5-Aza-2′-Deoxycytidine Ameliorates Choroidal Neovascularization by Inhibiting the Wnt/β-Catenin Signaling Pathway

**Background:** Choroidal neovascularization (CNV) is a key pathological feature of age-related macular degeneration (AMD) and other ocular diseases, leading to severe vision loss. While anti-VEGF therapy is the standard treatment, only 30–35% of patients show improved vision, and long-term injections carry risks. DNA methylation, an epigenetic modification mediated by DNA methyltransferases (DNMTs), is implicated in retinal development and disease, but its role in CNV is unclear. 5-Aza-2′-deoxycytidine (5-aza-dC), a nonselective DNMT inhibitor, has shown protective effects in various conditions, including diabetic retinopathy. This study investigates the effects and mechanisms of 5-aza-dC on CNV.

**Methods:** Laser-induced CNV was created in C57BL/6J mice (6–8 weeks old). DNA methylation profiles of RPE/choroidal complexes were assessed using Arraystar Mouse RefSeq Promoter Arrays. Mice received daily intraperitoneal injections of 5-aza-dC (1 mg/kg) or DMSO for 7 days. CNV area, blood flow density and intensity were measured by optical coherence tomography angiography (OCTA), and leakage by fundus fluorescein angiography (FFA). In vitro, bEnd.3 murine endothelial cells were stimulated with VEGFA (10 ng/mL) or Wnt-3a (200 ng/mL) with or without 5-aza-dC (50 ng/mL). Notum promoter methylation was analyzed by bisulfite sequencing PCR. DNMT and Wnt pathway gene expression were measured by qRT-PCR and western blot. Endothelial functions (proliferation, migration, tube formation, permeability) were assessed by CCK-8, scratch, tube formation, FITC-Na leakage, and TEER assays.

**Key Results:** In laser-induced CNV mice, DNMT1, DNMT3A, and DNMT3B mRNA and protein were significantly upregulated (P < 0.05 to P < 0.001). Microarray analysis identified 1581 differentially methylated fragments (1094 hypermethylated, 487 hypomethylated). KEGG analysis showed hypermethylation of Wnt pathway genes. Notum, a Wnt antagonist, was downregulated in CNV tissues (P < 0.001), while Wnt targets AXIN2, CCND1, and LEF1 were upregulated (P < 0.05 to P < 0.001). In bEnd.3 cells, VEGFA increased DNMT expression and activity, and induced Notum promoter methylation (15 CpG sites in the –2000/–1877 region). 5-aza-dC reversed these effects, reducing methylation from ~70% to ~30% (P < 0.001). 5-aza-dC also reversed VEGFA- or Wnt-3a-induced suppression of Notum and activation of β-catenin and its targets. Functionally, 5-aza-dC inhibited VEGFA-induced hyperpermeability (FITC-Na leakage reduced, TEER increased, P < 0.01), restored ZO-1 junctional localization, and reduced tube formation, migration, and proliferation (P < 0.05 to P < 0.001). In vivo, 5-aza-dC significantly reduced CNV lesion area, blood flow intensity and density (OCTA), and fluorescein leakage (FFA grade, P < 0.001). Western blot confirmed increased Notum and decreased β-catenin and VEGFA in treated mice.

**Clinical Implications:** This study provides evidence that DNA methylation, particularly hypermethylation of the Notum promoter, contributes to CNV pathogenesis by activating Wnt/β-catenin signaling. 5-aza-dC, a clinically used demethylating agent, effectively attenuates CNV in a mouse model by restoring Notum expression and inhibiting Wnt signaling. These findings suggest that epigenetic modulation, targeting the Notum gene, could be a novel therapeutic strategy for CNV, potentially complementing or providing an alternative to anti-VEGF therapy. However, further studies are needed to establish causality and explore cell-type-specific effects.