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Cardiovascular Research Advance Access first published online on March 1, 2008
This version [Corrected Proof] published online on March 25, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn056
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Cocoa procyanidins inhibit expression and activation of MMP-2 in vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP activities

Ki Won Lee1,2,{dagger}, Nam Joo Kang1,{dagger}, Min-Ho Oak3, Mun Kyung Hwang1,2, Jong Hun Kim2, Valérie B Schini-Kerth3,* and Hyong Joo Lee1,*

1 Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea
2 Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
3 Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, Université Louis Pasteur de Strasbourg F-67401, Illkirch, France

* Corresponding author. Tel: +82 2 880 4860; fax: +82 2 873 5095. E-mail address: leehyjo{at}snu.ac.kr(H.J.L)/Tel: +33 3 90 24 41 27; fax: +33 3 90 24 43 13. E-mail address: schini{at}aspirine.u-strasbg.fr(V.B.S.)

Received 15 August 2007; revised 26 February 2008; accepted 27 February 2008

Time for primary review: 28 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC.

Methods and results: Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 µg/mL and 5 µM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1).

Conclusion: Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

KEYWORDS Atherosclerosis; MAP kinase; Matrix metalloproteinase; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
The development of atherosclerotic lesions is characterized by excessive vascular remodelling with accumulation of cells and lipids within the intimal layer of the pathological artery.1 The structural reorganization of the blood vessel wall during atherogenesis is controlled by matrix metalloproteinases (MMPs), which degrade most of the extracellular matrix (ECM).1,2 The essential members of this family in human vascular smooth muscle cells (VSMC) are the gelatinases MMP-2 and MMP-9,2 which are thought to be major MMPs that degrade most collagens in human atherosclerotic plaques.2 MMP-2 is expressed as a latent zymogen, pro-MMP-2, by VSMC, endothelial cells, and macrophages, and is activated by membrane type MMPs (MT-MMPs),2,3 especially MT1-MMP (which is also the most studied). Thus, gelatinases are likely to play an important role in atherosclerotic remodelling as well as in clinical complications of atherosclerosis (e.g. fissure and rupture) that lead to thrombosis by inducing a collagen-poor local environment.

Thrombin is a serine protease enzyme with different actions on blood cells and blood vessels. It regulates platelet aggregation, cascade coagulation, and VSMC activation. Previous studies have indicated that thrombin is a potent activator of pro-MMP-2 in VSMC and that this effect requires MT1-MMP activity in endothelial cells.4 The mitogen-activated protein kinases (MAPKs) including extracellular-signal-regulated protein kinase (ERK) and p38 MAPK are present in the most common signalling pathways known to mediate activation or expression of MMP-2.5 Recent studies have linked thrombin-stimulated migration of VSMC to reactive oxygen species (ROS) production and p38 MAPK activation.6 It is also known that thrombin and activation of the ERK signalling pathway control MMP-2 expression in arterialized vein grafts.5 MAPK kinase (MEK) is a dual-specificity protein kinase that phosphorylates its downstream target ERK on specific threonine and tyrosine residues. Although the proteins have some redundancy in function, the MEK/ERK signalling pathway generally plays a critical role in transmitting signals initiated by angiogenesis or atherosclerosis promoters such as thrombin, platelet-derived growth factor, and cytokines.7,8 However, there has been no report on whether the MEK/ERK signalling pathway is involved in thrombin-induced invasion and migration, and MMP-2 expression in VSMC.

Cocoa and chocolate are excellent sources of dietary phenolic substances, and their major phenolic phytochemicals are procyanidins.9 Epidemiological studies suggested that cocoa consumption exerts beneficial effects on cardiovascular diseases and atherosclerosis.10–12 A recent cohort study demonstrated that constant intake of cocoa can lower blood pressure and reduce the risk of cardiovascular diseases.13 However, the underlying molecular mechanisms and molecular target(s) are poorly understood. In the present work, we studied the possible inhibitory effects of the procyanidin-rich fraction prepared from commercially available cocoa (cocoa procyanidin fraction, CPF) and the main procyanidin compound of cocoa, procyanidin B2, on thrombin-induced expression and activation of pro-MMP-2 using human VSMC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Chemicals
Procyanidin B2 was purchased from Funakoshi. MCDB131, basal medium Eagle, streptomycin/penicillin, insulin, and L-glutamine were purchased from GIBCO BRL. Fetal bovine serum (FBS) was purchased from Gemini Bio-Products; and EGF and FGF were purchased from InVitrogen. The antibodies against phosphorylated MEK (Ser217/221), phosphorylated ERK (Thr202/Tyr204), and total ERK were purchased from Cell Signal Biotechnology. The antibodies against total MEK1 were purchased from Santa Cruz Biotechnology. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and the antibodies against β-actin and thrombin were purchased from Sigma Chemical. The MEK1 and MT1-MMP kit was obtained from Upstate Biotechnology and Amersham, respectively. CNBr-Sepharose 4B, glutathione-Sepharose 4B, and [{gamma}-32P]ATP were purchased from Amersham Pharmacia Biotech, and the protein assay kit was obtained from Bio-Rad Laboratories.

2.2 Preparation of cocoa procyanidin fraction, red wine polyphenolic compounds, and green tea polyphenolic extract
The procedures used to prepare CPF, red wine polyphenolic comounds (RWPCs), and green tea polyphenol extract (GTE) have been described previously.14–16

2.3 Determination of total flavonoid content
The total flavonoid content of CPF was measured by a colourimetric assay method developed by Zhishen et al.17 The assay was performed in five replications for each sample. The total flavonoid content is expressed as milligrams per gram of epicatechin equivalent (ECE).

2.4 Cell culture
Human aortic VSMC originated from normal human tissue were obtained by permission according to the Declaration of Helsinki and supplied by BioWhittaker (Walkersville, USA). They were cultured in monolayers at 37°C in a 5% CO2 incubator in MCDB131 containing 10% FBS, 2 mM L-glutamine, EGF, bFGF, insulin, and streptomycin/penicillin. All experiments were performed with VSMC from passages 5 to 15.

2.5 Cell proliferation assay
To estimate cell proliferation, VSMC (1.5 x 104) were seeded into 96-well plates and cultured for 24 h. The cells were treated with different concentrations of CPF (0–100 µg/mL), procyanidin B2 (0–100 µM), or U0126 (0–10 µM), and their viability was determined by reading the absorbance at 492 nm at 12-h intervals up to 48 h.

2.6 Gelatin zymographic assay for matrix metalloproteinases
VSMC (5 x 105) were plated on 6-well dishes and grown to 90% confluence in 2 mL of growth medium. The cells were maintained in serum-free medium for an additional 24 h with 2 U/mL thrombin alone or together with CPF, procyanidin B2, or U0126. Gelatin zymographic assay was performed as described in our previous study.15 Areas of gelatinase activity were detected as clear bands against the blue-stained gelatin background.

2.7 Invasion assay
VSMC (5 x 104) were resuspended with 2 U/mL thrombin alone or together with CPF, procyanidin B2, or U0126 in 100 µL of medium and placed in the upper part of the Transwell plate. The cells were then incubated for 24 h in a humidified atmosphere of 5% CO2 at 37°C. The ability of VSMCs to invade matrix was determined with a commercial cell invasion assay kit (Chemicon International) as described in our previous study.15

2.8 Wound migration assay
VSMC (5 x 105) were plated on 6-well dishes and grown to 90% confluence in 2 mL of growth medium. The cells were damaged using a 2-mm-wide tip. The cells were treated with 2 U/mL thrombin alone or together with CPF, procyanidin B2, or U0126. The cells were allowed to migrate, and photographs were taken through an inverted microscope (x40 magnification).

2.9 MT1-MMP activity assay
A commercially available MT1-MMP activity assay kit (Amersham) was used for determining the MT1-MMP activity. In brief, solubilized membrane-bound MT1-MMP was bound to a specific antibody coating a 96-well microplate, and activity was assayed with a chromogenic peptide substrate.

2.10 Western blotting
After VSMC (1.5 x 106) were cultured in a 10-cm-diameter dish for 48 h, they were starved in serum-free medium for another 24 h to eliminate the influence of FBS on the activation of kinases. The cells were then treated with CPF (0–30 µg/mL) or procyanidin B2 (0–50 µM) for 1 h before being exposed to 2 U/mL thrombin for 15 min. Western blot analysis was performed as described in our previous study.15

2.11 MEK1 assay
The kinase assays were performed in accordance with the instructions provided by Upstate Biotechnology. The effects of CPF (0–30 µg/mL) and procyanidin B2 (0–30 µM) were evaluated by separately incubating each compound with the reaction mixtures at 30°C for 30 min.

2.12 Pull-down assay
Recombinant MEK1 (2 µg) was incubated with CPF–Sepharose 4B (or Sepharose 4B alone as a control) beads (100 µL, 50% slurry) in reaction buffer comprising 50 mM Tris–HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, 2 µg/mL BSA, 0.02 mM PMSF, and 1x protease inhibitor mixture. After incubation with gentle rocking overnight at 4°C, the beads were washed five times with buffer [50 mM Tris–HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, and 0.02 mM PMSF], and proteins bound to the beads were analyzed by immunoblotting.

2.13 Statistical analysis
Data are expressed as mean ± SD values. One-way analysis of variance was used for comparisons in the experiments with multiple time points and concentrations. Probability value of P < 0.05 was used as the criterion for statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 CPF and procyanidin B2 inhibit thrombin-induced expression and activation of pro-MMP-2 in vascular smooth muscle cells
CPF was extracted from commercial cocoa powder as described in our previous study and contained 413 mg/g ECE flavonoids. We first measured the viability of VSMC treated with CPF or procyanidin B2.14 Treatment with CPF or procyanidin B2 (up to a concentration of 30 µg/mL or 50 µM, respectively) did not affect the viability of VSMC for 48 h (Figure 1A and B). We next examined the inhibitory effects of CPF or procyanidin B2 on thrombin-induced activation of pro-MMP-2 in VSMC. Thrombin activated pro-MMP-2 to MMP-2 in VSMC, and this was inhibited dose-dependently by treatment with CPF at 1, 3, 10, and 30 µg/mL. CPF at 30 µg/mL also inhibited pro-MMP-2 expression without thrombin induction (Figure 1C). The thrombin-induced expression and activation of pro-MMP-2 in VSMC were significantly inhibited by procyanidin B2 even at a concentration of 5 µM, and procyanidin B2 at 50 µM also inhibited basal pro-MMP-2 expression without thrombin induction (Figure 1D). These results indicate that cocoa procyanidins have inhibitory effects on thrombin-mediated activation and expression of MMP-2 in VSMC.


Figure 1
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  		Figure 1 Effects of CPF and procyanidin B2 on cell viability and pro-MMP-2 activity in VSMC. (A and B) CPF and procyanidin B2 did not show cytotoxicity at concentrations of 0–30 µg/mL and 0–50 µM, respectively, in VSMC. VSMC were treated with 10, 30, 50, or 100 µg/mL CPF (A) or with 10, 30, 50 or 100 µM procyanidin B2 (B) for 12, 24, and 48 h. Results are expressed as cell viability relative to untreated controls, as determined from three independent experiments. Data are mean ± SD values: black circle, 12 h; black diamond, 24 h; black square, 48 h. *P < 0.05 vs. untreated control. (C and D) CPF and procyanidin B2 suppressed thrombin-induced expression and activation of pro-MMP-2 in VSMC. VSMC were exposed to either solvent (0.1% ethanol), or 1, 3, 10, or 30 µg/mL CPF (C) or 5, 10, 30, or 50 µM procyanidin B2 (D) for 30 min before the addition of 2 U/mL thrombin for 24 h. Data are representative of three independent experiments that gave similar results.

 
3.2 CPF and procyanidin B2 attenuate thrombin-induced invasion and migration of vascular smooth muscle cells
Previous study has suggested that expression and activation of MMP-2 play pivotal roles in the invasion and migration of VSMC, which are strongly linked to atherosclerosis.18 We next examined whether the inhibition of MMP-2 by CPF and procyanidin B2 is involved in the inhibition of invasion and migration of VSMC. Treatment with CPF (at concentrations of 10 and 30 µg/mL) or procyanidin B2 (at concentrations of 10 and 30 µM) strongly inhibited the thrombin-induced invasion (Figure 2A and B) and migration (Figure 2C and D) of VSMC.


Figure 2
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  		Figure 2 Effects of CPF and procyanidin B2 on thrombin-induced invasion and migration of VSMC. (A and B) VSMC seeded into the inner chamber in serum-free medium were exposed to 0, 10, or 30 µg/mL CPF (A) or 0, 10, or 30 µM procyanidin B2 (B) for 30 min before the addition of 2 U/mL thrombin for 48 h. Results are expressed as the number of invaded cells relative to untreated controls, as determined from three independent experiments. #P < 0.05 vs. untreated control; *P < 0.05 vs. thrombin treatment. (C and D) The confluent VSMC in serum-free medium were exposed to 0, 10, or 30 µg/mL CPF (C) or 0, 10, or 30 µM procyanidin B2 (D) for 30 min before the addition of 2 U/mL thrombin. Thereafter, the widths of injury lines made in cells were measured at 0, 12, 24, and 48 h. Results are expressed as the widths of injury lines relative to untreated controls at 0 h, as determined from three independent experiments. Data are mean ± SD values. Open circle, untreated control; filled circle, only thrombin treatment; open square, thrombin and 10 µg/mL CPF (or 10 µM procyanidin B2) treatment; filled square, thrombin and 30 µg/mL CPF (or 30 µM procyanidin B2) treatment. #P < 0.05 vs. untreated control; *P < 0.05 vs. thrombin treatment.

 
3.3 CPF and procyanidin B2 suppress thrombin-induced MT1-MMP activity in vascular smooth muscle cells
A previous study has shown that the thrombin-induced activation of MMP-2 on the cell surface is mediated by MT-MMPs, and especially by MT1-MMP in endothelial cells.4 This suggests that the inhibitory effects of CPF and procyanidin B2 on thrombin-induced activation of pro-MMP-2 are related to MT1-MMP. Thus, we next examined whether CPF and procyanidin B2 directly inhibited MT1-MMP activity. The data revealed that thrombin increased the activity of MT1-MMP in VSMC, and this was effectively suppressed by CPF or procyanidin B2 (Figure 3A and B). In particular, MT1-MMP activity was inhibited more strongly by CPF than by RWPC. The inhibitory activity of CPF on MT1-MMP activity was similar to that of GTE (Figure 3C).


Figure 3
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  		Figure 3 Effects of CPF and procyanidin B2 on thrombin-induced MT1-MMP activity and comparison of inhibitory effects of CPF, RWPC, and GTE. (A and B) MT1-MMP was extracted from untreated-control and thrombin-treated VSMC using a commercial kit, and thereafter the activity of MT1-MMP was determined in the presence of 0, 1, 3, 10, or 30 µg/mL CPF (A) or 0, 1, 3, 10, or 30 µM procyanidin B2 (B). Results are expressed as MT1-MMP activity relative to untreated controls, as determined from three independent experiments. Data are mean ± SD values. #P < 0.05 vs. untreated control; *P < 0.05 vs. thrombin treatment. (C) MT1-MMP was extracted from controls, RWPC-treated (followed by washing out) VSMC, or GTE-treated (followed by washing out) VSMC, and thereafter the activity of MT1-MMP was determined relative to controls in the absence or presence of RWPC, GTE or CPF (30 µg/mL). Results are expressed as MT1-MMP activity relative to untreated controls. Data are mean ± SD values. *P < 0.05 vs. untreated control.

 
3.4 CPF and procyanidin B2 inhibit thrombin-induced phosphorylation of ERK but not that of MEK in vascular smooth muscle cells
Recent studies have shown that ERK is strongly involved in thrombin-induced MMP-2 activation in human coronary artery smooth muscle cells,5 and that p38 MAPK is strongly involved in thrombin-induced migration of VSMC.6 Thus, we next investigated the effect of CPF on the ERK and p38 MAPK signalling pathway, and found that CPF suppressed thrombin-induced phosphorylation of ERK in VSMC (Figure 4A). However, CPF had no effect on thrombin-induced phosphorylation of p38 MAPK in VSMC (Figure 4A). Similarly, procyanidin B2 suppressed thrombin-induced phosphorylation of ERK in VSMC (Figure 4B). Importantly, CPF and procyanidin B2 did not inhibit thrombin-induced phosphorylation of MEK, an upstream kinase of ERK, indicating that MEK may be a molecular target responsible for the inhibitory effects of CPF and procyanidin B2 on thrombin-induced phosphorylation of ERK in VSMC (Figure 4A and B).


Figure 4
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  		Figure 4 Effects of CPF and procyanidin B2 on thrombin-induced phosphorylation of MEK and ERK, and effects of CPF on thrombin-induced phosphorylation of p38 MAPK. (A) Cells were treated with 10 or 30 µg/mL CPF for 1 h, then stimulated with 2 U/mL thrombin and harvested 15 min later. The levels of phosphorylated ERK and total ERK, phosphorylated p38 MAPK and total p38 MAPK, and phosphorylated MEK and total MEK proteins were then determined by Western blot analysis. (B) Cells were treated with 10, 30, or 50 µM procyanidin B2 for 1 h, then stimulated with 2 U/mL thrombin and harvested 15 min later. The levels of phosphorylated MEK and total MEK, and phosphorylated ERK and total ERK were then determined by Western blot analysis. Data are representative of two independent experiments that gave similar results. Quantification of proteins (p-MEK, p-ERK, or p-p38) was normalized to total proteins (MEK, ERK, or p38) using a Sicon Image (NIH, Bethesda, MD).

 
3.5 CPF and procyanidin B2 suppress MEK1 activity, and directly bind with MEK1
We next investigated the effects of CPF and procyanidin B2 on MEK1 activity to determine whether MEK1 is a target of CPF and procyanidin B2. The data indicated that both CPF and procyanidin B2 strongly suppressed MEK1 activity in a dose-dependent manner (Figure 5A and B), indicating that the inhibition of MMP-2 expression by CPF and procyanidin B2 is associated with the suppression of MEK1 activity and its downstream ERK signalling pathway. To further confirm whether CPF and procyanidin B2 directly interact with MEK1, we next performed pull-down assays with CPF and procyanidin B2. The data revealed that MEK1 bound to CPF–Sepharose 4B beads (Figure 5C, lane 3) and to procyanidin B2–Sepharose 4B beads (Figure 5D, lane 3), but not to Sepharose 4B beads alone (Figure 5C and D, lane 2), indicating that MEK1 could directly bind with CPF or procyanidin B2. Overall, the results indicate that MEK1 may be an important target molecule of CPF and procyanidin B2 for the inhibition of thrombin-induced cell invasion and migration, and MMP-2 expression.


Figure 5
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  		Figure 5 Effects of CPF and procyanidin B2 on MEK1 kinase activity and binding ability with MEK1. (A and B) The MEK1 kinase assay was performed with various concentrations of CPF (1, 3, 10, or 30 µg/mL) (A) or procyanidin B2 (1, 3, 10, or 30 µM) (B) as described in the Materials and Methods, and MEK1 kinase activity is expressed as the percentage inhibition relative to the MEK1 kinase activity of untreated controls. The mean 32P count was determined from three independent experiments, and is expressed as mean ± SD values. *P < 0.05 vs. active MEK1 added. (C and D) MEK1–CPF binding (C) and MEK1–procyanidin B2 binding (D) were confirmed by immunoblotting using an antibody against MEK1: lane 1, MEK1 protein standard served as input control; lane 2, as a negative control, Sepharose 4B was used to pull down MEK1 as described in the Materials and Methods; and lane 3, MEK1 was pulled down using CPF–Sepharose 4B and procyanidin B2–Sepharose 4B affinity beads. Data are representative of two independent experiments that gave similar results.

 
3.6 Activation of MEK is required for thrombin-induced expression and activation of pro-MMP-2, and invasion and migration of vascular smooth muscle cells
Since CPF and procyanidin B2 inhibited the phosphorylation of ERK via binding with MEK1, we further investigated the functional role of MEK activation in the thrombin-induced invasive and migrative phenotypes of VSMC using U0126, which is a specific inhibitor of MEK. The thrombin-induced increases in the invasion (Figure 6A) and migration (Figure 6B) of VSMC were reduced by treatment with U0126. The inhibitory effects of U0126 were not caused by its cytotoxicity because the concentration range used with U0126 did not affect cellular viability (Figure 6C). The thrombin-induced expression and activation of pro-MMP-2 were also inhibited by U0126 at both 5 and 10 µM. U0126 at 10 µM inhibited basal pro-MMP-2 expression without thrombin induction (Figure 6D). These results confirm that activation of MEK is essential for thrombin-induced expression and activation of pro-MMP-2 in VSMC, which affects the invasive and migrative phenotypes of VSMC. These results support that MEK is a major molecular target of CPF for inhibiting invasion and migration, and the activation and expression of pro-MMP-2 in VSMC.


Figure 6
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  		Figure 6 Effects of the MEK inhibitor U0126 on thrombin-induced invasion and migration and the activation of pro-MMP-2 in VSMC. (A) VSMC seeded into the inner chamber in serum-free medium were exposed 0, 5 or 10 µM U0126 for 30 min before the addition of 2 U/mL thrombin for 48 h. Results are expressed as the number of invaded cells relative to untreated controls, as determined from three independent experiments. Data are mean ± SD values. #P < 0.05 vs. untreated control; *P < 0.05 vs. thrombin treatment. (B) VSMC that fully filled dishes containing serum-free medium were exposed to 0, 5, or 10 µM U0126 for 30 min before the addition of 2 U/mL thrombin. Thereafter, the widths of injury lines made in cells were measured at 0, 12, 24, and 48 h. Results are expressed as the widths of injury lines relative to untreated controls at 0 h, as determined from three independent experiments. Data are mean ± SD values: Open circle, untreated control; filled circle, only thrombin treatment; open square, thrombin and 5 µM U0126 treatment; filled square, thrombin and 10 µM U0126 treatment. #P < 0.05 vs. untreated control; *P < 0.05 vs. thrombin treatment. (C) VSMC were treated with 5 or 10 µM U0126 for 12, 24 and 48 h. Results are expressed as cell viability relative to untreated controls, as determined from three independent experiments. Data are mean ± SD values: Filled circle, 12 h; filled diamond, 24 h; filled square. (D) VSMC were exposed to 0, 5 or 10 µM U0126 for 30 min before the addition of 2 U/mL thrombin for 24 h. Data are representative of three independent experiments that gave similar results.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
There are multiple lines of evidence that MMP-2 and MT1-MMP play pivotal roles in ECM degradation, and in the invasion and migration of VSMC.3 Thrombin has been identified as a potential physiological inducer of the formation of MMP-2 in VSMC and endothelial cells.19 Although the mechanisms of activation by thrombin remain unclear, in endothelial cells they are usually categorized as being either dependent on or independent of MT-MMP.20 Thrombin markedly induces pro-MMP-2 activation in the presence of VSMC, and this effect is attributed to MT-MMP. Previous findings indicate that thrombin does not affect either the mRNA or protein expression levels of MT1-MMP.15 Thrombin markedly increases cell-associated MT1-MMP activity in VSMC. Our data demonstrated that CPF and procyanidin B2 exerted a strong inhibitory effect on the thrombin-induced MT1-MMP activity in VSMC. Moreover, MT1-MMP is inhibited more strongly by CPF than by RWPC. The inhibitory activity of CPF on MT1-MMP activity is similar to that of GTE. These findings suggest that CPF and procyanidin B2 suppress the activation of pro-MMP-2 by directly inhibiting MT1-MMP activity, which may in part explain their inhibition of the invasive and migrative phenotypes of VSMC.

It is also known that the formation of MMP-2 requires the proteolytic activity of thrombin and involves a protease-activated receptor-independent mechanism in VSMC. A recent study has shown that the thrombin-induced migration of VSMC is linked to the generation of intracellular ROS by NADPH oxidase via p38 MAPK signals.6 Inhibitors of NADPH oxidase and antioxidants suppress the phosphorylation of p38 MAPK and migration of VSMC. However, in the present study, CPF did not inhibit the thrombin-induced phosphorylation of p38 MAPK, which led us to predict that different mechanisms underlie the suppression of thrombin-induced migration of VSMC by CPF, independent of inhibiting the generation of ROS and the p38 MAPK signalling pathway. MEK1 and MEK2 exhibit 79% amino acid homology and are equally effective at phosphorylating ERK substrates.21 No substrates for MEK1 have been identified other than ERK1 and ERK2, and activated MEK1 catalyzes the phosphorylation of ERK on both a threonine (Thr183) and a tyrosine (Tyr185) residue.21 This tight selectivity, coupled with a unique ability to phosphorylate both threonine and tyrosine residues, indicates that this kinase is essential in integrating signals into the MAPK signalling pathway. The constitutive activation of MEK1 results in induction of angiogenesis and atherosclerosis, while a small-molecule inhibitor of MEK is capable of inhibiting angiogenesis and atherosclerosis in both cell culture and animal models.5 CPF and procyanidin B2 bind to MEK1, inhibit its activity, and subsequently suppress phosphorylation of ERK. Our results also demonstrated that activation of MEK is required for the thrombin-induced expression and activation of pro-MMP-2, and in the invasion and migration of VSMC. Recent reports suggest that most of the mechanisms underlying antiatherosclerosis by phenolic phytochemicals are not related to their direct antioxidant activities but instead are due to the direct binding of the polyphenols to target molecules, including in the inhibition of selected protein kinases, MMPs, and cyclooxyenases.15,22 Our results support that the MEK1 is mainly responsible for the protective effects of CPF and procyanidin B2 on thrombin-induced expression and activation of pro-MMP-2 in VSMC.

Our recent study demonstrated that RWPC inhibited thrombin-induced invasion by directly inhibiting MT1-MMP activity in VSMC.15 However, the compounds responsible for these effects of red wine are still unknown. We found that resveratrol (a well-known bioactive non-flavonoid compound in red wine) did not inhibit the thrombin-induced MT1-MMP activity (unpublished observation). The flavonoid content of red wines is dominated by oligomeric procyanidins, myricetin, and quercetin, with the content of resveratrol being much lower. Our results also suggest that flavonoids including procyanidin B2 may contribute to the inhibition of invasion and migration of VSMC treated with RWPC. Recent epidemiological studies suggested that a regular intake of cocoa exerts beneficial effects on cardiovascular diseases and atherosclerosis.10–13 Overall, the present study indicates that both CPF and procyanidin B2 inhibit the conversion of the inactive form pro-MMP-2 to the active form MMP-2, and that this is partially due to a direct inhibition of its physiological activator MT1-MMP. Both CPF and procyanidin B2 inhibited the expression of pro-MMP-2 by directly inhibiting MEK1, and may have contributed to inhibiting the invasion and migration of VSMC through the ECM degradation. It is notable that the inhibitory effects were significant when CPF and procyanidin B2 were used at low concentrations (1–3 µg/mL and 1–3 µM, respectively), with this level of procyanidin being considered achievable in the blood circulation after the oral ingestion of 80 g of black chocolate.23,24 Thus, our results might partially account for the chemoprotective effects of cocoa on cardiovascular diseases and atherosclerosis.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by research grants from the Biofood Research Program, the Korea Science and Engineering Foundation, Republic of Korea and Technology, Republic of Korea, and the French Ministry of Agriculture (ONIVINS), France.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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