Medical School, Nanjing University, Nanjing 210093, China; Department of Ophthalmology, Affiliated Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
Department of Ophthalmology, Affiliated Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
Cell Signal. 2023 Sep;109:110784. doi: 10.1016/j.cellsig.2023.110784. Epub 2023 Jun 23.
Corneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N-methyladenosine (mA) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and mA in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo.
A PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA mA abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo.
METTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased mA levels. METTL3 knockdown or inhibition by STM2457 further reduced mA levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an mA-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo.
Our findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.
角膜新生血管（CNV）是单纯疱疹性角膜炎（HSK）的一种症状，可导致失明。单纯疱疹病毒 1 型（HSV-1）引起的角膜血管生成受血管内皮生长因子 A（VEGFA）的强烈影响。甲基转移酶样 3（METTL3）催化的 N-甲基腺苷（mA）修饰是血管生成特性的关键表观遗传调控过程。然而，METTL3 和 mA 在 HSK 诱导的 CNV 中的作用尚不清楚。在这里，我们在体外和体内研究了这些作用。
使用 HSV-1 感染的人脐静脉内皮细胞（HUVEC）中的 PCR 阵列筛选出 METTL3 等转录后基因。管形成和划痕实验用于研究细胞迁移能力。通过斑点印迹分析评估全局 mRNA mA 丰度。通过 RT-qPCR、western blot 和荧光免疫染色评估基因表达。此外，进行生物信息学分析以鉴定 HUVEC 中 METTL3 的下游分子。METTL3 敲低和 STM2457 处理阐明了影响体外 HSV-1 诱导血管生成的特定潜在分子机制。建立急性 HSK 小鼠模型，以检查 METTL3 敲低或使用 STM2457 抑制对体内病理性血管生成发展的影响。
METTL3 在 HSV-1 感染的 HUVEC 中高度上调，并导致 mA 水平升高。METTL3 敲低或 STM2457 抑制进一步降低 mA 水平和 VEGFA 表达，并损害 HSV-1 感染后 HUVEC 的迁移和管形成能力。在机制上，METTL3 通过依赖 mA 的转录后 mRNA 修饰调节 LRP6 表达，增加其稳定性，上调 VEGFA 表达，并促进 HSV-1 感染的 HUVEC 中的血管生成。此外，METTL3 敲低或 STM2457 抑制减少了体内的 CNV。
我们的研究结果表明，METTL3 通过体外和体内的经典 Wnt 和 VEGF 信号通路促进病理性血管生成，为预防 HSK 中 CNV 的进展提供了潜在的药物靶点。
