METTL14 promotes intimal hyperplasia through m6A-mediated control of vascular smooth muscle dedifferentiation genes
Grace Chensee, Bob S.L. Lee, Immanuel D. Green, Jessica Tieng, Renhua Song, Natalia Pinello, Quintin Lee, Majid Mehravar, David A. Robinson, Mian Wang, Mary M. Kavurma, Jun Yu, Justin J.L. Wong, Renjing Liu
Grace Chensee, Bob S.L. Lee, Immanuel D. Green, Jessica Tieng, Renhua Song, Natalia Pinello, Quintin Lee, Majid Mehravar, David A. Robinson, Mian Wang, Mary M. Kavurma, Jun Yu, Justin J.L. Wong, Renjing Liu
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Research Article Therapeutics Vascular biology

METTL14 promotes intimal hyperplasia through m6A-mediated control of vascular smooth muscle dedifferentiation genes

Abstract

Vascular smooth muscle cells (VSMCs) possess significant phenotypic plasticity, shifting between a contractile phenotype and a synthetic state for vascular repair/remodeling. Dysregulated VSMC transformation, marked by excessive proliferation and migration, primarily drives intimal hyperplasia. N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotes, plays a critical role in gene expression regulation; however, its impact on VSMC plasticity is not fully understood. We investigated the changes in m6A modification and its regulatory factors during VSMC phenotypic shifts and their influence on intimal hyperplasia. We demonstrate that METTL14, crucial for m6A deposition, significantly promoted VSMC dedifferentiation. METTL14 expression, initially negligible, was elevated in synthetic VSMC cultures, postinjury neointimal VSMCs, and human restenotic arteries. Reducing Mettl14 levels in mouse primary VSMCs decreased prosynthetic genes, suppressing their proliferation and migration. m6A-RIP-seq profiling showed key VSMC gene networks undergo altered m6A regulation in Mettl14-deficient cells. Mettl14 enhanced Klf4 and Serpine1 expression through increased m6A deposition. Local Mettl14 knockdown significantly curbed neointimal formation after arterial injury, and reducing Mettl14 in hyperplastic arteries halted further neointimal development. We show that Mettl14 is a pivotal regulator of VSMC dedifferentiation, influencing Klf4- and Serpine1-mediated phenotypic conversion. Inhibiting METTL14 is a viable strategy for preventing restenosis and halting restenotic occlusions.

Authors

Grace Chensee, Bob S.L. Lee, Immanuel D. Green, Jessica Tieng, Renhua Song, Natalia Pinello, Quintin Lee, Majid Mehravar, David A. Robinson, Mian Wang, Mary M. Kavurma, Jun Yu, Justin J.L. Wong, Renjing Liu

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Figure 6

Mettl14 silencing suppresses injury-induced VSMC dedifferentiation in vitro.

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Mettl14 silencing suppresses injury-induced VSMC dedifferentiation in vi...
(A) Immunoblotting and quantification of Mettl14, Klf4, and Serpine1 levels in VSMCs pretreated with PDGF-BB or not for 72 hours, followed by transduction with shCtrl or shMettl14-2 viruses. n = 4 independent repeats. (B) MTS cell proliferation assay performed on murine VSMCs treated with PDGF-BB for 72 hours and then transduced with shCtrl or shMettl14-2 viruses over 148 hours. n = 3 independent repeats. (C) Representative Western blot and quantification of Ccnd1 and Cdkn1a in shCtrl- and shMettl14-2–transduced VSMCs following 72 hours of PDGF-BB pretreatment. n = 3 independent repeats. (D) Representative images of scratch assay assessing cellular migration of PDGF-B–pretreated murine VSMCs transduced with shCtrl or shMettl14-2 at 0, 24, and 48 hours. Lines mark the wound edges of the cultures. Scale bars: 50 μm. Quantification of percentage of wounded area shown on the right. n = 3 independent repeats. (E) Representative image of gelatin zymography stained with Coomassie blue assessing Mmp2 activity in PDGF-BB–pretreated murine VSMCs transduced with shCtrl or shMettl14-2. Each lane represents 1 individual sample. n = 5 biologically independent samples. Quantification of enzymatic activity in 3D plot shown below. (F) Representative Western blot and quantification of p-Stat1 (Tyr701) and Stat1 in PDGF-BB–pretreated murine VSMCs transduced with shCtrl or shMettl14-2. n = 4 independent repeats. (G) Representative Western blot and quantification of contractile VSMC markers in PDGF-BB–pretreated murine VSMCs transduced with shCtrl or shMettl14-2. n = 4 independent repeats. (H) Representative images of collagen gel contraction assay of PDGF-BB–pretreated murine VSMCs transduced with shCtrl or shMettl14-2. n = 3 independent repeats. Scale bars: 5 mm. *P < 0.05, ***P < 0.005 by 2-way ANOVA with Šidák’s multiple-comparison test (A–C and G), repeated measures 2-way ANOVA (D), 1-way ANOVA with Tukey’s multiple-comparison test (F), or unpaired, 2-tailed Student’s t test (H). Data are presented as mean ± SD.