Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: (a) In vitro waveforms recreate athero-protective unidirectional flow (UF) mimicking in vivo hemodynamics in human distal carotid artery or athero-prone disturbed flow (DF) mimicking hemodynamics in human carotid sinus. (b and c) LC-MS/MS detected a significant increase of internal m 7 G but not in the cap-m 7 G of mRNA in HAEC subjected to 24-hr UF compared to cells exposed to 24-hr DF. (n = 6 biological replicates; p values were obtained using two-tailed Student’s t-test using GraphPad Prism. * p ≤0.05 and n.s. indicates non-significance ( p >0.05)). (d) Whole-genome RNA-seq in HAEC, illustrated by a volcano plot, detected a significant increase of endothelial METTL7A but not METTL1 , METTL3 , and METTL14 by UF. UF-induced endothelial METTL7A but not METTL1 , METTL3 , and METTL14 was validated by real-time PCR (n = 6 biological repeats, ** p ≤0.01 and n.s. indicates non-significance ( p >0.05)). (e) 48-hr acute disturbed flow in the partially-ligated left carotid artery markedly reduced Mettl7a1 transcripts in endothelium-enriched intima but not in non-endothelial media/adventitia (M+A) (n = 6 biological repeats, ** p ≤0.01 and n.s. indicates non-significance ( p >0.05)). Experiments were performed on 8-week-old male C57BL/6 mice. PCAL: partial carotid artery ligation, LCA: left carotid artery, RCA: right carotid artery, ECA: external carotid artery, ICA: internal carotid artery, OA: occipital artery and STA: superior thyroid artery. Different flow patterns were indicated in the LCA (DF) and RCA (UF). (f) Suppression of elevated METTL7A by siRNA in HAEC under UF significantly reduced mRNA internal m 7 G quantified by LC-MS/MS (n = 5 biological replicates, **p ≤0.01). (g) Reduced internal m 7 G in METTL7A-knockdowned HAEC was restored by METTL7A mRNA replenishment via overexpression (OE) of in vitro transcribed METTL7A mRNA without the 3’UTR targeted site by siMETTL7A (n = 6 biological repeats, * p ≤0.05). (h) LC-MS/MS detected a significant decrease of mRNA internal m 7 G in lung vascular endothelial cells isolated from Mettl7a1 -/- mice compared to endothelial cells isolated from wild-type Mettl7a1 +/+ mice (n = 6 biological repeats, * p ≤0.05).

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: In Vitro, In Vivo, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, RNA Sequencing, Real-time Polymerase Chain Reaction, Ligation, Over Expression, Isolation

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: METTL7A knockdown has no effect on the endothelial expression of METTL1, METTL3, and METTL14. (a) METTL7A-targeting siRNA significantly decreased the METTL7A protein expression in HAEC under unidirectional flow (n = 6 biological repeats, ** p ≤0.01). (b) METTL7A knockdown had no effect on the expression of METTL1 , METTL3 , and METTL14 in HAEC under unidirectional flow (n = 3 biological repeats). p values were obtained using two-tailed Student’s t-test using GraphPad Prism. n.s. indicates non-significance ( p >0.05). Data were presented as mean±SD.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: Knockdown, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: METTL7A knockdown has no effect on m 6 A/A, C m /C, A m /A, m 1 A/A and G m /G of endothelial mRNA. (a) METTL7A-targeting siRNA significantly decreased the METTL7A protein expression in HAEC (n = 3 biological repeats, * p ≤0.05). (b) METTL7A knockdown had no effect on m 6 A and cap-m 7 G of mRNA in HAEC (n = 6 biological repeats for m 6 A measurement and 3 biological repeats for cap-m 7 G measurement). (c) METTL7A knockdown had no effect on C m /C, A m /A, m 1 A/A and G m /G of mRNA in HAEC (n = 4). p values were obtained using two-tailed Student’s t-test using GraphPad Prism. n.s. indicates non-significance ( p >0.05). Data were presented as mean±SD.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: Knockdown, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: METTL7A overexpression significantly increases internal m 7 G but not the m 6 A of endothelial mRNA. (a) Adenoviruses (10 MOI/multiplicity of infection) effectively overexpressed HA-tagged METTL7A in HAEC as shown in the western blot. (b) Adenovirus-mediated METTL7A overexpression significantly increased the internal m 7 G but not the m 6 A of endothelial mRNA in HAEC (n = 9 biological repeats, ** p ≤0.01 and n.s. indicates non-significance ( p >0.05)). Data were presented as mean±SD.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: Over Expression, Infection, Western Blot

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: (a) RNA-seq analysis revealed significantly lower METTL7A mRNA expression in ischemic atherosclerotic coronary artery tissues (n = 35) compared to non-atherosclerotic coronary arteries (n = 23), Adjusted p = 4.91E-03. (b–c) Immunostaining of human coronary artery samples showed reduced METTL7A protein expression in CD31⁺ endothelial cells within atherosclerotic lesions, with the lowest levels observed in calcified plaques relative to non-lesion regions. Samples were pathologically classified as non-lesion, atherosclerotic lesion, or atherosclerotic lesion with calcification. Image counts: n = 39 (non-atherosclerotic controls), n = 43 (atherosclerotic lesions), n = 15 (calcified lesions). ***p ≤ 0.005, ****p ≤ 0.0001.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: RNA Sequencing, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: (a) METTL7A CLIP-seq demonstrated that METTL7A preferentially binds to protein-coding mRNA in endothelial cells. (b) METTL7A CLIP-seq identified that U/A-AG-G/A is the top RNA binding motif of endothelial METTL7A. (c) Ingenuity Pathway Analysis (IPA) identified that endothelial cell movement, vasculogenesis, migration, and endothelial development are top annotated biological functions in the METTL7A-mediated endothelial transcriptome. (d) DAVID (the Database for Annotation, Visualization, and Integrated Discovery) identified that cell migration, cell motility, and cell adhesion are the top gene ontology (GO) biological processes regulated by endothelial METTL7A. (e) Comparison of the METTL7A CLIP-seq and METTL7A-mediated transcriptome identified a list of 115 endothelial mRNAs directly bound by METTL7A and significantly regulated by METTL7A. (f) METTL7A knockdown significantly reduced elevated mRNA and protein levels of KLF4 and NFKBIA in HAEC under unidirectional flow (n = 4 biological repeats; p values were obtained using two-tailed Student’s t-test using GraphPad Prism. ** p ≤0.01 and * p ≤0.05). (g) Adenovirus-mediated METTL7A overexpression markedly increased the mRNA and protein expression of KLF4 and NFKBIA in HAEC (n = 3 biological repeats, ** p ≤0.01 and * p ≤0.05). Data were presented as mean±SD.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: RNA Binding Assay, Migration, Comparison, Knockdown, Two Tailed Test, Over Expression, Expressing

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: METTL7A functions as a methyltransferase to confer internal m 7 G modifications of KLF4 and NFKBIA mRNAs and enhance their stability. (a) m 7 G-RIP-qPCR using anti-m 7 G-specific antibody and gene-specific primers demonstrated that METTL7A knockdown markedly reduced internal m 7 G in KLF4 and NFKBIA mRNAs in HAEC. No change of internal m 7 G of endothelial GAPDH mRNA was detected (n = 4 with 2 biological repeats and 2 technical replicates, ** p ≤0.01 and n.s.= non-significance). p values were obtained using two-tailed Student’s t-test using GraphPad Prism. (b) In vitro methylation assays using purified METTL7A protein and S-(5′-Adenosyl)-L-methionine-d3 (d3-SAM) demonstrated that METTL7A effectively conferred internal m 7 G methylation on KLF4 and NFKBIA transcripts (n = 5 independent reactions with KLF4 transcript, n = 5 independent reactions with NFKBIA transcript, ** p ≤0.01). Negative control (Neg) is the experimental control without METTL7A protein. (c) Actinomycin D-based mRNA stability assays showed that knockdown of elevated METTL7A in HAEC under unidirectional flow markedly decreased the half-life of KLF4 and NFKBIA transcripts (n = 4 biological repeats, * p ≤0.05). (d) Adenovirus-mediated METTL7A overexpression markedly increased the stability of KLF4 and NFKBIA transcripts in HAEC treated with Actinomycin D (n = 4 biological repeats, * p ≤0.05). (e) Site-targeted delivery of METTL7A by CRISPR-Cas-inspired RNA targeting system (CIRTS) markedly increased KLF4 and NFKBIA mRNA in HAEC. KLF4 or NFKBIA-targeting guide RNAs were designed against KLF4 or NFKBIA m 7 G sites validated by METTL7A CLIP-seq and m 7 G-RIP-PCR (n = 6 biological replicates for KLF4 targeting experiments and n = 5 biological replicates for NFKBIA targeting experiments, ** p ≤0.01). Data were presented as mean±SD.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: Knockdown, Two Tailed Test, In Vitro, Methylation, Purification, Negative Control, Control, Over Expression, CRISPR

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: Restoration of endothelial METTL7A using CDH5 promoter-driven plasmids delivered via polymer-based nanoparticles significantly upregulates Klf4 and Nfkbia expression and reduces disturbed flow-induced atherosclerosis in vivo . (a) Endothelial Klf4 and Nfkbia transcripts were significantly reduced in carotid intima in Mettl7a1 -/- mice compared to Mettl7a1 +/+ mice ( Mettl7a1 +/+ n = 7 individual mice, Mettl7a1 -/- n = 6 individual mice, *** p ≤0.001 and * p ≤0.05). p values were obtained using two-tailed Student’s t-test using GraphPad Prism. (b) Klf4 and Nfkbia protein expression was significantly reduced in the ligated carotid artery in hypercholesterolemic (PCSK-9 virus-injected) Mettl7a1 -/- mice compared to hypercholesterolemic Mettl7a1 +/+ mice. DAPI shows blue color; elastin shows green color; Mettl7a1 shows white color and Klf4 or Nfkbia shows red color in the images. Scale bars (150 and 20 μm) were indicated in the right-lower corner of immunostaining images. The zoomed-in area is 150 μm x 150 μm. (c) Atherosclerosis in the ligated carotid artery in hypercholesterolemic Mettl7a1 -/- mice is markedly lessened by an injection of nanoparticles encapsulating METTL7A-expressing plasmids driven by an endothelial-specific VE-cadherin/CDH5 promoter, when compared to lesions in hypercholesterolemic Mettl7a1 -/- mice injected with nanoparticles carrying control plasmids with CDH5 promoter but no expressed gene (Neg). (n = 6 individual mice, * p ≤0.05). Scale bar (100 μm) was shown in the right-lower corner of oil-red staining images. (d) An injection of CDH5-METTL7A plasmid-encapsulated nanoparticles effectively induced intimal expression of METTL7A in the ligated carotid artery of hypercholesterolemic ApoE −/− mice. DAPI shows blue color; elastin shows green color and FLAG-tagged METTL7A shows red color in the images. Scale bars (150 and 20 μm) were indicated in the right-lower corner of immunostaining images. The zoomed-in area is 150 μm x 150 μm. (e) Atherosclerosis in the ligated carotid artery in hypercholesterolemic ApoE -/- mice is markedly lessened by injections of nanoparticles encapsulating CDH5-METTL7A plasmids, when compared to lesions in hypercholesterolemic in ApoE -/- mice injected with nanoparticles carrying control plasmid (n = 9 individual mice for negative control and 7 individual mice for METTL7A overexpression condition, *** p ≤0.005). p values were obtained using two-tailed Mann-Whitney U test using GraphPad Prism. Scale bar (100 μm) was shown in the right-lower corner of oil-red staining images. (f) Intimal protein expression Klf4 and Nfkbia in the ligated carotid artery was increased in hypercholesterolemic ApoE -/- mice administered with nanoparticles encapsulating CDH5-METTL7A plasmids, when compared to hypercholesterolemic ApoE -/- mice subjected to nanoparticles carrying control plasmids. L indicates lumen area. DAPI shows blue color; elastin shows green color and klf4 or nfkbia shows red color in the images. Scale bars (150 and 20 μm) were shown in the right-lower corner of immunostaining images. The zoomed-in area is 150 μm x 150 μm.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: Polymer, Expressing, In Vivo, Two Tailed Test, Virus, Injection, Immunostaining, Control, Staining, Plasmid Preparation, Negative Control, Over Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mechanosensitive Endothelial METTL7A Regulates Internal m 7 G mRNA Methylation and Protects Against Atherosclerosis

doi: 10.1101/2025.05.22.655328

Figure Lengend Snippet: Restoration of METTL7A in inflamed endothelial cells using N1-methylpseudouridine (m1Ψ)-modified METTL7A mRNA delivered via VCAM1-targeting lipid nanoparticles attenuates atherosclerosis in vivo . (a) In hypercholesterolemic Mettl7a1 -/- mice, a single intravenous injection of VCAM1-targeting lipid nanoparticles encapsulating m1Ψ-modified METTL7A mRNA (METTL7A) significantly reduced disturbed flow-induced atherosclerosis in the ligated carotid artery compared to mice treated with PBS or nanoparticles carrying non-translatable mutant METTL7A mRNA (METTL7Amut) (n = 5 for PBS, n = 7 for METTL7A mRNA, n = 5 for METTL7Amut; ** p ≤ 0.01). Scale bar: 100 μm. (b) In hypercholesterolemic ApoE -/- mice, two intravenous injections of VCAM1-targeting lipid nanoparticles encapsulating m1Ψ-modified METTL7A mRNA significantly attenuated atherosclerotic lesion formation in the ligated carotid artery compared to PBS or control nanoparticle groups (n = 5 for PBS, n = 6 for METTL7A mRNA, n = 4 for METTL7Amut; ** p ≤ 0.01). Scale bar: 100 μm. Statistical analyses were performed using a two-tailed Mann– Whitney U test (GraphPad Prism). (c) Immunostaining demonstrated increased intimal expression of KLF4 and NFKBIA proteins in the ligated carotid arteries of ApoE -/- mice treated with METTL7A mRNA nanoparticles compared to METTL7Amut. Lumen (L) is indicated. Nuclei (DAPI) are shown in blue, elastin in green, and KLF4 or NFKBIA in red. Scale bars: 150 μm (overview) and 20 μm (zoomed-in panels). Zoomed-in regions measure 150 μm × 150 μm.

Article Snippet: The original METTL7A construct was purchased from Addgene (plasmid #62017, Watertown, MA).

Techniques: Modification, In Vivo, Injection, Mutagenesis, Control, Two Tailed Test, MANN-WHITNEY, Immunostaining, Expressing