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

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.

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.