Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2026 Apr 20;16:18248. doi: 10.1038/s41598-026-49496-x

Knockdown of DVL2 inhibits vascular smooth muscle cell proliferation and migration

Lei Li 1,2,3, Yujuan Yao 1, Yao Wang 1, Jing Dong 1, Pingyang Zhang 1,✉
PMCID: PMC13260992  PMID: 42010267

Abstract

Despite advancements in drug-eluting stent technology, the incidence of in-stent restenosis (ISR) remains around 5–10%. The complex mechanisms of ISR are primarily based on the proliferation and migration of vascular smooth muscle cells (VSMCs). The dishevelled gene (DVL2), a component of the WNT signaling pathway, is involved in tumorigenesis. However, the role of DVL2 in ISR remains unknown. In this study, we used the rat carotid balloon injury model in vivo and PDGF-BB-treated VSMCs in vitro. Intimal hyperplasia was assessed by Hematoxylin and eosin staining. VSMCs proliferation was measured using EdU and CCK-8 assays, and migration was evaluated via transwell and Wound-healing assays. DVL2 expression was significantly increased in both balloon-injured carotid arteries and PDGF-BB-stimulated VSMCs. Knockdown of DVL2 using adeno-associated virus (AAV) effectively reduced intimal hyperplasia after balloon injury. Similarly, siRNA-mediated DVL2 silencing suppressed PDGF-BB-induced VSMCs proliferation and migration, whereas plasmid-driven DVL2 overexpression enhanced these effects. Additionally, DVL2 knockdown in PDGF-BB-stimulated VSMCs markedly upregulated the contractile markers α-smooth muscle actin and calponin. Mechanistically, DVL2 silencing inhibited β-catenin activation in PDGF-BB-treated VSMCs. Collectively, these findings demonstrated that DVL2 promoted VSMCs proliferation and migration by activating β-catenin signaling, highlighting its potential as a candidate therapeutic target for ISR.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49496-x.

Keywords: In-stent restenosis, Dishevelled gene, Vascular smooth muscle cells, Proliferation, Migration

Subject terms: Cardiology, Cell biology, Diseases

Introduction

Coronary artery disease (CAD) is commonly treated with percutaneous coronary intervention (PCI); however, this procedure can trigger excessive proliferation of vascular smooth muscle cells (VSMCs), leading to in-stent restenosis (ISR) and the need for repeat revascularization1. Although drug-eluting stents have substantially reduced ISR rates, the condition still occurs in 5%-10% of cases, underscoring the need to elucidate its underlying mechanisms2,3.

ISR is associated with phenotypic changes in VSMCs. Under physiological conditions, VSMCs in the tunica media maintain a contractile phenotype with limited proliferative activity. However, pathological stimuli such as stent implantation, atherosclerotic plaque rupture, or exposure to growth factors like platelet-derived growth factor (PDGF) can induce a phenotypic switch to a synthetic state, marked by increased proliferation, migration, and reduced expression of differentiation markers4. This transition contributes to vascular pathologies, including ISR, atherosclerosis, and aortic aneurysm5,6.

Emerging evidence implicates Wnt signaling in VSMCs dysfunction, including foam cell formation, phenotypic switching, and migration, although the contributions of specific components require clarification7,8. Notably, the Dishevelled proteins (DVL1, DVL2, and DVL3), central mediators of Wnt signaling, regulate fundamental cellular processes including proliferation and differentiation, with DVL2 particularly implicated in cancer progression9–12. These findings imply that DVL2 may comparably regulate VSMC phenotypic modulation and migratory capacity, warranting further mechanistic investigation.

In this study, we demonstrated that DVL2 knockdown suppressed the transition of VSMCs from a contractile to a synthetic phenotype, thereby inhibiting their proliferation and migration. Furthermore, we identified β-catenin as a key downstream effector in DVL2-mediated vascular restenosis, providing new insights into potential therapeutic targets for ISR.

Materials and methods

Rat viral transfection and carotid artery balloon injury model

All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies. The experiment was approved by the Institutional Animal Care Committee of Nanjing First Hospital, Nanjing Medical University (Protocol #DWSY-23077381) and performed following ARRIVE guidelines (https://arriveguidelines.org). Male, 250–300 g, 10-week-old Sprague-Dawley (SD) rats from the Hangzhou Medical Lab Animal Center were housed on a 12-hour light/dark cycle and fed a standard diet. Rats were intravenously injected via the tail vein with either an adeno-associated virus (AAV) carrying DVL2-targeting shRNA (AAV9-SM22α-EGFP-sh-Dvl2-SV40PolyA, Genechem Co., Shanghai, China) or a negative control virus (AAV9-NC).

Three weeks post-injection, a carotid artery balloon injury model was established in accordance with Tulis’s research13. Briefly, rats were anesthetized (100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine, intraperitoneal), and a 2 F Fogarty balloon catheter (Edwards Lifesciences Germany, #120602F) was introduced through the external carotid artery into the common carotid artery. The balloon was inflated, rotated, and withdrawn to denude the endothelium; this procedure was repeated three times. Two weeks after injury, the animals were euthanized by overdosage of 100 mg/kg sodium pentobarbital and perfused in-situ with 4% paraformaldehyde solution. The injured and uninjured common carotid arteries were harvested, fixed in 4% paraformaldehyde, dehydrated, and paraffin-embedded. Transverse sections of 5 μm thickness were collected at 25 μm intervals from the bifurcation, stained with hematoxylin and eosin (H&E), and analyzed using ImageJ software version 1.38e (NIH, USA, https://imagej.net/ij/) to calculate the intima-to-media (I/M) ratio. A total of 50 rats were used, with five rats per group.

Rat aortic thoracic smooth muscle cells culture

Rat aortic thoracic smooth muscle cell line (A7r5) were obtained from the National Collection of Authenticated Cell Cultures and were cultivated in 10% fetal bovine serum (FBS; Austain, 0986180) supplemented with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, 30030). A7r5 cells were cultured in an incubator (Life Technologies, Baltimore, MD, USA) at 37 °C and 5% CO2. The investigations employed A7r5 cells from the first two passages.

Small interfering RNA (siRNA) transfection

Negative control siRNA (si-NC) and DVL2-targeting siRNA (si-DVL2) were obtained from GenePharma (Shanghai, China). Following the manufacturer’s instructions, lipo8000 (Beyotime, 103122230412) was used to transiently transfect si-DVL2 or si-NC into A7r5 cells.

DVL2 overexpression vector constructs

A DVL2-eukaryotic expression vector (DVL2-GV657) was produced by inserting the open reading frame of DVL2 into GV657 for DVL2 over-expression. A7r5 cells were transfected with DVL2 plasmid or the control vector using lipo8000 (Beyotime, 103122230412). After transfection, the cells were kept for 48 h for further experiments.

Cell counting kit-8 assay

The transfected A7r5 cells were incubated with 20 ng/ml PDGF-BB (MCE, HYP7278-290146) for 48 h. Then, the cells were washed twice in PBS, trypsinized, and counted. The treated A7r5 cells were deposited at a density of 5 × 103 cells/well into 96-well plates. After incubating for 48 h, 10 µl of Cell Counting Kit-8 (CCK-8) reagent (Vazyme, 7E632I2) was added to each well. The combination was then incubated at 37 °C for an extra 4 h. Subsequently, a microplate reader (Bio-Tek, Winooski, VT, USA) was used to measure the optical density (OD) at a wavelength of 450 nm.

EdU assay

The transfected A7r5 cells were incubated with PDGF-BB (30 ng/ml) for 48 h. Then, the EdU cell proliferation assay (Beyotime, C0071S) was performed in accordance with the manufacturer’s protocol. Images were captured using a fluorescence microscope (Axio Imager, Zeiss, Shanghai, China). EdU-positive cells were counted using ImageJ software version 1.38e (NIH, USA, https://imagej.net/ij/).

Wound-healing assay

The treated A7r5 cells were seeded in 6-well plates and cultured to > 90% confluence. A standardized wound was created in each well using a 200 µL pipette tip. Wound closure was monitored at 0- and 24-hours post-scratching using a phase-contrast microscope (Olympus CK30, Tokyo, Japan). The migration rate was quantified with ImageJ software version 1.38e (NIH, USA, https://imagej.net/ij/) by calculating: (0 h wound width − 24 h wound width)/0 h wound width×100%.

Transwell assay

Cell migration was assessed using Transwell chambers (Corning Incorporated, NY, USA). Briefly, treated A7r5 cells (300 µL serum-free medium) were seeded in the upper chamber, while the lower chamber contained 700 µL medium with 20% FBS as a chemoattractant. After 48 h, non-migrated cells on the upper membrane surface were removed with a cotton swab. Migrated cells on the lower surface were fixed with 4% paraformaldehyde (10 min), stained with 1% crystal violet (10 min), and quantified by counting five random fields per membrane under a microscope.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using RNA-Solv Reagent (Omega Bio-tek, R6830-02) and reverse transcribed into cDNA with HiScript III Reverse Transcriptase (Vazyme, R323-01). qPCR was performed using 10 µL 2× Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712-02), 2 µL cDNA, 0.8 µL primers, and 7.2 µL ddH₂O in a 20 µL reaction volume. Relative gene expression was calculated by the 2−ΔΔCt method using β-actin as the endogenous control.

Primer sequences:

DVL2:

Forward 5′-GCCCCTGCTGCCTACCTT-3′.

Reverse 5′-GCCCCCCCATCACTTCTT-3′.

β-actin:

Forward 5′-TATGCTCTCCCTCACGCCATCC-3′.

Reverse 5′-GTCACGCACGATTTCCCTCTCAG-3′.

Western blot analysis

Total protein was extracted from A7r5 cells using RIPA lysis buffer (Beyotime, P0013B) supplemented with protease inhibitors, followed by sonication and centrifugation (8,050 × g, 15 min, 4 °C). Protein concentration was determined using a Bicinchoninic Acid protein assay kit (Beyotime, 070721211214). Equal amounts of protein (50 µg) were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA).

After blocking with 5% non-fat milk in TBST for 1 h at room temperature, membranes were incubated overnight at 4 °C with the following primary antibodies:

DVL2 (Cell Signaling Technology, 3224, 1:1000).

α-SMA (Cell Signaling Technology, 19245, 1:1000).

Calponin (Cell Signaling Technology, 17819, 1:1000).

β-Catenin (Cell Signaling Technology, 8480, 1:1000).

GAPDH (Proteintech, 10494-1-AP, 1:10000).

β-Actin (Proteintech, 20536-1-AP, 1:6000).

Membranes were then incubated with HRP-conjugated secondary antibodies (Proteintech; goat anti-mouse, 20000838, 1:6000; goat anti-rabbit, 20000798, 1:10000) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (NCM Biotech) and quantified using ImageJ software version 1.38e (NIH, USA, https://imagej.net/ij/).

Statistical analysis

Data are presented as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were analyzed by two-tailed unpaired Student’s t-test, while multiple group comparisons were performed using one-way ANOVA. Statistical significance was set at p < 0.05. All analyses were conducted using SAS version 9.2 (Cary, NC, USA, https://www.sas.com).

Results

DVL2 knockdown prevents neointima formation in a rat carotid artery balloon injury model

The results revealed a significant upregulation of DVL2 protein expression in the rat carotid artery balloon injury model compared to the sham group (Fig. 1a). To assess the role of DVL2 in intimal hyperplasia, four experimental groups were established: sham, injury, injury + AAV9-NC (negative control), and injury + AAV9-sh-DVL2 (DVL2 knockdown). Successful transfection was confirmed by Western blot analysis (Fig. 1b). Morphometric analysis demonstrated a marked increase in the intima-to-media (I/M) thickness ratio in the injury group relative to the sham group (Fig. 1c). Conversely, the I/M ratio was significantly reduced in the injury + AAV9-sh-DVL2 group compared to the injury + AAV9-NC group (Fig. 1c), suggesting that DVL2 downregulation suppresses neointimal hyperplasia, potentially by inhibiting vascular smooth muscle cell proliferation.

Fig. 1.

Fig. 1

Open in a new tab

DVL2 down-regulation inhibited balloon injury-induced intimal hyperplasia. (a) The expression of DVL2 in balloon injured carotid arteries was determined using Western blot analysis. (b) The expression of DVL2 in carotid arteries was measured by Western blot in different groups. (c) Representative photos and quantitative analysis of H&E staining of carotid arteries in different groups. Scale bars: 400 and 100 µM. H&E, hematoxylin and eosin. *p < 0.05 versus the sham group; #p < 0.05 versus the Injury + AAV9‐NC group. Results are expressed as mean ± SD. The statistical significance of differences was assessed by a two-tailed unpaired Student’s t-test or two-way ANOVA wherever applicable, N = 5 animals/group.

DVL2 expression was significantly upregulated in PDGF-BB-stimulated A7r5 cells

To mimic smooth muscle cell proliferation in vitro, A7r5 cells were treated with PDGF-BB for 48 h. Western blot and quantitative analysis revealed that DVL2 levels in the PDGF-BB-stimulated group were markedly higher than those in the control group (Fig. 2a, b).

Fig. 2.

Fig. 2

Open in a new tab

DVL2 was highly expressed in cultured A7r5 cells stimulated by PDGF-BB. (a) the protein expression of DVL2 in PDGF-induced A7r5 cells was measured using Western blot analysis. (b) the mRNA levels of DVL2 in PDGF-induced A7r5 cells were measured using qRT-PCR analysis. *p < 0.05 versus the control group. Results are expressed as mean ± SD. The statistical significance of differences was assessed by a two-tailed unpaired Student’s t-test, N = 3.

Knockdown of DVL2 attenuates PDGF-BB-induced proliferation and migration in A7r5 cells

Transfection efficiency was confirmed by significantly reduced DVL2 expression in the si-DVL2 group compared to the si-NC group (Fig. 3a, b). To assess the functional role of DVL2, A7r5 cells were divided into four experimental groups: Control (untreated), PDGF-BB (stimulated), PDGF-BB + si-NC (negative control siRNA), and PDGF-BB + si-DVL2 (DVL2 knockdown). Proliferation assays (CCK-8 and EdU assays) revealed that PDGF-BB significantly enhanced A7r5 cell proliferation, whereas DVL2 knockdown (PDGF-BB + si-DVL2) markedly suppressed this effect compared to the PDGF-BB + si-NC group (Fig. 3c and d). Migration assays (Wound-healing and transwell assays) demonstrated that PDGF-BB robustly promoted cell migration, which was significantly attenuated by DVL2 knockdown (Fig. 3e, f). These findings indicate that DVL2 is critical for PDGF-BB-induced proliferation and migration in A7r5 cells, and its knockdown effectively inhibits these processes.

Fig. 3.

Fig. 3

Open in a new tab

Effect of si-DVL2 on the proliferation and migration of A7r5 cells stimulated by PDGF-BB. (a and b) A7r5 cells were transfected with si-DVL2 or si-NC. Expressions of DVL2 were detected using Western blot and qRT-PCR analysis, separately. (c and d) A7r5 cells proliferation was determined by CCK8 and EdU assay. Scale bar:100 μm. (e and f) A7r5 cells migration was determined by Wound-healing and transwell assay. Scale bars:100 µM. *p < 0.05 versus the control group; #p < 0.05 versus the PDGF + si-NC group. Results are expressed as mean ± SD. The statistical significance of differences was assessed by a two-way ANOVA, N = 3.

DVL2 overexpression enhances PDGF-BB-induced proliferation and migration in A7r5 cells

Successful transfection was confirmed by significantly higher DVL2 expression in the DVL2-GV657 group compared to the GV657 control group (Fig. 4a, b). To investigate DVL2 functional role, A7r5 cells were divided into four groups: Control, PDGF-BB stimulation alone, PDGF-BB + empty vector (GV657), and PDGF-BB + DVL2 overexpression (DVL2-GV657). Both CCK-8 and EdU assays demonstrated that PDGF-BB stimulation significantly increased A7r5 cell proliferation. Notably, DVL2 overexpression (PDGF-BB + DVL2-GV657) further enhanced this proliferative effect compared to the empty vector control (PDGF-BB + GV657) (Fig. 4c, d). Wound healing and transwell assays revealed that PDGF-BB markedly promoted cell migration. Importantly, DVL2-overexpressing cells (PDGF-BB + DVL2-GV657) exhibited significantly greater migratory capacity than vector control cells (Fig. 4e, f). These results demonstrate that DVL2 overexpression potentiates PDGF-BB-induced proliferation and migration in A7r5 cells, suggesting a critical role for DVL2 in these processes.

Fig. 4.

Fig. 4

Open in a new tab

Effects of DVL2 overexpression on the proliferation and migration of A7r5 cells stimulated by PDGF-BB. (a and b) A7r5 cells were transfected with DVL2-GV657 or GV657. Expressions of DVL2 were detected using Western blot and qRT-PCR, separately. (c and d) A7r5 cells proliferation was determined by CCK8 and EdU assay. Scale bar:100 μm. (e and f) A7r5 cells migration was determined by Wound-healing and transwell assay. Scale bars:100 µM.*p < 0.05 versus the control group; #p < 0.05 versus the PDGF + si-NC group. Results are expressed as mean ± SD. The statistical significance of differences was assessed by a two-way ANOVA, N = 3.

DVL2 knockdown suppresses contractile phenotype markers in PDGF-BB-stimulated A7r5 cells

To investigate DVL2’s role in phenotypic modulation, we assessed expression of contractile markers (α-SMA and calponin) by Western blotting. PDGF-BB stimulation significantly reduced the expression of both α-SMA and calponin compared to the control group (Fig. 5). Notably, DVL2 knockdown significantly restored contractile marker expression versus the negative control group (PDGF-BB + si-NC) (Fig. 5).

Fig. 5.

Fig. 5

Open in a new tab

Effect of DVL2 knockdown on the expressions of contractile markers in PDGF-BB-stimulated A7r5 cells. (a) the expression of α-SMA was determined using Western blot analysis. (b) the expression of calponin was determined using Western blot analysis. *p < 0.05 versus the control group; #p < 0.05 versus the PDGF + si-NC group. Results are expressed as mean ± SD. The statistical significance of differences was assessed by a two-way ANOVA, N = 3.

DVL2 knockdown attenuates PDGF-BB-induced β-catenin activation in A7r5 cells

To elucidate the mechanistic role of DVL2, we investigated its effect on β-catenin signaling. Western blot analysis revealed that PDGF-BB stimulation significantly upregulated β-catenin expression compared to the control group. Notably, DVL2 knockdown (PDGF-BB + si-DVL2) significantly inhibited this activation compared to the negative control (PDGF-BB + si-NC) (Fig. 6).

Fig. 6.

Fig. 6

Open in a new tab

Knockdown of DVL2 prevented the activation of β-catenin in A7r5 cells in response to PDGF-BB. *p < 0.05 versus the control group; #p < 0.05 versus the PDGF + si-NC group. Results are expressed as mean ± SD. The statistical significance of differences was assessed by a two-way ANOVA, N = 3.

Discussion

Percutaneous coronary intervention is a critical therapeutic approach for coronary artery disease14. However, stent implantation often induces intravascular restenosis, which compromises the efficacy of coronary stents and poses a significant clinical challenge2,3,15,16. In this study, we demonstrated that knockdown of DVL2 suppressed the proliferation and migration of VSMCs. Furthermore, we identified that DVL2 may exert this inhibitory effect by modulating its downstream target, β-catenin, suggesting a potential mechanism for mitigating restenosis.

Dishevelled is a key regulator of the Wnt signaling pathway, which governs diverse cellular processes, including proliferation, migration, differentiation, and stem cell renewal9. Previous studies have implicated DVL2 in tumorigenesis and metastasis12,17. In this study, we observed significant upregulation of DVL2 in both PDGF-BB-stimulated vascular smooth muscle cells and a carotid artery balloon injury model. DVL2 knockdown prevented neointima formation in a rat carotid artery balloon injury model, suggesting that DVL2 downregulation suppressed neointimal hyperplasia, potentially by inhibiting vascular smooth muscle cell proliferation. Knockdown of DVL2 using si-DVL2 markedly suppressed PDGF-BB-induced VSMC proliferation and migration, whereas DVL2 overexpression exacerbated these effects, underscoring its critical role in VSMC phenotypic modulation.

Under physiological conditions, VSMCs maintain a quiescent, contractile phenotype, which is essential for regulating vascular tone and hemodynamic stability18,19. However, pathological stimuli- vascular injury or stent implantation-trigger a phenotypic switch from a contractile to a synthetic state, characterized by reduced expression of contractile markers (e.g., α-SMA, SM22α, and calponin) and enhanced proliferative/migratory capacity4,20,21. In this study, PDGF-BB stimulation downregulated these contractile proteins in A7r5 cells, suggesting the transition to a synthetic phenotype. Conversely, DVL2 knockdown restored the expression of contractile markers in PDGF-treated A7r5 cells, suggesting that DVL2 suppression attenuated the PDGF-induced phenotypic switch.

In the canonical Wnt/β-catenin pathway, binding of Wnt ligands to Frizzled receptors activates DVL, which subsequently inhibits glycogen synthase kinase-3β (GSK-3β), leading to cytoplasmic accumulation of β-catenin22,23. This signaling cascade plays critical roles in diverse cellular processes, including embryonic development, tumorigenesis, and cancer progression24–27. Notably, Huang et al. demonstrated that knockdown of DVL2 and its downstream effector β-catenin suppresses hepatoblastoma cell proliferation and invasion12. In our study, we observed upregulated β-catenin protein expression in PDGF-BB-stimulated VSMCs. Importantly, DVL2 knockdown attenuated this PDGF-induced β-catenin upregulation, suggesting that the DVL2/β-catenin axis may mediate VSMC proliferation and migration during phenotypic modulation.

Despite the success of drug-eluting stents (DES), late restenosis remains a pertinent clinical challenge, primarily driven by the aberrant proliferation and migration of VSMCs28,29. Our study identifies a potential mechanism underlying this pathological VSMCs activity. This mechanism may serve as a potential therapeutic strategy for ISR after extensive mechanistic and translational validation.

This study has several limitations. First, although the A7r5 cells are widely used in cardiovascular disease experiments, we acknowledge that as an immortalized line, it may not fully recapitulate the phenotypic switching of primary VSMCs. Specifically, because A7r5 cells showed a more “synthetic” phenotype characteristic compared with primary VSMCs, our observations regarding phenotypic switching should be interpreted with caution30,31. Future studies utilizing primary VSMCs are essential to validate these observations. Second, the SM22α promoter was employed for VSMC-specific targeting, the absence of co-localization data to confirm the specificity remains a limitation. We cannot entirely exclude the possibility that unintended transduction of other vascular cells (endothelial cells or adventitial fibroblasts). Finally, the phenotypic marker panel is not entirely comprehensive. A more detailed analysis incorporating additional contractile markers (SM-MHC and SM22α) and synthetic markers (vimentin and osteopontin) would provide a more complete understanding of the phenotypic state32. Therefore, our conclusions regarding phenotypic switching should be interpreted given this limitation.

Conclusion

Our findings demonstrated that DVL2 played a critical role in intimal hyperplasia development, as evidenced by its significant upregulation in both PDGF-BB-stimulated VSMCs and the rat carotid artery injury model. Knockdown of DVL2 effectively suppressed PDGF-BB-induced VSMCs proliferation, migration, and phenotypic switching by inhibiting β-catenin signaling pathway activation. These finding indicated that targeting DVL2-mediated Wnt/β-catenin signaling may represent a potential regulator for preventing in-stent restenosis.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (458.9KB, pdf)

Abbreviations

ISR

In-stent restenosis

VSMCs

Vascular smooth muscle cells

DVL2

Dishevelled gene

PDGF-BB

Platelet-derived growth factor subunit B homodimer

α-SMA

α-smooth muscle actin

SM22α

smooth muscle 22α

SM-MHC

smooth muscle myosin heavy chain

PCI

Percutaneous coronary intervention

CAD

Coronary artery disease

MI

Myocardial infarction

miRNAs

microRNAs

SD

Sprague-Dawley

AAV

Adeno-associated virus

H&E

Hematoxylin and Eosin

I/M

Intima/media

DMEM

Dulbecco’s modified Eagle’s medium

FBS

Fetal bovine serum

siRNA

Small interfering RNA

qRT-PCR

Quantitative real-time polymerase chain reaction

PVDF

Polyvinylidene fluoride

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

NCM

Enhanced chemiluminescence

SD

Standard deviation

ANOVA

Analysis of variance

DES

Drug-eluting stent

GSK-3β

Glycogen synthase kinase 3β

OD

Optical density

Author contributions

LL and YJY carried out cell experiments. LL and YW performed animal experiments. JD provided reagents. LL and PYZ wrote the manuscript. All authors reviewed and edited the manuscript.

Funding

This study was supported by the Key Medical Research Program of Jiangsu Provincial Health and Wellness Commission, Grant Number: ZD2021048.

Data availability

The datasets generated and analyzed during the current study are not publicly available due to the Nanjing First Hospital regulations, but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Animal rights

All the experiments involving animals were approved by theInstitutional Animal Care Committee of Nanjing First Hospital, Nanjing Medical University (Protocol #DWSY-23077381) and performed following ARRIVE guidelines (https://arriveguidelines.org).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Kim, M. S. & Dean, L. S. In-Stent Restenosis. Cardiovasc. Ther.29, 190–198. 10.1111/j.1755-5922.2010.00155.x (2011). [DOI] [PubMed] [Google Scholar]
  • 2.Giustino, G. et al. Coronary In-Stent Restenosis. J. Am. Coll. Cardiol.80, 348–372. 10.1016/j.jacc.2022.05.017 (2022). [DOI] [PubMed] [Google Scholar]
  • 3.Madhavan, M. V. et al. Stent-Related Adverse Events > 1 Year After Percutaneous Coronary Intervention. J. Am. Coll. Cardiol.75, 590–604. 10.1016/j.jacc.2019.11.058 (2020). [DOI] [PubMed] [Google Scholar]
  • 4.Cao, G. et al. How vascular smooth muscle cell phenotype switching contributes to vascular disease. Cell. Communication Signal.2010.1186/s12964-022-00993-2 (2022). [DOI] [PMC free article] [PubMed]
  • 5.Alencar, G. F. et al. Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis. Circulation142, 2045–2059. 10.1161/circulationaha.120.046672 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lu, H. et al. Vascular Smooth Muscle Cells in Aortic Aneurysm: From Genetics to Mechanisms. J. Am. Heart Association. 1010.1161/jaha.121.023601 (2021). [DOI] [PMC free article] [PubMed]
  • 7.Akoumianakis, I., Polkinghorne, M. & Antoniades, C. Non-canonical WNT signalling in cardiovascular disease: mechanisms and therapeutic implications. Nat. Reviews Cardiol.19, 783–797. 10.1038/s41569-022-00718-5 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zhuang, Y. et al. Hyperlipidemia induces vascular smooth muscle cell proliferation involving Wnt/β-catenin signaling. Cell. Biol. Int.40, 121–130. 10.1002/cbin.10543 (2015). [DOI] [PubMed] [Google Scholar]
  • 9.Sharma, M., Castro-Piedras, I., Simmons, G. E., Pruitt, K. & Dishevelled A masterful conductor of complex Wnt signals. Cell. Signal.47, 52–64. 10.1016/j.cellsig.2018.03.004 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Rasha, F. et al. Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer. BMC Cancer. 2310.1186/s12885-023-10647-2 (2023). [DOI] [PMC free article] [PubMed]
  • 11.Liu, R. et al. Potential role and prognostic importance of dishevelled-2 in epithelial ovarian cancer. Int. J. Gynecol. Obstet.138, 304–310. 10.1002/ijgo.12218 (2017). [DOI] [PubMed] [Google Scholar]
  • 12.Huang, S. et al. Down-regulation of dishevelled-2 inhibits cell proliferation and invasion in hepatoblastoma. Pediatr. Blood Cancer. 65, e27032. 10.1002/pbc.27032 (2018). [DOI] [PubMed] [Google Scholar]
  • 13.Tulis, D. A. Rat carotid artery balloon injury model. Methods Mol. Med.139, 1–30. 10.1007/978-1-59745-571-8_1 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lawton, J. S. et al. 2021 ACC/AHA/SCAI Guideline for Coronary Artery Revascularization: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation14510.1161/cir.0000000000001038 (2022). [DOI] [PubMed]
  • 15.Huang, C., Zhao, J. & Zhu, Y. Drug-Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation. J. Am. Heart Association. 910.1161/jaha.119.014103 (2020). [DOI] [PMC free article] [PubMed]
  • 16.Shlofmitz, E., Iantorno, M. & Waksman, R. Restenosis of Drug-Eluting Stents. Circulation: Cardiovasc. Interventions. 1210.1161/circinterventions.118.007023 (2019). [DOI] [PubMed]
  • 17.Metcalfe, C. et al. Dvl2 promotes intestinal length and neoplasia in the ApcMin mouse model for colorectal cancer. Cancer Res.70, 6629–6638. 10.1158/0008-5472.CAN-10-1616 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Chistiakov, D. A., Orekhov, A. N. & Bobryshev, Y. V. Vascular smooth muscle cell in atherosclerosis. Acta Physiol. (Oxf). 214, 33–50. 10.1111/apha.12466 (2015). [DOI] [PubMed] [Google Scholar]
  • 19.Furmanik, M. et al. Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification. Circul. Res.127, 911–927. 10.1161/circresaha.119.316159 (2020). [DOI] [PubMed] [Google Scholar]
  • 20.Alexander, M. R. & Owens, G. K. Epigenetic Control of Smooth Muscle Cell Differentiation and Phenotypic Switching in Vascular Development and Disease. Annu. Rev. Physiol.74, 13–40. 10.1146/annurev-physiol-012110-142315 (2012). [DOI] [PubMed] [Google Scholar]
  • 21.Worssam, M. D. et al. Cellular mechanisms of oligoclonal vascular smooth muscle cell expansion in cardiovascular disease. Cardiovascular. Res.119, 1279–1294. 10.1093/cvr/cvac138 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kioussi, C. et al. Identification of a Wnt/Dvl/β-Catenin → Pitx2 Pathway Mediating Cell-Type-Specific Proliferation during Development. Cell111, 673–685. 10.1016/s0092-8674(02)01084-x (2002). [DOI] [PubMed] [Google Scholar]
  • 23.Pai, V. C. et al. ASPM promotes prostate cancer stemness and progression by augmenting Wnt – Dvl-3 – β-catenin signaling. Oncogene38, 1340–1353. 10.1038/s41388-018-0497-4 (2018). [DOI] [PubMed] [Google Scholar]
  • 24.Sarode, P. et al. Reprogramming of tumor-associated macrophages by targeting beta-catenin/FOSL2/ARID5A signaling: A potential treatment of lung cancer. Sci. Adv.6, eaaz6105. 10.1126/sciadv.aaz6105 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Valenta, T., Hausmann, G. & Basler, K. The many faces and functions of β-catenin. EMBO J.31, 2714–2736. 10.1038/emboj.2012.150 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.MacDonald, B. T., Tamai, K. & He, X. Wnt/β-Catenin Signaling: Components, Mechanisms, and Diseases. Dev. Cell. 17, 9–26. 10.1016/j.devcel.2009.06.016 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Clevers, H. & Nusse, R. Wnt/beta-catenin signaling and disease. Cell149, 1192–1205. 10.1016/j.cell.2012.05.012 (2012). [DOI] [PubMed] [Google Scholar]
  • 28.Mori, H. et al. Very Late Pathological Responses to Cobalt–Chromium Everolimus-Eluting, Stainless Steel Sirolimus‐Eluting, and Cobalt–Chromium Bare Metal Stents in Humans. J. Am. Heart Association. 610.1161/jaha.117.007244 (2017). [DOI] [PMC free article] [PubMed]
  • 29.Nakamura, D. et al. Failure Mechanisms and Neoatherosclerosis Patterns in Very Late Drug-Eluting and Bare-Metal Stent Thrombosis. Circulation: Cardiovasc. Interventions. 910.1161/circinterventions.116.003785 (2016). [DOI] [PubMed]
  • 30.Wu, G. et al. Activation of AMP-activated Protein Kinase by Metformin Inhibits Dedifferentiation of Platelet-derived Growth Factor-BB-induced Vascular Smooth Muscle Cells to Improve Arterial Remodeling in Cirrhotic Portal Hypertension. Cell. Mol. Gastroenterol. Hepatol.1910.1016/j.jcmgh.2025.101487 (2025). [DOI] [PMC free article] [PubMed]
  • 31.Chi, J. et al. Primary Culture of Rat Aortic Vascular Smooth Muscle Cells: A New Method. Med. Sci. Monit.23, 4014–4020. 10.12659/msm.902816 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Jain, M. et al. Smooth muscle cell–specific fibronectin-EDA mediates phenotypic switching and neointimal hyperplasia. J. Clin. Invest.130, 295–314. 10.1172/jci124708 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (458.9KB, pdf)

Data Availability Statement

The datasets generated and analyzed during the current study are not publicly available due to the Nanjing First Hospital regulations, but are available from the corresponding author on reasonable request.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES