RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice
- Tianjin Key Laboratory of Metabolic Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Collaborative Innovation Center of Tianjin for Medical Epigenetics and Department of Physiology and Pathophysiology, Department of Cardiovascular Surgery, Tianjin Medical University General Hospital, Tianjin Medical University, China
- CAS Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, College of Future Technology, Beijing Institute of Genomics, Chinese Academy of Sciences, China
- China National Center for Bioinformation, China
- University of Chinese Academy of Sciences, China
- Key Laboratory of Remodeling-Related Cardiovascular Diseases (Ministry of Education), Beijing Collaborative Innovation Center for Cardiovascular Disorders, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing Anzhen Hospital, Capital Medical University, China
Figures
Figure 1 with 1 supplement
Methyltransferase like 3 (METTL3)-dependent N6-methyladenosine (m6A) methylation is decreased in atheroprone regions.
Human umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (mAECs) were exposed to OS (0.5 ± 4 dyn/cm2) for 6 hr. Cells with static treatment (ST) were a control. (A) …
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Figure 1—source data 1
METTL3-dependent m6A methylation is decreased in atheroprone regions.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig1-data1-v2.zip
Figure 1—figure supplement 1
Methyltransferase like 3 (METTL3) is decreased in atheroprone regions.
(A–B) Protein was extracted from the aortic arch (AA) and thoracic aorta (TA) of 8-week-old C57BL/6 mice. (A) Western blot analysis of expression of METTL3, METTL14, Wilms tumor 1-associated …
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Figure 1—figure supplement 1—source data 1
METTL3 is decreased in atheroprone regions.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig1-figsupp1-data1-v2.zip
Figure 2 with 1 supplement
Methyltransferase like 3 (METTL3) deficiency induces endothelial activation in atheroprone regions.
(A–C) Protein was extracted from the AA and TA of 8-week-old EC-Mettl3KO and Mettl3flox/flox mice. (A) Western blot analysis of the expression of METTL3, vascular adhesion molecule (VCAM-1), CD31, …
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Figure 2—source data 1
Mettl3 deficiency induces endothelial activation in atheroprone regions.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig2-data1-v2.zip
Figure 2—figure supplement 1
Identification of EC-specific methyltransferase like 3 (Mettl3)-deficient mice.
(A) Genotyping of mice. PCR indicated that tamoxifen-stimulated Cdh5-Cre recombination in tail preparations of mice as follows: wild-type mice and Mettl3flox/-, Mettl3flox/flox, and EC-Mettl3KO …
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Figure 2—figure supplement 1—source data 1
Identification of EC-specific Mettl3-deficient mice.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
Oscillatory stress (OS)-abolished N6-methyladenosine (m6A) prevents epidermal growth factor receptor (EGFR) mRNA degradation.
(A) Distribution of m6A peaks along the 5’ untranslated regions (5’UTR), CDS (coding sequence), and 3’UTR regions of mRNA in static treatment (ST) and OS. (B) m6A motif identified from human …
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Figure 3—source data 1
OS-abolished m6A prevents EGFR mRNA degradation.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig3-data1-v2.zip
Figure 3—figure supplement 1
N6-methyladenosine (m6A) profiling in human umbilical vein endothelial cells treated with static treatment (ST) and oscillatory stress (OS).
(A) Pie charts showing the m6A peak distribution in different RNA regions (CDS, stop codon, TSS (transcriptional start site), 5’ untranslated regions [5′ UTR], 3′ UTR) in ST and OS. (B) Scatter …
Figure 4 with 1 supplement
The thrombospondin-1/epidermal growth factor receptor (TSP-1/EGFR) pathway participates in EC inflammation induced by methyltransferase like 3 (METTL3) inhibition in response to oscillatory stress (OS).
(A) Western blot analysis of p-EGFR, EGFR, p-AKT, t-AKT, p-ERK, t-ERK, FLAG (tag of METTL3), and vascular adhesion molecule 1 (VCAM-1) expression. GAPDH is the protein loading control. Human …
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Figure 4—source data 1
The TSP-1/EGFR pathway participates in EC inflammation in response to OS.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig4-data1-v2.zip
Figure 4—figure supplement 1
Thrombospondin-1 (TSP-1) and epidermal growth factor receptor (EGFR) are involved in methyltransferase like 3 (METTL3)-mediated EC dysfunction in response to oscillatory stress (OS).
(A) Quantification of expression of vascular adhesion molecule 1 in human umbilical vein endothelial cells (HUVECs) upon different treatments. Cells were infected with the indicated adenoviruses for …
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Figure 4—figure supplement 1—source data 1
TSP-1 and EGFR are involved in METTL3-mediated EC dysfunction in response to OS.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
Epidermal growth factor receptor (EGFR) contributes to EC activation in endothelial methyltransferase like 3 (Mettl3)-deficient mice.
(A–B) EC-Mettl3KO and Mettl3flox/flox mice underwent partial ligation of the carotid artery for 2 weeks were infused with the indicated adeno-associated virus. Immunofluorescence staining for …
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Figure 5—source data 1
EGFR contributes to EC activation in endothelial Mettl3-deficient mice.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig5-data1-v2.zip
Figure 5—figure supplement 1
Verification of disturbed flow in the partially ligated left common carotid artery.
(A) EC-Mettl3KO and Mettl3flox/flox mice underwent partial ligation of the carotid artery for 2 weeks. En face immunofluorescence staining of methyltransferase like 3 (METTL3) expression in ECs of …
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Figure 5—figure supplement 1—source data 1
Verification of disturbed flow in the partially ligated left common carotid artery.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig5-figsupp1-data1-v2.zip
Figure 6
EC-specific methyltransferase like 3 (METTL3) deficiency accelerates atherogenesis in Apoe-/- mice.
Apoe-/-EC-Mettl3KO and Apoe-/-Mettl3flox/flox mice were fed a Western-type diet for 12 weeks. (A) Oil Red O staining of aortas. (B) Plaque area as a percentage of total area. AA, aortic arch; TA, …
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Figure 6—source data 1
EC-specific METTL3 deficiency accelerates atherogenesis in Apoe-/- mice.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig6-data1-v2.zip
Figure 7 with 1 supplement
Thrombospondin-1/epidermal growth factor receptor (TSP1/EGFR) signaling is involved in atherosclerosis.
(A) An 8-week-old male Apoe–/– Mettl3flox/flox and Apoe–/– EC-Mettl3KO mice with 2 weeks of partial ligation were infused with the indicated lentiviruses or pretreated with AG1478 (AG, 10 mg/kg/day) …
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Figure 7—source data 1
TSP1/EGFR signaling is involved in atherosclerosis.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig7-data1-v2.zip
Figure 7—figure supplement 1
AG1478 decreases phosphorylation of epidermal growth factor receptor (EGFR) induced by oscillatory stress.
(A–B) EC-Mettl3KO and Mettl3flox/flox mice underwent partial ligation of the carotid artery for 2 weeks. During ligation, carotid arteries were infused with the indicated lentiviruses. En face GFP …
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Figure 7—figure supplement 1—source data 1
AG1478 decreases phosphorylation of EGFR induced by OS.
- https://cdn.elifesciences.org/articles/69906/elife-69906-fig7-figsupp1-data1-v2.zip
Author response image 1
TSP1 participates in endothelial atherogenic responses to disturbed flow.
(A-B) 8-week-old male Apoe–/– Mettl3flox/flox and Apoe–/– EC-Mettl3KO mice with 2 weeks of partial ligation were infused with the indicated lentiviruses, and arterial tissue cross sections …
Author response image 2
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(A-C) UHPLC-MRM-MS analysis of m6A levels in mRNA extracted from HUVECs (A) and mAECs (C) exposed to ST and OS, and infected with the indicated adenoviruses (B). Data are shown as the mean ± SEM, *p<…
Author response image 3
METTL16 remains unchanged upon Mettl3 knockdown.
HUVECs and mAECs were infected with METTL3 siRNA for 24 hr, Western blot analysis of METTL3, METTL16 and GAPDH. (B and D) Quantification of the expression of the indicated proteins in (A and C). …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-METTL3(Rabbit monoclonal) | Cell Signaling Technology | Cat# 96,391 | WB (1:1000) |
| Antibody | anti-METTL14(Rabbit monoclonal) | Cell Signaling Technology | Cat# 51,104 | WB (1:1000) |
| Antibody | anti-METTL16(Rabbit monoclonal) | Cell Signaling Technology | Cat# 17,676 | WB (1:1000) |
| Antibody | anti-WTAP(Rabbit monoclonal) | Cell Signaling Technology | Cat# 56,501 | WB (1:1000) |
| Antibody | anti-Virillizer(Rabbit monoclonal) | Cell Signaling Technology | Cat# 88,358 | WB (1:1000) |
| Antibody | anti-phospho-EGFR(Rabbit monoclonal) | Cell Signaling Technology | Cat# 3,777 | WB (1:1000) |
| Antibody | anti-EGFR(Rabbit monoclonal) | Cell Signaling Technology | Cat# 4,267 | WB (1:1000) |
| Antibody | anti-VCAM-1(Rabbit monoclonal) | Cell Signaling Technology | Cat# 15,631 | WB (1:1000) |
| Antibody | anti-VCAM-1(Rabbit monoclonal) | Cell Signaling Technology | Cat# 39,036 | IF (1:100) |
| Antibody | anti-thrombospondin-1(Rabbit monoclonal) | Cell Signaling Technology | Cat# 37,879 | WB (1:1000) |
| Antibody | anti-αSMA(Rabbit monoclonal) | Cell Signaling Technology | Cat# 19,245 | WB (1:1000) |
| Antibody | anti-phospho-ERK(Rabbit monoclonal) | Cell Signaling Technology | Cat# 8,544 | WB (1:1000) |
| Antibody | anti-phospho-AKT(Rabbit monoclonal) | Cell Signaling Technology | Cat# 5,012 | WB (1:1000) |
| Antibody | anti-ERK(Mouse monoclonal) | Santa Cruz | Cat# sc-271269 | WB (1:1000) |
| Antibody | anti-AKT(Mouse monoclonal) | Santa Cruz | Cat# sc-5298 | WB (1:1000) |
| Antibody | anti- METTL3(Rabbit monoclonal) | Proteintech | Cat# 15073–1-AP | IF (1:100) |
| Antibody | anti- GFP(Rabbit monoclonal) | Proteintech | Cat# 50430–2-AP | IF (1:100) |
| Antibody | anti- GAPDH (Rabbit monoclonal) | Proteintech | Cat# 60004–1-Ig | WB (1:5000) |
| Antibody | anti- EGFR (Rabbit monoclonal) | Abcam | Cat# ab52894 | IF (1:100) |
| Antibody | anti- VE-cadherin (Rat monoclonal) | Abcam | Cat# ab33168 | IF (1:100) |
| Antibody | anti- CD31(Rabbit monoclonal) | Abcam | Cat# ab24590 | IF (1:100) |
| Antibody | anti- CD68(Rabbit monoclonal) | Abcam | Cat# ab955 | IF (1:100) |
| Antibody | anti- vWF(Sheep monoclonal) | Abcam | Cat# ab11713 | IF (1:100) |
| Antibody | anti-thrombospondin-1(Mouse monoclonal) | Abcam | Cat# ab1823 | IF (1:100) |
| Antibody | anti- thrombospondin-1 (Mouse monoclonal) | Abcam | Cat# ab1823 | IF (1:100) |
| Antibody | Alex 488-conjugated goat anti-rabbit antibody | Thermo Fisher Scientific | Cat# A-11008 | IF (1:200) |
| Antibody | Alex 594-conjugated goat anti-mouse antibody | Thermo Fisher Scientific | Cat# A-11008 | IF (1:200) |
| Antibody | Alex 488-conjugated goat anti-rabbit antibody | Thermo Fisher Scientific | Cat# A-11005 | IF (1:200) |
| Antibody | Alex 594-conjugated donkey anti-sheep antibody | Thermo Fisher Scientific | Cat# A-11016 | IF (1:200) |
| Chemical compound, drug | AG1478 | Selleck | Cat# S2728 | |
| Chemical compound, drug | Recombinant Human Thrombospondin-1 | Absin | Cat# abs 04665 | 1.03 mg/ml |
| sequence-based reagent | Human EGFR-3utr | This paper | N/A | Sequences in Supplementary file 3 |
| sequence-based reagent | Human EGFR | This paper | N/A | Sequences in Supplementary file 3 |
| sequence-based reagent | Human THBS1 | This paper | N/A | Sequences in Supplementary file 3 |
| sequence-based reagent | Human GAPDH | This paper | N/A | Sequences in Supplementary file 3 |
| software, algorithm | Ingenuity Pathway Analysis | National Clinical Research Center for Blood Diseases | http://www.ingenuity.com/ | |
| software, algorithm | Prism version 8.0 | GraphPadSoftware Inc | https://www.graphpad.com/scientific-software/prism/ |
Additional files
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Supplementary file 1
Identified m6A peaks in ST and OS conditions.
- https://cdn.elifesciences.org/articles/69906/elife-69906-supp1-v2.xlsx
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Supplementary file 2
Identified candidate genes with decreased m6A and increased expression levels in response to OS.
- https://cdn.elifesciences.org/articles/69906/elife-69906-supp2-v2.xlsx
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Supplementary file 3
Information of primers used in this study.
- https://cdn.elifesciences.org/articles/69906/elife-69906-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69906/elife-69906-transrepform1-v2.docx
