Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe East Road, Erqi District, Zhengzhou, 450000, Henan, China.
Sci Rep. 2024 Oct 5;14(1):23226. doi: 10.1038/s41598-024-72388-x.
Upregulation of vascular endothelial growth factor (VEGF) and enhanced angiogenesis have been implicated in the severe progression of age-related macular degeneration (AMD). Abnormal arachidonate 5-lipoxygenase (ALOX5) is associated with AMD pathogenesis. However, no reports have shown the causal role of ALOX5 in angiogenesis during AMD. In the present study, ARPE-19 cells were exposed to hypoxia, an inducer of VEGF expression. Potential proteins implicated in AMD progression were predicted using bioinformatics. RNA affinity antisense purification-mass spectrometry (RAP-MS) was applied to identify the binding proteins of ALOX5 3'UTR. Expression of ALOX5 and YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) was detected by qRT-PCR and western blotting. VEGF expression and secretion were assessed by immunofluorescence and ELISA, respectively. The chicken embryo chorioallantoic membrane (CAM) was used to analyze the effect of ALOX5 on angiogenesis. RNA stability was assayed using the Actinomycin D assay. The results show that hypoxia promoted cell growth and increased VEGF expression in ARPE-19 cells. ALOX5 was associated with AMD progression, and hypoxia upregulated ALOX5 expression in ARPE-19 cells. ALOX5 silencing reduced VEGF expression induced by hypoxia in ARPE-19 cells. Moreover, the conditioned medium of ALOX5-silenced ARPE-19 cells could suppress the viability and migration of HUVECs and diminish angiogenesis in the CAM. Furthermore, YTHDF1 was validated to bind to ALOX5 3'UTR, and YTHDF1 promoted ALOX5 expression by elevating the stability of ALOX5 mRNA. In conclusion, our findings demonstrate that YTHDF1-regulated ALOX5 increases VEGF expression in hypoxia-exposed ARPE-19 cells and enhances the viability, migration, and angiogenesis of vascular endothelial cells.
血管内皮生长因子（VEGF）的上调和血管生成的增强与年龄相关性黄斑变性（AMD）的严重进展有关。异常的花生四烯酸 5-脂氧合酶（ALOX5）与 AMD 的发病机制有关。然而，尚无报告显示 ALOX5 在 AMD 期间在血管生成中的因果作用。在本研究中，将 ARPE-19 细胞暴露于缺氧，这是 VEGF 表达的诱导剂。使用生物信息学预测与 AMD 进展相关的潜在蛋白质。应用 RNA 亲和反义纯化-质谱（RAP-MS）鉴定 ALOX5 3'UTR 的结合蛋白。通过 qRT-PCR 和 Western blot 检测 ALOX5 和 YTH N6-甲基腺苷 RNA 结合蛋白 1（YTHDF1）的表达。通过免疫荧光和 ELISA 分别评估 VEGF 的表达和分泌。鸡胚绒毛尿囊膜（CAM）用于分析 ALOX5 对血管生成的影响。使用放线菌素 D 测定法测定 RNA 稳定性。结果表明，缺氧促进 ARPE-19 细胞的生长并增加 VEGF 的表达。ALOX5 与 AMD 的进展有关，并且缺氧上调了 ARPE-19 细胞中的 ALOX5 表达。ALOX5 沉默减少了 ARPE-19 细胞中缺氧诱导的 VEGF 表达。此外，ALOX5 沉默的 ARPE-19 细胞的条件培养基可抑制 HUVEC 的活力和迁移，并减少 CAM 中的血管生成。此外，验证了 YTHDF1 与 ALOX5 3'UTR 结合，并且 YTHDF1 通过提高 ALOX5 mRNA 的稳定性来促进 ALOX5 的表达。总之，我们的研究结果表明，YTHDF1 调节的 ALOX5 增加了缺氧暴露的 ARPE-19 细胞中 VEGF 的表达，并增强了血管内皮细胞的活力、迁移和血管生成。
