m7G Methyltransferase METTL1 Promotes Post-ischemic Angiogenesis via Promoting VEGFA mRNA Translation

Abstract

Post-transcriptional modifications play pivotal roles in various pathological processes and ischemic disorders. However, the role of N7-methylguanosine (m7G), particularly m7G in mRNA, on post-ischemic angiogenesis remains largely unknown. Here, we identified that methyltransferase like 1 (METTL1) was a critical candidate responsible for a global decrease of m7G within mRNA from the ischemic tissues. The in vivo gene transfer of METTL1 improved blood flow recovery and increased angiogenesis with enhanced mRNA m7G upon post-ischemic injury. Increased METTL1 expression using plasmid transfection in vitro promoted HUVECs proliferation, migration, and tube formation with a global increase of m7G in mRNA. Mechanistically, METTL1 promoted VEGFA mRNA translation in an m7G methylation-dependent manner. Our findings emphasize a critical link between mRNA m7G and ischemia and provide a novel insight of targeting METTL1 in the therapeutic angiogenesis for ischemic disorders, including peripheral arterial disease.

Keywords: N7-methylguanosine; methyltransferase like 1; peripheral artery disease; post-ischemic angiogenesis; vascular endothelial growth factor A.

FIGURE 1

m7G and METTL1 are decreased in ischemic gastrocnemius. (A) Quantitative LC-MS/MS analysis of m7G/G of mRNA from the tissues post-ischemia. (B) Quantification of METTL1 and WDR4 mRNA expression and β-actin was used as a loading control. (C) Representative blot image (left panel) and quantitative analysis (right panel) of METTL1 protein expression upon ischemia or non-ischemia. β-actin was used as a loading control. N = 6 and all data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ns indicate no significance.

FIGURE 2

Improved blood flow recovery and increased angiogenesis with enhanced m7G upon METTL1 overexpression post-ischemic injury. (A) Schematic illustration of hind-limb ischemia design with METTL1 overexpression. The blood flow recovery was scanned at indicated time points. Twenty-one days post-ischemia, samples were collected for subsequent experiments. (B) Quantification of RT-qPCR data for METTL1 mRNA expression. β-actin was used as a loading control. (C) Representative blot image showing METTL1 protein expression. Protein expression was normalized to β-actin. (D) Quantitative LC-MS/MS analysis of mRNA m7G levels. (E) Representative Laser Doppler images of blood flow recovery scan. (F) Quantitative analysis of blood flow recovery data. (G) Representative frozen section immunofluorescence images of the angiogenesis markers CD31 and α-SMA expression. (H) Quantitative analysis of capillary density (CD31/DAPI) and small artery density (α-SMA/mm²). N = 6 and all data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ns indicate no significance compared to the OE-Ctrl.

FIGURE 3

Increased HUVECs angiogenesis with enhanced m7G upon METTL1 overexpression post-hypoxic injury. (A) Quantification of HUVECs viability in different hypoxic time points without FBS supplemented in the culture medium. (B) Quantitative LC-MS/MS analysis of HUVECs mRNA m7G post-hypoxia. (C) Quantification of RT-qPCR data for HUVECs METTL1 and WDR4 mRNA expression post-hypoxia. (D) Representative blot image showing METTL1 protein expression post-hypoxia. (E) Representative blot image showing METTL1 protein overexpression efficacy post-hypoxia. (F) Quantitative LC-MS/MS analysis of mRNA m7G levels upon METTL1 overexpression post-hypoxia. (G) Quantification of HUVECs viability upon METTL1 overexpression post-hypoxia. (H) Representative EdU staining (left panel) and quantitative analysis (right panel) of relative EdU positive HUVECs proportion (EdU⁺/DAPI) upon METTL1 overexpression post-hypoxia. (I) Representative scratch closure images (left panel) and quantitative analysis (right panel) of relative scratch area upon METTL1 overexpression post-hypoxia. (J) Representative transwell images (left panel) and quantitative analysis (right panel) of relative migrated HUVECs numbers upon METTL1 overexpression post-hypoxia. (K) Representative tube formation images (left panel) and quantitative analysis (right panel) of relative tube formation upon METTL1 overexpression post-hypoxia. All experiments were from 4 independent replicates, and data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ns indicate no significance.

FIGURE 4

METTL1 promoted HUVECs angiogenesis via increasing VEGFA translation. (A) Quantification of MeRIP-qPCR shows the m7G fold enrichment of VEGFA mRNA. (B) Quantification of relative METTL1 and VEGFA mRNA expression. (C) Representative Western blot images of VEGFA protein expression. (D) Quantification of VEGFA concentration in the supernatant of HUVECs culture media. (E) Representative EdU staining images (left panel) and quantitative analysis (right panel) of EdU positive HUVECs proportion. (F) Representative scratch closure images (upper panel) and quantitative analysis (lower panel) of relative scratch area alteration. (G) Representative transwell images (upper panel) and quantitative analysis (lower panel) of relative migrated HUVECs numbers. (H) Representative tube formation images (upper panel) and quantitative analysis (lower panel) of relative tube formation. All experiments were from 4 independent replicates, and data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ns indicate no significance.