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. 2022 Feb 3:12:790345.
doi: 10.3389/fphys.2021.790345. eCollection 2021.

Myricanol Inhibits Platelet Derived Growth Factor-BB-Induced Vascular Smooth Muscle Cells Proliferation and Migration in vitro and Intimal Hyperplasia in vivo by Targeting the Platelet-Derived Growth Factor Receptor-β and NF-κB Signaling

Siyuan Fan  1   2 , Cheng Wang  2   3 , Kai Huang  1   2   3 , Minglu Liang  2
Affiliations

Myricanol Inhibits Platelet Derived Growth Factor-BB-Induced Vascular Smooth Muscle Cells Proliferation and Migration in vitro and Intimal Hyperplasia in vivo by Targeting the Platelet-Derived Growth Factor Receptor-β and NF-κB Signaling

Siyuan Fan et al. Front Physiol. .

Abstract

The abnormal proliferation and migration of Vascular smooth muscle cells (VSMCs) are related to many cardiovascular diseases, including atherosclerosis, restenosis after balloon angioplasty, hypertension, etc. Myricanol is a diarylheptanoid that can be separated from the bark of Myrica rubra. It has been reported that myricanol can anti-inflammatory, anti-cancer, anti-neurodegenerative, promote autophagic clearance of tau and prevent muscle atrophy. But its potential role in the cardiovascular field remains unknown. In this study, we investigated the effect of myricanol on the proliferation and migration of VSMCs in vitro and on the intimal hyperplasia in vivo. In vitro experiments, we found myricanol can inhibit the proliferation and migration of VSMCs induced by PDGF-BB. In terms of mechanism, the preincubation of myricanol can suppress the PDGF-BB induced phosphorylation of PDGFRβ and its downstream such as PLCγ1, Src, and MAPKs. In addition, NF-kB p65 translocation was also suppressed by myricanol. In vivo experiments, we found myricanol can suppress the intimal hyperplasia after wire ligation of the carotid artery in mice. These results may provide a new strategy for the prevention and treatment of coronary atherosclerosis and post-stent stenosis in the future.

Keywords: PDGF-BB; PDGFRβ; intimal hyperplasia; myricanol; vascular smooth muscle cells.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Myricanol inhibits PDGF-BB induced VSMCs proliferation. (A) VSMCs were stained with EdU (red) and DAPI (blue). Scale bar, 100 μm. (B) Quantification of the proliferative cells (normalized to vehicle). Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Vehicle group. *P < 0.05, **P < 0.01 vs. the Vehicle + PDGF-BB group. (C) After being pretreated with indicated concentrations of myricanol or vehicle for 30 min, the cells were stimulated by PDGF-BB (30 ng/ml) for 24 h. The protein level of CCND1, CCNE1, PCNA, E2F1, P21, P27, P53, Rb, and Phosphorylated Rb were determined by Western blot analysis. (D) Quantification of the Western blot analysis. Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Vehicle group. *P < 0.05, **P < 0.01 vs. the Vehicle + PDGF-BB group.
FIGURE 2
FIGURE 2
Myricanol inhibits VSMCs migration. (A) In starvation conditions, cells were treated with indicated concentrations myricanol or not for 30 min, then scratched and treated with PDGF-BB (30 ng/ml) for 24 h. Scale bar, 100 μm. (B) Quantification of the area of wound closure (%). Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Vehicle group. **P < 0.01 vs. the Vehicle + PDGF-BB group. (C) After treated with PDGF-BB and myricanol for 24 h, a transwell migration assay of VSMCs was performed and representative images were shown. Scale bar, 100 μm. (D) Quantification of migrated cells. Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Vehicle group. **P < 0.01 vs. the Vehicle + PDGF-BB group. (E) After being pretreated with indicated concentrations of myricanol or vehicle for 30 min, the cells were stimulated by PDGF-BB (30 ng/ml) for 24 h. The protein level of MMP2 and MMP9 were determined by Western blot analysis. (F) Quantification of the Western blot analysis. Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Vehicle group. *P < 0.05, **P < 0.01 vs. the Vehicle + PDGF-BB group.
FIGURE 3
FIGURE 3
Effect of myricanol on the phosphorylation of PDGFRβ, PLCγ1, Src and downstream MAPKs. (A) The protein level of phosphorylated PDGFRα, PDGFRβ, PLCγ1, Src, JNK, ERK1/2 and p38 were determined by Western blot analysis. (B) Quantification of the Western blot analysis. Data are represented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. the Vehicle + PDGF-BB group.
FIGURE 4
FIGURE 4
Effects of myricanol on PDGF-BB-induced NF-κB signaling in VSMCs. (A) After being pretreated with indicated concentrations of myricanol or vehicle for 30 min, the cells were stimulated by PDGF-BB (30 ng/ml) for 5 min. The protein level of p-IκBα, IκBα were determined by Western blot analysis. After being pretreated with indicated concentrations of myricanol or vehicle for 30 min, the cells were stimulated by PDGF-BB (30 ng/ml) for 30 min. The protein level of p-p65 and p65 were determined by Western blot analysis. (B) p65 nuclear translocation was analyzed by Western blot analysis. (C) p65 nuclear translocation was analyzed by immunocytochemistry. Scale bar, 50 μm. Data are represented as mean ± SEM (n = 3). #P < 0.05, ##p < 0.01 vs. the Vehicle group. *P < 0.05, **p < 0.01 vs. the Vehicle + PDGF-BB group.
FIGURE 5
FIGURE 5
Effect of myricanol on carotid artery ligation induced neointimal hyperplasia for 14 days. (A) myricanol (5 mg/kg/day) or hydration medium (PEG 400) was intraperitoneally injected for 14 days after carotid artery ligation. H&E-stained and elastic Masson trichrome-stained sections of all groups were shown. Scale bar, 200 μm. (B) Quantification of the mean intimal thickness. Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Sham + Vehicle group. **P < 0.01 vs. the Injured + Vehicle group. (C) Quantification of the ratio of intima to media (I/M ratio). Data are represented as mean ± SEM (n = 3). ##P < 0.01 vs. the Sham + Vehicle group. **P < 0.01 vs. the Injured + Vehicle group.
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