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. 2021 Jan 26:11:514494.
doi: 10.3389/fphys.2020.514494. eCollection 2020.

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression

Yi-Qi Li  1   2 , Ye-Li Li  1 , Xiao-Tong Li  1 , Jun-Yuan Lv  3 , Yang Gao  1 , Wen-Na Li  2 , Qi-Hai Gong  1 , Dan-Li Yang  1
Affiliations

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression

Yi-Qi Li et al. Front Physiol. .

Abstract

Percutaneous coronary intervention (PCI) is the most widely used therapy for treating ischemic heart disease. However, intimal hyperplasia and restenosis usually occur within months after angioplasty. Modern pharmacological researchers have proven that osthole, the major active coumarin of Cnidium monnieri (L.) Cusson, exerts potent antiproliferative effects in lung cancer cells, the human laryngeal cancer cell line RK33 and TE671 medulloblastoma cells, and its mechanism of action is related to cell cycle arrest. The goal of the present study was to observe the effect of osthole on vascular smooth muscle cell (VSMC) proliferation using platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs isolated from rats and vascular balloon injury as models to further elucidate the molecular mechanisms underlying this activity. We detected the relative number of VSMCs by the MTT assay and EdU staining and examined cell cycle progression by flow cytometry. To more deeply probe the mechanisms, the protein expression levels of PCNA, the cyclin D1/CDK4 complex and the cyclin E1/CDK2 complex in balloon-treated rat carotid arteries and the mRNA and protein expression levels of the cyclin D1/CDK4 and cyclin E1/CDK2 complexes in VSMCs were detected by real-time RT-PCR and western blotting. The data showed that osthole significantly inhibited the proliferation of VSMCs induced by PDGF-BB. Furthermore, osthole caused apparent VSMC cycle arrest early in G0/G1 phase and decreased the expression of cyclin D1/CDK4 and cyclin E1/CDK2. Our results demonstrate that osthole can significantly inhibit PDGF-BB-induced VSMC proliferation and that its regulatory effects on cell cycle progression and proliferation may be related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase. The abovementioned mechanism may be responsible for the alleviation of neointimal hyperplasia in balloon-induced arterial wall injury by osthole.

Keywords: CDK2; CDK4; cyclin D1; cyclin E1; osthole; platelet-derived growth factor-BB; proliferation; 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
Effects of osthole on neointimal formation in rat carotid arteries. Representative sections from the sham, model, Osthole-20, and Osthole-40 groups. The neointimal thickness and the ratio of tunica intima/media of each group is expressed as the mean ± SEM. Data are presented as the mean ± SEM, n = 6. **Significant difference compared to the sham group at p < 0.01, #Significant difference compared to the model group at p < 0.05, ##Significant difference compared to the model group at p < 0.01.
Figure 2
Figure 2
Effects of osthole on PCNA, cyclinD1, CDK4, cyclin E1, and CDK2 protein expression in balloon-treated rat carotid arteries. (A) PCNA protein expression. (B) CDK4 protein expression. (C) Cyclin D1 protein expression. (D) CDK2 protein expression. (E) Cyclin E1 protein expression. Data are presented as the mean ± S.E.M. n = 4. **Significant difference compared with the sham group at p < 0.01, ##Significant difference compared with the model group at p < 0.01.
Figure 3
Figure 3
Cells were rendered quiescent and treated with PDGF-BB, after which osthole (20, 40, and 80μM) was added and incubated for 24h. (A) Fluorescence images of EdU in staining are VSMCs. (B) Histograms showing the ratio of EdU-positive cells to total cells. (C) The MTT assay was used to evaluate the viability of VSMCs. Data are presented as the mean ± S.E.M. **Significant difference compared to the control group at p < 0.01, ##Significant difference compared to the PDGF-BB group at p < 0.01.
Figure 4
Figure 4
Distribution of cell cycle stages of VSMCs as determined by flow cytometry. Representative images of (A) the control group, (B) the PDGF-BB group, (C) the Osthole-20 group, (D) the Osthole-40 group, and (E) the Osthole-80 group. (F) Percentage of cells in each phase of the cell cycle. Data are expressed as the mean ± S.E.M. n = 5~7. In (F), *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the PDGF-BB group.
Figure 5
Figure 5
Effect of osthole on cyclin D1/CDK4 mRNA and protein expression in VSMCs. (A) Relative mRNA and protein expression of cyclin D1 in VSMCs. (B) Relative mRNA and protein expression of CDK4 in VSMCs. Data are expressed as the mean ± S.E.M. n = 5. **Significant difference compared to the control group at p < 0.01, ##Significant difference compared to the PDGF-BB group at p < 0.01.
Figure 6
Figure 6
Effect of osthole on cyclin E1/CDK2 mRNA and protein expression in VSMCs. (A) Relative mRNA and protein expression of cyclin E1 in VSMCs. (B) Relative mRNA and protein expression of CDK2 in VSMCs. Data are expressed as the mean ± S.E.M. n = 5. **Significant difference from the control group at p < 0.01, #Significant difference from the PDGF-BB group at p < 0.05, ##Significant difference from the PDGF-BB group at p < 0.01.
Figure 7
Figure 7
Osthole alleviates neointimal hyperplasia in balloon-induced arterial wall injury and inhibits PDGF-BB-induced VSMC proliferation, and the mechanisms are related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase.
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