Methyltransferase‐like 3 suppresses phenotypic switching of vascular smooth muscle cells by activating autophagosome formation

Abstract Prevention of neointima formation is the key to improving long‐term outcomes after stenting or coronary artery bypass grafting. RNA N6‐methyladenosine (m6A) methylation has been reported to be involved in the development of various cardiovascular diseases, but whether it has a regulatory effect on neointima formation is unknown. Herein, we revealed that methyltransferase‐like 3 (METTL3), the major methyltransferase of m6A methylation, was downregulated during vascular smooth muscle cell (VSMC) proliferation and neointima formation. Knockdown of METTL3 facilitated, while overexpression of METTL3 suppressed the proliferation of human aortic smooth muscle cells (HASMCs) by arresting HASMCs at G2/M checkpoint and the phosphorylation of CDC2 (p‐CDC2) was inactivated by METTL3. On the other hand, the migration and synthetic phenotype of HASMCs were enhanced by METTL3 knockdown, but inhibited by METTL3 overexpression. The protein levels of matrix metalloproteinase 2 (MMP2), MMP7 and MMP9 were reduced, while the expression level of tissue inhibitor of metalloproteinase 3 was increased in HASMCs with METTL3 overexpression. Moreover, METTL3 promoted the autophagosome formation by upregulating the expression of ATG5 (autophagy‐related 5) and ATG7. Knockdown of either ATG5 or ATG7 largely reversed the regulatory effects of METTL3 overexpression on phenotypic switching of HASMCs, as evidenced by increased proliferation and migration, and predisposed to synthetic phenotype. These results indicate that METTL3 inhibits the phenotypic switching of VSMCs by positively regulating ATG5‐mediated and ATG7‐mediated autophagosome formation. Thus, enhancing the level of RNA m6A or the formation of autophagosomes is the promising strategy to delay neointima formation.

Herein, we revealed that methyltransferase-like 3 (METTL3), the major methyltransferase of m 6

| INTRODUCTION
Neointima formation is one of the main causes of poor prognosis after stenting or coronary artery bypass graft. 1 It is well known that excessive proliferation of vascular smooth muscle cells (VSMCs) is the major cause of neointima formation. Paclitaxel-eluting stents and limus-eluting stents are the two common types of stents coated with anti-proliferative drugs, which significantly improve the long-term outcomes of patients after stenting. 2 However, the challenge of developing more effective drugs remains formidable. The proliferation of VSMCs is regulated by a variety of molecular mechanisms, and our recently published results showed that autophagy is involved in the growth of VSMCs. 3,4 Moreover, autophagy has also been reported to be participated in the phenotypic switching of VSMCs. 5 However, the regulatory mechanisms need to be further elucidated.
Various epigenetic modifications have been reported to be contributed to the regulation of VSMC proliferation or autophagy. [6][7][8] N 6 -methyladenosine (m 6 A) RNA methylation is an RNA epigenetic modification that occurs in the N6-position of adenosine, which has been shown to be involved in the occurrence and development of various cardiovascular diseases. 9 Methyltransferase-like 3 (METTL3) is the major methyltransferase that mediates RNA m 6 A modification. 9 Although METTL3 has been studied in atherosclerosis, 10,11 abdominal aortic aneurysm 12 and tumour angiogenesis, 13 it is unknown whether METTL3 regulates VSMC proliferation and neointima formation.
Recently, METTL3 was reported to participate in the regulation of autophagy in cardiomyocytes and tumour cells, but its role in autophagy is still controversial. 14,15 For example, Song et al. 14 demonstrated that METTL3 suppressed autophagic flux in cardiomyocytes with hypoxia/reoxygenation treatment, while Liu et al. 15 showed that METTL3 facilitated autophagy in non-small cell lung cancer (NSCLC) cells. These results indicate that METTL3 may regulate autophagy in a context-dependent or cell-type-dependent manner. However, it is unclear whether METTL3 affects VSMC phenotypic switching by regulating autophagy. Autophagy has been reported to play an important role in neointima formation and its role is a double-edged sword. For example, Grootaert et al. 16 demonstrated that defective autophagy in VSMCs accelerated senescence and promoted neointima formation, whereas Ouyang et al. 17 showed that SMC-specific deletion of Ulk1 suppressed autophagy and impeded neointima hyperplasia. Our previous studies also showed that excessive activation of autophagy inhibited VSMC growth and even led to autophagic cell death. 3,4 Thus, it is imperative to clarify whether METTL3 regulates autophagy, proliferation, as well as phenotypic switching of VSMCs.
In the present study, we found that the expression of METTL3 was negatively correlated with the proliferation of VSMCs. METTL3 inhibited HASMC proliferation by inhibiting the p-CDC2 and arresting cells at the G2/M checkpoint. METTL3 also suppressed the migration of HASMCs and helped maintain the contractile phenotype of HASMCs. Moreover, METTL3 promoted the expression of autophagy-related 5 (ATG5) and ATG7, thereby increasing the formation of autophagosomes. Knockdown of either ATG5 or ATG7 to reduce autophagosome formation largely reversed the effects of METTL3 overexpression on VSMCs. Therefore, METTL3 may restrain neointima formation by activating the autophagy in VSMCs.

| Cell culture and treatments
The primary human aortic smooth muscle cells (HASMCs) were cultured as described previously. 4,18,19 This study was approved by the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Review Board in Wuhan, China. The aortic tissues were soaked in pre-chilled DME/F12 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics, and transfer it to the laboratory as soon as possible. Furtherly, the intima and media of aortic tissues were stripped with tweezers under a stereo microscope after removal of blood stains and connective tissue with DME/F12 medium. Then, the media was peeled off layer by layer with micro tweezers, and in the case of ensuring that each layer of the media is thin enough to minimize the stretch to avoid smooth muscle cell damage. The dissected media of the vessels was transferred to cell-culture flask and cut into small pieces (1 Â 1 mm). The tissue pieces are evenly spread on the side wall of the culture flask, and the distance is kept about 2 mm. The flasks were placed upright in a 37 C cell incubator with 5% carbon dioxide to dry for half an hour. After the tissue completely attached to the wall, 8 ml of DME/F12 medium containing 10% FBS was added along the opposite side wall, and the culture flask was placed horizontally to ensure medium can infiltrate the tissue.
Long spindle-shaped smooth muscle cells were observed around the tissue pieces in 1 week. After the cells grew, pay great attention to the growth state of the cells under the microscope, and pass the cells when the growth density reaches 80% for the flowing experiments.

| Plasmids construction
The full-length human METTL3 CDS sequence was amplified by polymerase chain reaction (PCR) and cloned into the pHAGE lentiviral vector with a Flag tag. The primers used to amplify the CDS of METTL3 were as follows:

| Western blot analysis
The total protein from HASMCs was extracted by RIPA as previously described. 3,4 The Pierce™ BCA Protein Assay Kit (23,225, Thermo Fisher Scientific) was used to determine the protein concentration. The protein was denatured at 95 C with 5 Â Loading buffer, 20 μg of total protein was loaded and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Then, the protein was transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010). Furtherly, the membrane was incubated with indicated primary antibody overnight at 4 C after blocked by 5% non-fat milk. Subsequently, incubating with the peroxidase-conjugated secondary antibody (Jackson Immu-noResearch Laboratories, 111-035-003, at 1:25,000 dilution), the protein signals were detected by using the ChemiDocTM XRS + system
After incubating with 50 μM EdU medium (300 μl per well) for 2 h, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with 2 mg/ml glycine for 5 min to neutralize paraformaldehyde.
Cells were incubated with 1Â Apollo staining solution for 30 min at room temperature in the dark after washing with phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 10 min. Then, washing the cells with 0.5% Triton X-100 PBS solution again, and cells were incubated with 1Â Hoechst 33342 for 30 min at room temperature in the dark. Last, cells were washed with PBS for three times. The fluorescence microscope was used to collect fluorescence images of cells.

| Immunofluorescence assay
The porcine model of restenosis after stenting was generated as our previously described. 22,23 After porcine were sacrificed, the coronary arteries were removed from the heart and fixed with 10% formalin, then paraffin Olympus light microscope BX53 system was applied for images capture.

| Detection of autophagic flux
The autophagic flux was dynamically observed in HASMCs with mCherry-GFP-LC3B overexpression as described previously. 14 To further evaluate the effect of METTL3 on autophagic flux, we further knocked down or overexpressed METTL3 in mCherry-GFP-LC3B-overexpressing HASMCs. Chloroquine (CQ) (20 μM; C6628; Sigma-Aldrich), a lysosomotropic agent, was used to inhibit the content degradation of autolysosome. After treated with indicated stimulus, the HASMCs were fixed with 4% paraformaldehyde in PBS for 10 min. The fluorescence images were acquired by using a fluorescence microscope. Yellow and red colour indicate autophagosomes or autolysosomes, respectively.

| Transwell assay
After starving with serum-free medium for 24 h, the HASMCs were digested and resuspend in 0.5% FBS medium. A total of 100 μl of cell suspension (3 Â 10 4 ) was added to the upper chambers of a transwell culture plate. After the cells adhered for 2.5 h, 800 μl of medium containing 10% FBS was added to the bottom chamber. The plate was incubated in cell incubator with 5% CO 2 for 12 h. After that, discarding the culture medium in the well, and the cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet for 30 min. The nonmigrated cells on the upper surface of the well were carefully removed with wet cotton swabs. Then, the cells were observed with a microscope.

| Flow cytometry
After treated with indicated stimulus, the HASMCs were collected by trypsin digestion and then washed with PBS for twice. After centrifugation for 10 min, the cells were resuspended with 500 μl PBS, then 5 ml pre-cooled ethanol was added and put it at 4 C overnight.
After discarding the ethanol, the cells were washed twice with PBS.
Finally, the cells were treated with 0.3 mg of Ribonuclease A (R5125; Sigma-Aldrich) and stained by 0.015 mg of PI (P4864; Sigma-Aldrich) for 2 h in the dark. A BD FACS Aria™ III sorter was used to analyse the cycle of the target cells.

| Statistical analyses
The data were analysed by GraphPad Prism 9 software in this study.
All the results were represented as mean ± SD. Student's t-test was used to analyse the means of two groups. Multiple group comparisons were performed by using one-way ANOVA with post hoc analysis. A p-value < 0.05 is considered to be statistically significant. was also significantly down-regulated in HASMCs with 5% and 10% FBS, and the markers of proliferation, PCNA (proliferating cell nuclear antigen) and p-H3 (phosphorylation of histone H3) were robustly elevated in HASMCs with 2%, 5% and 10% FBS treatment, while the expression level of P21 significantly reduced ( Figure 1B,C). In addition, we generated a pig model of restenosis after coronary stenting. 22 Our results showed that obvious coronary stenosis with smaller lumen was observed after stenting ( Figure 1D). However, METTL3 was downregulated in the stented coronary arteries ( Figure 1D). These results indicated that METTL3 may involve in the proliferation of VSMCs and neointima formation.

| METTL3 inhibited the proliferation of HASMCs
To investigate the role of METTL3 on VSMC proliferation, we first knockdown of METTL3 in HASMCs by infecting with lentiviruses containing targeting sequences (Figure 2A,B). The result of the growth curve showed that compared with lenti-shRNA, the cell counts of lenti-shMETTL3-1 and lenti-shMETTL3-2 groups were obviously

| METTL3 repressed phenotypic switching of HASMCs
It is known that inhibition of VSMC proliferation is often accompanied by changes in phenotypic switching. 26 As we have shown that METTL3 inhibits HASMC proliferation, we were therefore very curious whether METTL3 also affects the phenotypic switching of HASMCs. We first examined the effects of METTL3 on HASMC migration which was evaluated by transwell assay. The results showed that compared with con- It is reported that the phenotypic switching of VSMCs is characterized by decreased expression levels of contractile markers but increased expression levels of synthetic markers, which contributes to VSMC proliferation and migration. 28  MYH10 and COL1A1 showed an opposite expression pattern to that of the contractile markers ( Figure 5K-N). Therefore, these results indicate that METTL3 negatively regulates migration and switching from contractile to synthetic phenotype of HASMCs.

| METTL3 facilitated autophagosome formation in HASMCs
METTL3 is known to regulate autophagy, but its effect on autophagy remains controversial. 14,15 Therefore, we first examined whether METTL3 regulates autophagy in HASMCs and showed that METTL3 knockdown inhibited the protein levels of ATG5, ATG7, LC3II and p-ULK1, while enhanced the p-mTOR ( Figure 6A,B). On the contrary, METTL3 overexpression significantly promoted the expression of ATG5, ATG7, LC3II and p-ULK1, but suppressed p-mTOR level ( Figure 6C,D). These results indicated that METTL3 might enhance autophagy by regulating the expression of multiple molecules involving in autophagy initiation.
As autophagy is a multistep biological process, 8 thus, to further verify which step was affected by METTL3, the autophagic flux was monitored with mRFP-GFP-LC3 assay. 4 Our results showed that  Figure 6G,H). Therefore, these results indicate that METTL3 remarkably accelerates the formation of autophagosome in HASMCs.

| Inhibition of autophagosome formation largely reversed the effects of METTL3 on HASMCs
To determine whether autophagy activation plays a causative role in the METTL3-mediated inhibition of proliferation, migration and phenotypic switching in HASMCs, ATG5 and ATG7 were knocked down in the HASMCs with METTL3 overexpression (Figure 7A-D). Consistent with previous reports, 3 ATG5 or ATG7 knockdown significantly

| DISCUSSION
In this study, we used both gain-of-function and loss-of function approaches to decipher the potential role of METTL3 in proliferation, migration and phenotypic switching of VSMCs (Figure 9). We A growing evidence demonstrated that RNA m 6 A modification plays an important role in the regulation of cell proliferation. 29,30 METTL3, as the most important m 6 A methyltransferase, has also been extensively studied for its effect on cell proliferation, especially in tumour cells, and showed that METTL3 promotes the proliferation and migration of various tumour cells, such as gastrointestinal cancer, bladder cancer, and colorectal cancer. 29,31 However, the role of METTL3 in VSMC proliferation remains unknown. In the present study, we found that METTL3 knockdown facilitated, while overexpression of METTL3 suppressed the proliferation of VSMCs, which contributed to heart regeneration after myocardial infarction via facilitating cardiomyocytes to re-enter the cell cycle. 35 Thus, based on the potential opposed effects of METTL3 on tumour cells and normal cells, if targeting METTL3 or m 6 A to treat tumours, its side effects on normal cells should be carefully considered.
Cell proliferation is controlled by cell cycle checkpoints, and we found that METTL3 arrested HASMCs at G2/M checkpoint by downregulating p-CDC2 and upregulating p-CHK1. Binding of CDC2 to Cyclin B1 is required for its activity, which is responsible for entering mitosis. 24 On the other hand, CHK1 and CHK2 inhibit CDC2 by inactivating the phosphatase CDC25. 24 It is well known that contractile VSMCs have weak, while synthetic VSMCs have enhanced proliferative and migratory abilities. 36 METTL3 inhibited the proliferation of VSMCs, implying a phenotypic switching. Our further results revealed that METTL3 knockdown accelerated migration and synthetic phenotype of VSMCs. The loss of contractile properties of smooth muscle cells to a synthetic phenotype is the cause of their hyperproliferation, which will lead to neointima formation. 37 Although studies have shown that METTL3 has participated in the regulation of autophagy, its role in autophagy remains controversial. 14,15 In cardiomyocytes, METTL3 has been reported to inhibit autophagy, whereas in NSCLC cells, METTL3 promotes autophagy. 14,15 In our present study, we found that METTL3-activated autophagy by upregulating ATG5, ATG7 and p-ULK1, and downregulating p-mTOR. Autophagy has been reported to regulate the phenotypic switching of VSMCs, but its effect is context-dependent. 38 For example, PDGF-BB treatment induced VSMC proliferation and synthetic phenotype, as well as autophagy. 39 In contrast, rapamycinbased drugs (e.g., sirolimus and everolimus) which were known inhibitors of the mTOR pathway and inducers of autophagy prevented VSMC phenotypic switching and hyperproliferation. 38,40 Therefore, is there a causal relationship between autophagy and phenotypic switching or a concomitant phenomenon? To answer this question, we reduced autophagosome formation by knocking down ATG5 or ATG7 in METTL3-overexpressing HASMCs and found that the inhibitory effects of METTL3 on proliferation and migration were significantly reversed by either ATG5 or ATG7 knockdown. Thus, our results indicated that autophagy activation is responsible for attenuated proliferation and migration of VSMCs, at least indispensable for METTL3 overexpression-mediated maintenance of the contractile phenotype of VSMCs.
METTL3 is the most important RNA m 6 A methyltransferase, which can affect the fate of mRNA by regulating the m 6 A methylation modification of mRNA, such as translation, stability, and splicing. 9 We found that METTL3 could promote the protein expression of ATG5 and ATG7 to regulate autophagy and phenotypic switching. Many studies have also confirmed that METTL3 usually increases the stability of targeted mRNAs by enhancing their m 6 A modification. 15,41,42 For example, METTL3 has been reported to promote lung cancerassociated transcript 3 expression by increasing its mRNA m 6 A level. 42 We recently reported that METTL3 facilitates ferroptosis of HASMCs by promoting the degradation of SLC7A11 and FSP1 F I G U R E 9 methyltransferase-like 3 (METTL3) promotes autophagy to inhibit the proliferation, migration and phenotypic switching of vascular smooth muscle cells (VSMCs). The expression of METTL3 was inhibited by proliferation inducers in VSMCs. Overexpression of METTL3 promotes autophagy-related 5 (ATG5) and ATG7 protein expression to facilitate autophagosome formation, which subsequently inhibits VSMC proliferation, migration and switching from contractile to synthetic phenotype. Knockdown of either ATG5 or ATG7 largely reversed the inhibitory effects of METTL3 on proliferation, migration and phenotypic switching of VSMCs. These findings indicate that METTL3 may inhibit neointima formation by accelerating the formation of autophagosomes. mRNAs. 43 The limitation of this study is that although we found that METTL3 can increase the protein levels of ATG5 and ATG7, but whether METTL3 directly facilitates the m 6 A of the ATG5 and ATG7 mRNAs and their m 6 A methylation sites are not clear. In addition, the role of METTL3 on neointima formation was not validated by in vivo animal experiments.
In conclusion, we revealed that METTL3 is downregulated during VSMC proliferation, and METTL3 knockdown accelerates the proliferation and migration of VSMCs. Moreover, we further found that METTL3 promotes the formation of autophagosomes, thereby inhibiting the phenotypic switching of VSMCs. These results suggest that activation of autophagy or RNA m 6 A modification may protect against neointima formation after stenting or after coronary artery bypass grafting.