Procyanidin B2-induced LKB1-AMPK activation mitigates vascular smooth muscle cell proliferation through inhibition of mTOR signaling

Hee Young Park; Seul Gi Kim; Gyeong Ju Bae; Jin Young Sung; Hyoung Chul Choi

doi:10.4196/kjpp.25.108

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Park, Kim, Bae, Sung, and Choi: Procyanidin B2-induced LKB1-AMPK activation mitigates vascular smooth muscle cell proliferation through inhibition of mTOR signaling

Original Article

Korean J. Physiol. Pharmacol. 2025;29(5):659-667.

Published online: 1 September 2025

DOI: https://doi.org/10.4196/kjpp.25.108

Procyanidin B2-induced LKB1-AMPK activation mitigates vascular smooth muscle cell proliferation through inhibition of mTOR signaling

Hee Young Park¹, Seul Gi Kim^1,², Gyeong Ju Bae^1,², Jin Young Sung^1,², Hyoung Chul Choi^1,^2,^*

¹Department of Pharmacology, College of Medicine, Yeungnam University, Daegu 42415, Korea

²Senotherapy-based Metabolic Disease Control Research Center, College of Medicine, Yeungnam University, Daegu 42415, Korea

^*Correspondence Hyoung Chul Choi, E-mail: hcchoi@med.yu.ac.kr

Contributed by:

Author contributions: H.Y.P. investigated the study and wrote the manuscript. S.G.K. performed immunohistochemistry and wrote the manuscript. G.J.B. helped with calculations and making figure. J.Y.S. and H.C.C. supervised the study and critically revised the manuscript.

Received 3 April 2025 Revised 28 April 2025 Accepted 29 April 2025

(open-access):

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Vascular smooth muscle cell (VSMC) proliferation contributes to intimal thickening in atherosclerosis and restenosis diseases. As a proanthocyanidin type B, procyanidin B2 (PB2) is abundantly found in cocoa, apples, and grapes and is reported to have vascular protective effects. However, the mechanisms by which PB2 inhibits proliferation of VSMCs are not clearly understood. Therefore, the purpose of this study was to investigate the underlying mechanism of PB2-induced inhibition of cell proliferation in VSMCs. We found that PB2 dose- and time-dependently increased phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) in VSMCs. AMPK is a serine-threonine kinase and serves as a key sensor of cellular energy. PB2 induced LKB1 translocation from nucleus to cytosol which led to AMPK activation. In addition, PB2-induced AMPK activation decreased cell proliferation and cell cycle progression by inhibiting mammalian target of rapamycin signaling pathway. Transfection with LKB1 or AMPK siRNA and transduction of dominant-negative isoforms of the α1 and α2 subunits of AMPK eliminated anti-proliferative effects of PB2. These results demonstrate that PB2 might be a preventive agent for cardiovascular disorders such as atherosclerosis and hypertension.

Keywords: AMP-activated protein kinase, Liver kinase B1, Mammalian target of rapamycin, Procyanidin B2, Vascular smooth muscle cell

INTRODUCTION

Atherosclerosis is one of the most significant causes in vascular dysfunction and is initiated by endothelial dysfunction, which is caused by hypertension, disturbed blood flow, and reactive oxygen species (ROS) [1]. Vascular smooth muscle cells (VSMCs) play a role in arterial wall to maintain vascular function and structure. However, the abnormal proliferation and migration of VSMCs accelerates the formation of atherosclerotic plaque, leading to an important factor in the pathogenesis of vascular disorders including atherosclerosis [2]. Therefore, the inhibition of cell proliferation is a potential therapy for vascular disease.

As a major sensor of cellular energy, AMP-activated protein kinase (AMPK) is a serine-threonine kinase that recognizes the ATP:AMP ratio [3]. It has been reported that AMPK is involved in the regulation of cell metabolism [4,5] and inhibits VSMC proliferation through induction of cell cycle arrest and regulation of mitosis [6 -8]. Furthermore, previous study has indicated that antioxidant effect by AMPK attenuates bleomycin-induced lung fibrosis [9]. AMPK is activated by liver kinase B1 (LKB1) which triggers α-subunit phosphorylation of AMPK [10]. As a main regulator in synthesis of protein and lipid, mammalian target of rapamycin (mTOR) activation induces proliferation and migration, and is inhibited by AMPK activation [11].

Procyanidin is a polyphenol which found abundantly in fruits and vegetables such as cocoa, apples, and grapes [12]. Procyanidin B2 (PB2) is the most ubiquitous of the procyanidin dimers [13]. It is well known that procyanidin has a beneficial effect on blood pressure, insulin resistance, and anti-oxidative activity [14 -16]. Previous study has reported that a polyphenol-rich extract from the mulberry leaf inhibits VSMC proliferation by inducing G₀/G₁ phase arrest [17]. Moreover, procyanidin extracted from grape seed suppresses cell proliferation and ROS production and increases apoptosis in pancreatic cancer cells [18]. Cocoa procyanidins significantly suppress the invasion and migration of VSMCs by inhibiting the activation of mitogen-activated protein kinase kinase and membrane type 1-matrix metalloproteinase (MMP) [19].

Although it has been reported that several procyanidins are involved in cell proliferation and cell cycle progression [20,21], the correlation between PB2 and cell proliferation has not been demonstrated in VSMCs. Furthermore, the underlying mechanism of PB2 to regulate cell proliferation is not well understood. Accordingly, we hypothesized that PB2 might exert beneficial effects through AMPK activation and investigated whether PB2 induces the LKB1-AMPK signaling pathway to inhibit VSMC proliferation.

METHODS

Reagents and antibodies

PB2 was purchased from Sigma-Aldrich. Antibodies against LKB1, p-LKB1 (Ser428), AMPK, p-AMPK (Thr172), p-acetyl-CoA carboxylase (ACC) (Ser79), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), and p-eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) (Ser65) were obtained from Cell Signaling Technology. Antibodies against proliferating cell nuclear antigen (PCNA) and p-mTOR were acquired from Santa Cruz Biotechnology. Compound C was obtained from Calbiochem, and platelet-derived growth factor-BB (PDGF-BB) was purchased from Cell Signaling Technology. AMPK, LKB1, and control siRNAs were obtained from Santa Cruz Biotechnology.

Cell culture

A549 cells (LKB1 deficient lung cancer cell line) were obtained from American Type Culture Collection. To apply primary VSMCs in our study, we performed primary culture using Sprague–Dawley rats. The rats were euthanized using 95% CO₂. The thoracic aortas were then extracted and placed in serum-free, high-glucose Dulbecco's Modified Eagle's Medium (DMEM, WELGENE). Fatty tissues and adventitia were then removed, and the aortas were subsequently cut longitudinally. The lumens were meticulously scoured using cotton swabs to remove the intima, and subsequently chop into 3–5 mm-long pieces. These explanted pieces were then placed on new 10 cm dishes containing DMEM, which had been supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% antibiotics. The dishes were then placed in an incubator, which was set to 5% CO₂/95% air at 37°C, and the explanted pieces were incubated for 3 days. The sprouted VSMCs were then collected and cultured in growth media (DMEM, supplemented with 10% FBS and 1% antibiotics). For experimental purposes, primary VSMCs were utilized from passages 5 to 8, subsequent to achieving 70%–90% confluence.

Western blot analysis

Whole cell extracts were prepared by lysis in a pro-prep protein extract buffer. The protein concentration was measured using a protein assay reagent from Bio-Rad. Equal amounts of protein were mixed with 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and heated for 5 min at 95°C. Subsequently, total protein samples (30 μg) were subjected to SDS-PAGE using 8%–15% gels. The separated proteins were then electrophoretically transferred to a PVDF membrane for 80 min at 100 V. The membranes were then blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated with primary antibodies overnight at 4°C. Following the washing step with phosphate-buffered saline with Tween 20, the membranes were incubated with anti-rabbit or anti-mouse IgG (GeneTex) in 5% non-fat milk for a duration of 1 h at room temperature. The detection of protein bands was accomplished through the utilization of enhanced chemiluminescence (ECL) and ECL Plus Western blot detection reagents (Millipore).

Transfection of siRNA

VSMCs were transfected with siRNA using Lipofectamine 2000 reagent (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. VSMCs were cultured in 6-well plates to approximately 70% confluence and then subjected to transfection with 10 μM control, LKB1, or AMPK siRNAs in Opti-MEM reduced-serum medium (Thermo Fisher Scientific) for a period of 6 h. Thereafter, the medium was changed with growth medium, and the cells were cultured for a further 48 h.

Adenoviral transduction

Adenoviruses expressing the control gene of green fluorescent protein (GFP), a constitutively active form of AMPK (AMPK-CA), or the dominant-negative isoforms of the α1 and α2 subunits of AMPK (AMPK-DN α1 and α2) were amplified in AD293 cells using standard methodologies. VSMCs were transduced with the adenoviruses in serum-free DMEM for a period of 6 h.

Cell proliferation assay

VSMCs were seeded in 24-well plates at a density of 1 × 10⁴ cells/well in growth media. Cells were pretreated with PB2 for 1 h and then were incubated with PDGF-BB (10 ng/ml) for 24 h. Subsequently, 50 μl of 1 mg/mL MTT solution (VWR) was added to each well (0.1 mg/well) and incubated for 4 h at 37°C. The media were removed from the cells, and the formazan crystals within each well were solubilized with 200 μl of dimethyl sulfoxide. A 100 μl aliquot was transferred to a 96-well plate, and cell proliferation was assessed by measuring the absorbances at 570 nm using a microplate reader (Bio-Rad). For cell counting, VSMCs were seeded in 24-well plates at a density of 1 × 10⁴ cells/well and cultured in growth media at 37°C. Following the completion of the treatments, the cells were trypsinized and counted using a hemocytometer.

Flow cytometry for cell cycle analysis

VSMCs were pretreated with PB2 (5, 10, and 20 μM) for 1 h and then stimulated with PDGF-BB (10 ng/ml) for 24 h. The cells were harvested and fixed in 95% ethanol for a duration of 24 h. Following this, the fixed cells were stained with propidium iodide (PI, 50 μg/ml) for a period of 30 min at a temperature of 37°C, allowing PI to enter the cells and stain the nucleus. Prior to the analysis by flow cytometry, the PI-stained cells were filtered using a 5 ml polystyrene round-bottom tube with a cell-strainer cap. Flow cytometry analysis was performed using a FACS Canto instrument (Becton Dickinson), and cell cycle analysis was conducted using CellQuest Pro software.

Immunofluorescence analysis

VSMCs were seeded on coverslips in 35 mm dishes, fixed with 4% formaldehyde (Thermo Fisher Scientific) for 10 min, and permeabilized with 0.2% Triton X-100 (Daejung) for 5 min. Following blocking with 2% bovine serum albumin (GenDEPOT) for 1 h, the cells were incubated with anti-LKB1 (1:125) or p-mTOR (1:100) antibodies overnight at 4°C. The cells were then incubated with a rabbit FITC-conjugated secondary antibody (Thermo Fisher Scientific; 1:100) for 1 h at room temperature. The nuclei were stained with To-Pro-3 (Thermo Fisher Scientific, 1:150) or DAPI (Thermo Fisher Scientific, 1:500). Subsequent to the immunofluorescent staining, the cells were imaged with a confocal microscope (Leica) or a fluorescence microscope (Olympus).

Statistical analysis

Data are expressed as the mean ± standard error of the mean from a minimum of three independent experiments. The differences between the data sets were assessed using one-way analysis of variance followed by Bonferroni's t-test. p values < 0.05 were considered to be statistically significant.

RESULTS

PB2 increased the phosphorylation of AMPK in VSMCs

Treatment with different concentrations of PB2 (5, 10, and 20 μM) for 1 h led to a dose-dependent increase in AMPK phosphorylation (Fig. 1A). As previous studies have suggested that LKB1 is an upstream kinase of AMPK, we examined whether PB2 affected LKB1 phosphorylation. PB2 (5, 10, and 20 μM) treatment for 1 h increased the phosphorylation of not only LKB1 but also ACC, which is located downstream of AMPK in dose-dependent manner (Fig. 1C). Moreover, compared with the control, PB2 (20 μM, 1 h) caused a drastic increase in the phosphorylation of LKB1, AMPK, and ACC (Fig. 1B, D). Taken together, these results show that PB2 dose- and time-dependently induced the activation of AMPK.

PB2 induced AMPK activation through LKB1 in VSMCs

To confirm whether LKB1 is an upstream kinase of PB2-induced AMPK activation, we used A549 cells which are naturally LKB1-deficient [22]. PB2 induced AMPK phosphorylation in VSMCs, but not in A549 cells (Fig. 2A). Next, we conducted transfection with control or LKB1 siRNA in VSMCs. As shown in Fig. 2B, LKB1 siRNA-transfected cells inhibited the phosphorylation of LKB1, AMPK, and ACC. It has been reported that LKB1, which is mostly localized in the nucleus under normal condition, is translocated to cytoplasm and it means activated LKB1 [23,24]. Therefore, we investigated whether PB2 induced LKB1 translocation from the nucleus to the cytosol, leading to increased AMPK activation. VSMCs were stained with anti-LKB1 (green) and To-Pro-3 (red). The results showed that enhanced cytosolic localization of LKB1 is responsible for PB2-stimulated AMPK activation in VSMCs (Fig. 2C). In addition, PB2 inhibited PDGF-BB-induced cell proliferation, but this was restored by transfection with LKB1 siRNA (Fig. 2D). Taken together, these results suggest that PB2 induces the activation of the LKB1-AMPK signaling pathway in VSMCs.

PB2 attenuated cell proliferation and cell cycle progression in VSMCs

First, we examined whether PB2 regulated PDGF-BB-induced cell proliferation in VSMCs. Compared with the controls, PDGF-BB (10 ng/ml) alone significantly induced cell proliferation. However, PB2 decreased this effect in a dose-dependent manner (Fig. 3A). Similarly, cell counting data showed that PB2 significantly inhibited PDGF-BB-induced cell proliferation (Fig. 3B). We next investigated the effect of PB2 on cell cycle progression using flow cytometry. As shown in Fig. 3C, D, PB2 dose-dependently induced cell cycle arrest in G₀/G₁ phase. Furthermore, we examined the expression of PCNA by western blot analysis. In contrast to PDGF-BB-treated cells, PB2 decreased the expression of PCNA (Fig. 3E). These data show that PB2 has anti-proliferative effects and inhibits cell proliferation by increasing G₀/G₁ phase arrest in VSMCs.

Inhibition of AMPK activation decreased the anti-proliferative effect of PB2

mTOR promotes cell proliferation and cell cycle progression by activating the downstream proteins, p70S6K and 4EBP1 [11]. To further demonstrate the relationship between PB2 and AMPK, cells were transfected with control or AMPK siRNA (Fig. 4) or were transduced with an adenovirus-mediated overexpression system (Fig. 5). As shown in Fig. 4A, B, PB2-activated AMPK decreased the mTOR signaling pathway. However, transfection with AMPK siRNA restored the mTOR signaling pathway compared with control siRNA-transfected cells. In contrast to the GFP, the transduction of AMPK-DN α1 and α2 not only inhibited the PB2-induced AMPK phosphorylation but also increased the mTOR signaling pathway (Fig. 5A, B). Next, we investigated whether PB2-induced AMPK activation regulates the VSMC proliferation. Although treatment of PB2 decreased PDGF-BB-induced cell proliferation, transfection with AMPK siRNA restored the cell proliferation (Fig. 4C). Moreover, compared with adenovirus-GFP, the transduction of AMPK DN α1 and DN α2 increased the cell proliferation (Fig. 5C). Finally, immunofluorescence analysis was consistent with the results of Western blot analysis (Fig. 4D). These data suggest that PB2 has anti-proliferative effect through the AMPK-mTOR signaling pathway in VSMCs.

DISCUSSION

It has been proved that procyanidins induce anti-cancer effect in various cancer cells [25 -27] but cytotoxic effect is not reported on normal cells [28,29]. As a promising anti-cancer agent, the anti-proliferative and cell cycle arrest effects of PB2 have not been demonstrated. Previous studies have been indicated that PB2-induced Nrf2 upregulation ameliorates endothelial dysfunction and angiogenesis in human umbilical vein endothelial cells [30] and mitigates oxidative stress in epithelial cells and nucleus pulposus cells [31,32]. Furthermore, PB2 has an anti-inflammatory effect via PPARγ-dependent signaling pathway in macrophages and human hepatocytes [33,34]. Although it is well known that PB2 has many effects on different cell types, there are few reports regarding the effect of PB2 in VSMCs. Therefore, the purpose of this study is to investigate the effect and underlying mechanism of PB2 in VSMCs. Similar to our results, previous studies have confirmed that PB2 treatment alleviated high glucose-induced mitochondrial dysfunction and apoptosis through AMPK-SIRT1-PGC-1α signaling pathway in podocytes [35] and promotes skeletal slow-twitch myofiber gene expression by AMPK activation [36,37].

Previous studies have demonstrated that excessive cell proliferation, migration, and biophysical stress of VSMCs play a major cause in development of vascular diseases and complications [38,39]. mTOR which is activated by growth factor such as PDGF-BB regulates cell growth and cell cycle progression by inducing the phosphorylation of p70S6K and 4EBP1 [11,40]. In our previous studies, AMPK activation suppressed protein synthesis and cell growth through downregulation of the mTOR signaling pathway [41,42]. Similarly, present study showed that PB2 activated the LKB1-AMPK signaling pathway which inhibited PDGF-BB-induced cell proliferation and cell cycle progression by inhibiting the mTOR signaling pathway.

In previous studies, procyanidin extract dose-dependently inhibited the cell growth by inducing cell cycle arrest at G₁ phase in human bladder cancer cells [43]. Moreover, procyanidin inhibited cell proliferation by apoptotic induction, and decreased invasiveness by suppressing activation of MMP-2 or MMP-9 in pancreatic carcinoma cells [44]. Similarly, our study determined that PB2 attenuated cell proliferation and cell cycle progression via LKB1-AMPK signaling pathway in VSMCs.

In present study, we demonstrated that PB2 activated the LKB1-AMPK signaling pathway to suppress VSMC proliferation, and the effect of PB2 did not show in LKB1-deficient A549 cells. PB2 dose-dependently decreased PDGF-BB-induced cell proliferation and induced cell cycle arrest in G₀/G₁ phase. Finally, PB2-induced AMPK activation inhibited the mTOR signaling pathway which regulated cell proliferation and cell cycle progression. Moreover, the inhibition of the LKB1-AMPK signaling pathway using transfection with AMPK siRNA or transduction of AMPK DN α1 and DN α2 restored PDGF-BB-induced activation of the mTOR signaling pathway. These results suggest that PB2 directly affects AMPK activation, resulting in inhibited VSMC proliferation.

In conclusion, PB2 inhibits PDGF-BB-induced cell proliferation and cell cycle progression in VSMCs. These effects are due to the inhibition of the mTOR signaling pathway through PB2-induced activation of the LKB1-AMPK signaling pathway. Furthermore, our findings provide evidence of the protective effect of PB2 against vascular proliferative disorders such as atherosclerosis.

ACKNOWLEDGEMENTS

None.

Notes

FUNDING

This work was supported by the Medical Research Center Program (2022R1A5A2018865) through the National Research Foundation of Korea (NRF) funded by the Korean government.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

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Fig. 1

The effect of procyanidin B2 (PB2) on the activation of AMP-activated protein kinase (AMPK) in vascular smooth muscle cells.

(A, C) Cells were incubated with different concentrations of PB2 (5, 10, and 20 μM) for 1 h. (B, D) Cell were incubated with PB2 (20 μM) for various times (1, 6, and 12 h). The protein levels of p-liver kinase B1 (LKB1), p-AMPK, AMPK, and p-acetyl-CoA carboxylase (ACC) were determined by western blot analysis. Values are mean ± standard error of the mean (n = 3); *p < 0.05 vs. control (0 μM PB2 or time 0).

Fig. 2

The effect of procyanidin B2 (PB2) through liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) signaling pathway in vascular smooth muscle cells (VSMCs).

(A) A549 and VSMCs were treated with PB2 (20 μM) for 1 h. Protein levels of p-LKB1, LKB1, p-AMPK, and AMPK were determined by Western blot analysis. (B) After transfection with control or LKB1 siRNA, cells were pretreated PB2 (20 μM, 1 h) and then incubated with platelet-derived growth factor-BB (PDGF-BB) (10 ng/ml, 24 h). (C) Cells stained with anti-LKB1 (green) and To-Pro-3 (red) were observed under a confocal laser-scanning microscope. Yellow staining indicates the overlap of the two colors (×200). (D) Transfected cells with control or LKB1 siRNA were measured cell proliferation by MTT assay. Values are mean ± SEM (n = 3); *p < 0.05 vs. control, **p < 0.05 vs. PDGF-BB alone, #p < 0.05 vs. PDGF-BB + PB2.

Fig. 3

The procyanidin B2 (PB2)-mediated regulation of cell proliferation and cell cycle progression in vascular smooth muscle cells.

Cells were pretreated with PB2 (5, 10, and 20 μM) for 1 h and were incubated with platelet-derived growth factor-BB (PDGF-BB) (10 ng/ml) for 24 h. (A) Cell proliferation was determined by MTT assay and (B) cell counting. (C, D) Cell cycle progression was examined by FACS analysis. PB2 induced cell cycle arrest. (E) Expression of proliferating cell nuclear antigen (PCNA) was determined by Western blot analysis. Representative results are shown from three independent experiments. *p < 0.05 vs. control, **p < 0.05 vs. PDGF-BB alone.

Fig. 4

The inhibitory effect of AMP-activated protein kinase (AMPK) siRNA transfection on procyanidin B2 (PB2)-decreased cell proliferation.

Cells were transfected with control or AMPK siRNA for 48 h. After pre-treatment with PB2 (20 μM) for 1 h, cells were stimulated with platelet-derived growth factor-BB (PDGF-BB) (10 ng/ml) for 24 h. (A, B) Results of Western blot analysis indicated that AMPK activation inhibited the mammalian target of rapamycin (mTOR) signaling pathway. (C) PB2-activated AMPK decreased cell proliferation. (D) Cells were stained with anti-p- mTOR (green) and DAPI (blue). PDGF-BB-induced mTOR phosphorylation was inhibited by PB2 and transfection with AMPK siRNA restored the mTOR phosphorylation. Representative results are shown from three independent experiments (×200). *p < 0.05 vs. control, **p < 0.05 vs. PDGF-BB alone, #p < 0.05 vs. PDGF-BB + PB2.

Fig. 5

The regulation of procyanidin B2 (PB2) on mammalian target of rapamycin (mTOR) signaling pathway and cell proliferation through AMP-activated protein kinase (AMPK).

Cells were transduced with adenovirus-green fluorescent protein (GFP), constitutively active form of AMPK (AMPK-CA), or dominant-negative isoforms of the α1 and α2 subunits of AMPK (AMPK-DN α1 and α2) for 6 h. After pre-treatment with PB2 (20 μM) for 1 h, cells were stimulated with platelet-derived growth factor-BB (PDGF-BB) (10 ng/ml) for 24 h. (A, B) Results of western blot analysis indicated that PB2-activated AMPK inhibited the mTOR signaling pathway. (C) PB2 decreased cell proliferation through AMPK activation. Representative results are shown from three independent experiments. *p < 0.05 vs. control, **p < 0.05 vs. PDGF-BB alone, #p < 0.05 vs. PDGF-BB + PB2.

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