角膜新生血管化（CNV）可能由化学烧伤引起。巨噬细胞在CNV期间参与血管生成和淋巴管生成。本研究的目的是调查Wilms肿瘤1相关蛋白（WTAP）是否通过N6-甲基腺苷（m6A）修饰参与巨噬细胞招募和VEGF分泌。角膜碱烧伤建立了CNV小鼠模型。使用肿瘤坏死因子α（TNF-α）刺激血管内皮细胞。使用m6A免疫共沉淀qPCR确定mRNA中m6A水平的富集。利用染色质免疫共沉淀法检测CC趋化因子配体2（CCL2）启动子区域中H3K9me3的富集。使用载脂体载体进行了体内WTAP抑制。在碱烧伤角膜组织中，通过CD31和LYVE-1表达上升增加了血管生成和淋巴管生成，巨噬细胞数量以及WTAP表达也增加了。在TNF-α刺激下，WTAP通过促进CCL2分泌促进内皮细胞对巨噬细胞的招募。在机械上，WTAP通过调节SUV39H1 mRNA的m6A水平影响CCL2启动子中H3K9me3的富集。体内实验表明，在WTAP干预后，巨噬细胞的VEGFA / C / D分泌减少了。 WTAP通过m6A介导的HIF-1α翻译调节影响巨噬细胞VEGFA / C / D的分泌。这两个途径都参与了WTAP在CNV期间调节血管生成和淋巴管生成的过程。版权所有©2023 Elsevier B.V.发表。

Corneal neovascularization (CNV) can be caused by chemical burns. Macrophages are involved in angiogenesis and lymphangiogenesis during CNV. The aim of this study was to investigate whether Wilms' tumor 1-associated protein (WTAP) is involved in macrophage recruitment and VEGF secretion via N6-methyladenosine (m6A) modification.A CNV mouse model was established by corneal alkali burn. Tumor necrosis factor alpha (TNF-α) was used to stimulate vascular endothelial cells. m6A immunoprecipitation qPCR was used to determine the enrichment of m6A levels in mRNAs. The H3K9me3 enrichment in the promoter region of CC motif chemokine ligand 2 (CCL2) was detected by chromatin immunoprecipitation assay. The WTAP inhibition in vivo was performed using the adeno-associated virus.In the alkali burn corneal tissues, angiogenesis and lymphangiogenesis were promoted as CD31 and LYVE-1 expressions were elevated, and the number of macrophages as well as WTAP expression were increased. Under the TNF-α stimulation, WTAP promoted the recruitment of endothelial cells to macrophages by promoting CCL2 secretion. Mechanistically, WTAP affected the enrichment of H3K9me3 at the CCL2 promoter by regulating the m6A level of SUV39H1 mRNA. The in vivo experiment showed that VEGFA/C/D secretion of macrophages was reduced after WTAP interference. Mechanistically, WTAP regulated the translational efficiency of HIF-1α via m6A modification.WTAP affected macrophage recruitment to endothelial cells via regulation of H3K9me3-mediated CCL2 transcription. WTAP also affected macrophage secretion of VEGFA/C/D via m6A-mediated translation regulation of HIF-1α. Both pathways were involved in the WTAP regulation of angiogenesis and lymphangiogenesis during CNV.Copyright © 2023. Published by Elsevier B.V.