜组织切片免疫染色进行验证。用针对特定Wnt受体的小干扰RNA（siRNA）转染培养的原代LEC，以评估它们在存在或不存在200 ng/mL Wnt-5a的情况下对划痕伤口愈合的作用。
单细胞RNA测序分析揭示了Wnt受体ROR2、MCAM、FZD5、FZD6和FZD7的差异基因表达。DNA甲基化阵列显示，与非糖尿病LEC相比，糖尿病LEC中ROR2基因启动子的低甲基化率为41.3%（**P < 0.01），导致ROR2蛋白表达增加。用siRNA转染非糖尿病细胞以敲低ROR2，但不敲低FZD5、FZD6、FZD7、MCAM和RYK，与对照siRNA相比，伤口愈合显著降低约50%（*P < 0.05）。在糖尿病LEC中，敲低ROR2显著抑制伤口愈合40%（*P < 0.05），敲低FZD5部分阻断伤口愈合，添加Wnt-5a不能恢复。
Wnt-5a似乎主要通过受体酪氨酸激酶样孤儿受体2介导糖尿病LEC的伤口愈合，卷曲蛋白5作为可能的共受体，作用较小。
