PARP1 contributes to intimal hyperplasia by regulating METTL3-mediated m6A methylation of TRAIL

Abstract

Poly (ADP-ribose) polymerase 1 (PARP1) promotes vascular intimal hyperplasia (IH) while contributing to N6-methyladenosine (m6A) methylation regulatory processes. The present study focuses on whether the PARP1 inhibitor PJ34 can improve vascular IH by regulating m6A methylation modification. Mice with femoral artery wire injury-induced IH and platelet-derived growth factor-BB (PDGF-BB)-challenged mouse vascular smooth muscle cells (VSMCs) were utilized in the study. PJ34 treatment significantly alleviated neointimal formation, suppressed VSMC proliferation and phenotypic switching, and reduced global m6A methylation and methyltransferase-like 3 (METTL3) expression in injured arteries. Dot blot, RT-qPCR, western blot, and immunohistochemistry confirmed these changes. In vitro, PJ34 impaired PDGF-BB-stimulated proliferation and migration in VSMCs, effects reversed by METTL3 overexpression but not observed in METTL3-deficient cells. Mechanistically, METTL3 regulated the m6A methylation and stability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA. PJ34 downregulated TRAIL expression via inhibition of METTL3-mediated m6A modification. TRAIL-knockout mice were resistant to the vascular protective effects of PJ34, highlighting the essential downstream role of TRAIL. Immunohistochemistry confirmed TRAIL localization in the neointima and media. Moreover, TRAIL deficiency did not lead to increased systemic inflammation, as TNF-α, IL-6, and IL-1β levels in plasma remained unchanged. In conclusion, PJ34 mitigates vascular IH by modulating METTL3-mediated TRAIL m6A methylation. This finding provides novel insight into epigenetic therapy for vascular remodeling.

Keywords: Intimal hyperplasia; METTL3; PJ34; VSMCs.

Fig. 1

The influence of PJ34 on neointimal hyperplasia in mice with FA wire injury. (A) Illustration of the creation of C57BL/6 J mice with FA wire injury and PJ34 treatment (6 mice/group). (B) HE staining sections of the full cross-section of the FA from different groups at 4 weeks after injury. All images were acquired under the same magnification. Scale bar = 100 μm; the magnified scale bar = 5 μm; I = intimal; M = Media. (C-E) Quantification of neointimal area, neointimal/media area ratio, and media area at 4 weeks after injury. (F-G) Representative IHC images of α-SMA and PCNA in FA tissues from each group. Scale bar = 50 μm. All data are shown as mean ± SEM. Results were obtained from tissue samples from at least three different mice. One-way or two-way ANOVA was conducted; ^***p < 0.001and ns = not significant

Fig. 2

PJ34 reduces global m6A levels and METTL3 expression in injured femoral arteries. (A) Global m6A level of RNA extracted from intimal samples was measured via m6A dot blot assays. Methylene blue staining (lower) was used to detect input RNA. (B) Relative mRNA levels of METTL3, METTL14, WTAP, FTO, and ALKBH5 in intimal samples were detected by RT-qPCR. (C) IHC staining and quantification of METTL3 expression in FA sections. Scale bar = 50 μm. All data are shown as mean ± SEM. Results were obtained from tissue samples from at least three different mice. One-way or two-way ANOVA was conducted; ^***p < 0.001 and ns = not significant

Fig. 3

Overexpression of METTL3 overturns PJ34-mediated suppressive effects on the proliferation and migration of mouse VSMCs. (A and B) The transfection efficiency of the METTL3 overexpression plasmid in mouse VSMCs were determined by RT-qPCR and western blot. (C-E) CCK-8, EdU, and transwell migration assays analyzed the viability, proliferation, and migration of mouse VSMCs with different treatments (control, PDGF-BB + vehicle, PDGF-BB + PJ34, PDGF-BB + PJ34 + METTL3). (F) Relative protein levels of MMP2 and MMP9 in mouse VSMCs were detected by western blot. (G) Global m6A abundance in mouse VSMCs was measured via m6A dot blot assays. All data are shown as mean ± SEM. Results were obtained at least three independent experiments. One-way or two-way ANOVA was conducted; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001

Fig. 4

Proliferation and migration of METTL3-knockdown mouse VSMCs stimulated by PDGF-BB are not affected by PJ34. (A and B) The interference efficiency of three siRNAs targeting METTL3 in mouse VSMCs was assessed by RT-qPCR and western blot. (C-E) The viability, proliferation, and migration of PDGF-BB-challenged mouse VSMCs in different groups (si-NC + vehicle, si-NC + PJ34, si-METTL3 + vehicle, si-METTL3 + PJ34) were evaluated by CCK-8, EdU, and transwell migration assays under the PDGF-BB treatment. (F) Relative MMP2 and MMP9 protein levels in PDGF-BB-challenged mouse VSMCs were detected by western blot. (G) M6A dot blot assays were carried out to analyze the global m6A abundance in PDGF-BB-challenged mouse VSMCs. All data are shown as mean ± SEM. Results were obtained at least three independent experiments. One-way or two-way ANOVA was conducted; ^**p < 0.01, ^***p < 0.001, and ns = not significant

Fig. 5

PJ34 represses TRAIL expression by controlling METTL3-mediated the methylation of TRAIL. (A) The predicted m6A modification sites on the TRAIL mRNA sequence based on the online SRAMP database (http://www.cuilab.cn/sramp). (B) Six m6A modification sites with very high confidence on the TRAIL mRNA sequence. (C) MeRIP assays was adopted to test m6A levels of TRAIL in intimal tissues from mice within control, injured, injured + vehicle, injured + PJ34-L, and injured + PJ34-H. (D) RT-qPCR was employed to detect TRAIL mRNA levels in intimal tissues. (E) The m6A levels of TRAIL in PDGF-BB-stimulated mouse VSMCs of control, vehicle, PJ34, and PJ34-METTL3 were estimated by MeRIP assays. (F) Relative TRAIL mRNA levels in PDGF-BB-stimulated mouse VSMCs were tested using RT-qPCR. (G) The stability of TRAIL in METTL3 knockdown and its corresponding control cells treated with or without PDGF-BB was estimated by actinomycin D assays combined with RT-qPCR. All data are shown as mean ± SEM. Results were obtained at least three independent experiments. One-way or two-way ANOVA was conducted; ^***p < 0.001 and ns = not significant

Fig. 6

METTL3 participates in PDGF-BB-induced proliferation and migration of mouse VSMCs via TRAIL. (A and B) The transfection efficiency of the TRAIL overexpression plasmid was evaluated using RT-qPCR and western blot. (C-G) Mouse VSMCs were grouped as follows: control, PDGF-BB + si-NC, PDGF-BB + si-METTL3, and PDGF-BB + si-METTL3 + TRAIL. (C-E) Cell viability, proliferation, and migration were tested using CCK-8, EdU, and transwell migration assays. (F) Relative protein levels of MMP2 and MMP9 were detected by western blot. (G) Relative METTL3 and TRAIL protein levels were analyzed by western blot. All data are shown as mean ± SEM. Results were obtained at least three independent experiments. One-way ANOVA was undertaken; ^***p < 0.001 and ns = not significant

Fig. 7

PJ34 improves intimal hyperplasia induced by FA wire injury via inhibition of TRAIL in mouse models. (A) Mice were processed in the following groups: uninjured (WT, WT + PJ34, TRAIL KO, and TRAIL KO + PJ34) and injured (WT, WT + PJ34, TRAIL KO, and TRAIL KO + PJ34) groups. HE staining analysis of the changes in intimal hyperplasia of the FA vessels. Scale bar = 100 μm; the magnified scale bar = 5 μm; I = intimal; M = Media. (B-D) Quantification of neointimal area, neointimal/media area ratio, and media area in the injured group at 4 weeks. (E–F) RT-qPCR and western blot analyses showing TRAIL expression in injured arteries and its downregulation by PJ34 in WT but not TRAIL KO mice. (G) IHC staining of TRAIL expression in vascular tissues. Scale bar = 50 μm. All data are shown as mean ± SEM. Results were obtained from tissue samples from at least three different mice. One-way was conducted; ^***p < 0.001 and ns = not significant