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(Circulation. 2004;110:1861-1867.)
© 2004 American Heart Association, Inc.

Vascular Medicine

Red Wine Polyphenolic Compounds Strongly Inhibit Pro-Matrix Metalloproteinase-2 Expression and Its Activation in Response to Thrombin via Direct Inhibition of Membrane Type 1–Matrix Metalloproteinase in Vascular Smooth Muscle Cells

Min-Ho Oak, PhD^*; Jasser El Bedoui, MS^*; Patrick Anglard, PhD; Valérie B. Schini-Kerth, PhD

From the Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034 (M.-H.O., J.E.B., V.B.S.-K.), and Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM-U 596 (P.A.), Université Louis Pasteur de Strasbourg, France; and Research and Development Center, Yangji Chemicals, An-San, South Korea (M.-H.O.).

Correspondence to Valérie B. Schini-Kerth, PhD, UMR CNRS 7034, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, BP 60024, F-67401 Illkirch, France. E-mail schini{at}aspirine.u-strasbg.fr

Received August 5, 2003; de novo received April 15, 2004; revision received June 9, 2004; accepted June 10, 2004.

	Abstract

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Background— Regular consumption of moderate amounts of red wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation have been involved in atherosclerotic plaque growth and instability. The present study examined whether red wine polyphenolic compounds (RWPCs) inhibit activation of MMP-2, a major gelatinase, in vascular smooth muscle cells (VSMCs).

Methods and Results— Expression of pro-MMP-2 was assessed by Western and Northern blot analyses; MMP-2 activity was assessed by zymography and cell invasion by a modified Boyden’s chamber assay. High levels of pro-MMP-2 and low levels of MMP-2 activity were found in conditioned medium from unstimulated VSMCs. Thrombin induced cell-associated pro-MMP-2 protein expression and MMP-2 activity in conditioned medium of VSMCs. The stimulatory effect of thrombin on MMP-2 activation was prevented by RWPCs in a concentration-dependent and reversible manner. Thrombin markedly increased cell-associated membrane type 1 (MT1)–MMP activity, the physiological activator of pro-MMP-2, and this response was not affected by RWPCs. However, addition of RWPCs directly to MT1-MMP abolished its metalloproteinase activity in a reversible manner. Finally, matrix invasion of VSMCs was stimulated by thrombin, and this response was prevented by RWPCs as efficiently as a broad-spectrum MMP inhibitor.

Conclusions— The present findings demonstrate that RWPCs effectively inhibit thrombin-induced matrix invasion of VSMCs, most likely by preventing the expression and activation of MMP-2 via direct inhibition of MT1-MMP activity. The inhibitory effect of RWPCs on the activation of pro-MMP-2 and matrix degradation might contribute to their beneficial effects on the cardiovascular system.

Key Words: thrombin • metalloproteinases • flavonoid • wine • extracellular matrix

	Introduction

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Epidemiological studies have suggested an inverse relation between moderate wine consumption, particularly red wine, and the risk of coronary heart disease.^1,2 The beneficial effect of red wine on coronary disease might be attributable in part to its ability to retard the progression of early atherosclerotic lesions, as observed in human coronary arteries at childhood, to advanced plaques, which are prone to rupture with superimposed thrombosis, as suggested by studies with experimental models of atherosclerosis.^3–5 Because nonalcoholic wine products also prevent the progression of atherosclerotic lesions, polyphenols present in wine should account, at least in part, for the protective effect of moderate wine consumption.^3–5

The development of atherosclerotic lesions is characterized by excessive vascular remodeling with accumulation of cells and lipids within the intimal layer of the pathological artery.⁶ The structural reorganization of the blood vessel wall during atherogenesis is controlled by matrix metalloproteinases (MMPs), which degrade most of the extracellular matrix.^7,8 The gelatinases MMP-2 and MMP-9 are thought to be major MMPs degrading most collagens in human atherosclerotic plaques.^8–10 MMP-2 is expressed as a latent zymogen, pro-MMP-2, by vascular smooth muscle cells (VSMCs), endothelial cells, and macrophages,¹⁰ and its activation occurs through membrane-type MMPs (MT-MMPs).⁷ Previous studies have indicated that thrombin is a potent activator of pro-MMP-2 in VSMCs and endothelial cells^11,12 and that this effect requires membrane type 1 (MT1)–MMP activity in endothelial cells.^11,12 Thus, gelatinases are likely to play an important role in atherosclerotic remodeling as well as in clinical complications of atherosclerosis, such as fissure and rupture, leading to thrombosis, by inducing a collagen-poor local environment. Therefore, the purpose of the present study was to determine whether red wine polyphenolic compounds (RWPCs) could affect gelatinase expression and activation in VSMCs and the invasion capacity of these cells, and, if so, to elucidate the underlying mechanism.

	Methods

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Preparation of RWPCs
RWPCs in dry powder form from red French wine (Corbières A.O.C.) were provided by Dr M. Moutounet (Institut National de la Recherche Agronomique, Montpellier, France) and analyzed by Dr P.-L. Teissedre (Département d’Oenologie, Université de Montpellier, Montpellier, France). The procedures used to prepare and analyze RWPCs have been described previously.¹³ One liter of red wine produced 2.7 g of RWPCs, which contained 471 mg/g of total phenolic compounds expressed as gallic acid.

Cell Culture
Human aortic VSMCs were obtained from Clonetics and cultured as recommended. All experiments were performed with VSMCs from passages 5 to 15, which were serum deprived for 24 hours.

Gelatin Zymography
MMP-2 and MMP-9 activities in conditioned medium of cultured VSMCs were analyzed by substrate-gel electrophoresis (zymography) with the use of SDS-PAGE (10%) containing 0.1% gelatin, as described previously.¹² Gelatinolytic activity appears as a clear band on a blue background. MMP markers were from Chemicon International.

Western Blot and Northern Blot Analyses
Total proteins (20 µg) were subjected to SDS-PAGE (10%) and blotted on polyvinylidene difluoride membrane. Immunodetection was performed with the use of an antibody directed against either MMP-2 (Chemicon International) or the catalytic domain of MT1-MMP, 3H7,¹⁴ and enhanced chemiluminescence (Amersham). Total cellular RNA from VSMCs was prepared by isothiocyanate and phenol extraction. MMP-2 and MT1-MMP mRNA levels were assessed by Northern blot analysis. RNA (10 µg) was resolved by electrophoresis on agarose gels and transferred to nylon membrane. Blots were hybridized overnight at 42°C with specific ³²P-labeled cDNA probes for MT1-MMP¹⁵ and for MMP-2.¹⁶ Membranes were exposed to Kodak films with intensifying screens (Q-biogen) at –70°C. Autoradiographs were analyzed by scanning densitometry. MMP-2 and MT1-MMP mRNA levels were normalized to the 28S ribosomal RNA levels and expressed in arbitrary units as a fold increase of the signal relative to untreated cells.

Thrombin and MT1-MMP Activity Assays
The serine protease activity of thrombin was assessed with the use of the chromogenic substrate S-2238 (Hemochrom Diagnostica). Optical densities were measured in a spectrophotometer at 405 nm. MT1-MMP activity was determined with the use of a commercial kit (Amersham).

Cell Invasion Assay
The ability of VSMCs to invade matrix was determined with a commercial cell invasion assay kit (Chemicon International).

Statistical Analysis
Results are shown as mean±SEM. Statistical analyses were performed by ANOVA followed by the Fisher protected least significant difference test to compare 2 treatments. A value of P<0.05 was considered statistically significant.

	Results

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Thrombin Stimulates Conversion of Pro-MMP-2 into MMP-2
Human VSMCs released high levels of the latent zymogen pro-MMP-2 into conditioned medium as well as low levels of the active form of MMP-2 and pro-MMP-9 (Figure 1). Thrombin increased the amount of MMP-2 in conditioned medium in a time- and concentration-dependent manner, whereas that of pro-MMP-2 and pro-MMP-9 was only minimally affected (Figure 1). Hirudin, a specific thrombin inhibitor, totally prevented its stimulatory effect, thereby demonstrating the requirement of thrombin catalytic activity (Figure 2A).

View larger version (57K): [in this window] [in a new window]   Figure 1. Thrombin causes the release and activation of pro-MMP-2 in human VSMCs. VSMCs were incubated with different concentrations of thrombin for the indicated time period. The conditioned medium was then analyzed by gelatin zymography. Representative gelatin zymography shows that thrombin activates MMP-2 in a time- (A) and concentration-dependent (B, 2 left panels) manner. Similar findings were obtained in 2 (A) and 3 (B) additional experiments. In B (2 right panels), the effect of direct treatment of conditioned medium containing pro-MMP-2 (obtained after a 24-hour incubation period of untreated VSMCs) with thrombin for either 1 or 24 hours is also shown. Similar findings were obtained in 2 additional experiments. C, Statistical analysis of the stimulatory effect of thrombin on MMP-2 activation in VSMCs after a 24-hour treatment period. Results are shown as mean±SEM of 4 different experiments. *P<0.05, treatment vs control.

View larger version (62K): [in this window] [in a new window]   Figure 2. A, Representative zymography shows the effect of hirudin, a direct inhibitor of thrombin, on thrombin-induced activation of MMP-2 in VSMCs. VSMCs were incubated with hirudin in amounts neutralizing 20 U/mL of thrombin for 30 minutes before the addition of thrombin for 24 hours. The conditioned medium was then analyzed by gelatin zymography. B, Effect of the thrombin receptor agonist TRAP1–7, a proteolytically inactive agonist of PAR-1, on the activation of MMP-2 in VSMCs. Similar observations were obtained in 2 additional experiments.

Next, the possibility that thrombin directly catalyzes the conversion of pro-MMP-2 into active MMP-2 was investigated. Exposure of pro-MMP-2–containing conditioned medium to thrombin in the absence of cells was not associated with the generation of MMP-2 after a 1-hour incubation period, whereas low amounts of active MMP-2 were observed after a 24-hour incubation period at high concentrations (Figure 1B) (3 U/mL of thrombin increased MMP-2/pro-MMP-2 levels by 147±42% and 5 U/mL by 221±35%; n=3).

To determine whether the stimulatory effect of thrombin involves the proteolytic activation of protease-activated receptor-1 (PAR-1), a major thrombin receptor expressed in VSMCs,¹⁷ the effect of TRAP_1–7, a proteolytically inactive agonist of PAR-1, was studied. TRAP_1–7 did not stimulate the formation of MMP-2 in conditioned medium even at high concentrations (Figure 2B). Together, these results indicate that the most significant activation of MMP-2 by thrombin requires the presence of VSMCs and the catalytic activity of thrombin but does not depend on PAR-1 activation.

RWPCs Prevent Thrombin-Induced Activation of Pro-MMP-2
RWPCs prevented the stimulatory effect of thrombin on MMP-2 activation in a concentration-dependent manner, with significant inhibition observed at concentrations >3 µg/mL and complete abolition of the response at concentrations >30 µg/mL (Figure 3A). Moreover, the inhibitory effect of RWPCs was reversible (Figure 3B). RWPCs alone only slightly affected the low levels of pro-MMP-9 and MMP-2 and the high levels of pro-MMP-2 (Figure 3). Because the catalytic activity of thrombin was found to be required for MMP-2 activation, the direct effect of RWPCs on thrombin activity was tested with the use of the chromogenic substrate S-2238. RWPCs did not affect the catalytic activity of thrombin (Figure 3C), indicating that the inhibitory effect of RWPCs is not due to the direct inhibition of thrombin activity. RWPC treatment did not affect VSMCs viability (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 3. RWPCs inhibit thrombin-induced activation of MMP-2 in a concentration-dependent and reversible manner in VSMCs without affecting the catalytic activity of thrombin. A, VSMCs were exposed to either solvent (0.1% ethanol) or different concentrations of RWPCs for 30 minutes before the addition of thrombin for 24 hours. Conditioned medium was analyzed by gelatin zymography. Top, Representative zymograph. Bottom, Corresponding cumulative data. B, VSMCs were exposed to either solvent (0.1% ethanol), RWPCs for 30 minutes, or RWPCs for 30 minutes followed by washout before the addition of thrombin for 12 hours. Similar observations were obtained in 1 additional experiment. C, RWPCs did not affect the catalytic activity of thrombin as assessed with the use of the chromogenic substrate S-2238. Thrombin was incubated with S-2238 (200 µmol/L) in absence or presence of RWPCs for 2 minutes, and thereafter optical densities were measured in a spectrophotometer at 405 nm. Results are shown as mean±SEM of 3 different experiments. *P<0.05, treatment vs thrombin alone.

Effect of Thrombin and RWPCs on Expression of Pro-MMP-2
The possibility that RWPCs and thrombin affect the expression of pro-MMP-2 in VSMCs was examined with the use of Northern and Western blot analyses. Untreated VSMCs have a high steady-state level of MMP-2 mRNA, which was not affected by thrombin for either 1 or 24 hours (Figure 4A and data not shown). RWPCs alone or in the presence of thrombin markedly reduced the expression of MMP-2 mRNA after a 1- or 24-hour incubation period (Figure 4A and data not shown). Consistent with MMP-2 mRNA levels, pro-MMP-2 protein was detected in VSMC lysates, and a 2-fold induction was observed in the presence of thrombin after a 24-hour incubation period (Figure 4B). Both the basal and the thrombin-induced expression of pro-MMP-2 were markedly reduced by RWPCs (Figure 4B).

View larger version (28K): [in this window] [in a new window]   Figure 4. RWPCs inhibit pro-MMP-2 expression in VSMCs. VSMCs were exposed to either solvent (0.1% ethanol) or RWPCs for 30 minutes before the addition of thrombin for 24 hours. Thereafter, the expression of MMP-2 mRNA was assessed by Northern blot analysis (A) and that of pro-MMP-2 protein in cell lysates by Western blot analysis (B). Cumulative data are shown as mean±SEM of 4 (A) and 3 (B) different experiments. *P<0.05, treatment vs respective control.

Effect of Thrombin and RWPCs on Expression of MT1-MMP
Previous studies have suggested that the thrombin-induced activation of MMP-2 on the cell surface is mediated by MT-MMPs¹² and especially by MT1-MMP in endothelial cells.^12,18 Therefore, the possibility that RWPCs could prevent thrombin-induced MMP-2 activation by inhibiting MT1-MMP expression in VSMCs was investigated. Expression of MT1-MMP mRNA was not affected by thrombin (Figure 5A). However, this expression was decreased by RWPCs with or without thrombin by approximately 30% and 26%, respectively (Figure 5A). At the protein level, MT1-MMP was not affected by either thrombin or RWPCs (Figure 5B).

View larger version (40K): [in this window] [in a new window]   Figure 5. RWPCs inhibit MT1-MMP expression and its activity directly. VSMCs were exposed to either solvent (0.1% ethanol) or RWPCs for 30 minutes before the addition of thrombin. Thereafter, the expression of MT1-MMP mRNA was assessed by Northern blot analysis (A) and that of pro-MT1-MMP protein in cell lysates by Western blot analysis (B). Similar observations were made in 2 additional experiments. C, MT1-MMP was extracted from control and thrombin-treated VSMCs, and thereafter the activity of MT1-MMP was determined in the presence of either RWPCs or GM6001. Results are shown as mean±SEM of 3 different experiments performed in triplicate. #P<0.05, treatment vs control; *P<0.05, treatment vs thrombin alone.

In addition, thrombin significantly increased the cell-associated MT1-MMP activity (Figure 5C). This effect was not observed with TRAP_1–7 (200 µmol/L for 24 hours), whereas the stimulatory effect of thrombin was prevented by hirudin (data not shown). Treatment of VSMCs with RWPCs did not affect the basal and thrombin-induced MT1-MMP activity after a 24-hour incubation period (data not shown). However, RWPCs markedly reduced thrombin-induced MT1-MMP activity in a dose-dependent manner when added directly to the MT1-MMP enzymatic assay at concentrations 1 µg/mL (Figure 5C). However, this effect was not as high as that observed with the broad-spectrum MMP inhibitor GM6001 (Figure 5C). This RWPC inhibitory effect was reversible because MT1-MMP activity recovered to 95.4±4.1% (n=5) after 3 sequential changes of the incubation medium to wash out RWPCs.

RWPCs Prevent Thrombin-Induced Extracellular Matrix Invasion by VSMCs
The possibility that RWPCs prevent thrombin-induced extracellular matrix invasion of VSMCs was next examined with the use of a cell invasion assay. VSMCs were stimulated for 72 hours with thrombin with or without either RWPCs or the GM6001 MMP inhibitor. Thrombin was found to stimulate matrix invasion of VSMCs, but this response was markedly reduced by either RWPCs or GM6001, whereas either agent alone did not affect cell invasion (Figure 6).

View larger version (24K): [in this window] [in a new window]   Figure 6. RWPCs prevent thrombin-induced VSMC matrix invasion, as assessed by a cell invasion kit. VSMCs, seeded into the inner chamber in serum-free medium, were exposed to either solvent, RWPCs, or GM6001 for 30 minutes before the addition of thrombin for 72 hours. Thereafter, cells from the inner chamber were removed, and cells on the lower surface of the polycarbonate membrane were stained and counted. Results are shown as mean±SEM of 3 different experiments. *P<0.05, treatment vs thrombin alone.

	Discussion

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Although regular consumption of moderate amounts of red wine has been associated with a decreased risk of coronary disease, the underlying mechanism of this beneficial effect remains unclear. Key components of red wine thought to be responsible for these protective effects include alcohol content and polyphenols. Experimental evidence has indicated that red wine polyphenols are able to prevent oxidation of LDL,¹⁹ activation of platelets,²⁰ and expression of prothrombotic and proatherosclerotic molecules such as monocyte chemoattractant protein-1,²¹ tissue factor,²² and vascular endothelial growth factor.¹³ The present study provides evidence for an additional mechanism by showing that RWPCs prevent the expression and activation of MMP-2, a major MMP expressed in the vascular wall that contributes to the degradation of basement membranes.

In blood vessels, the major cellular sources of MMP-2 are VSMCs, endothelial cells, and macrophages, which constitutively express and release high amounts of the latent enzyme pro-MMP-2 into the extracellular space (Galis et al,¹¹ Zucker et al,¹⁸ Shah et al,²³ and present findings). Potential physiological activators of MMP-2 include thrombin and the plasminogen activator–plasmin system.^11,24 Although the mechanism underlying the stimulatory effect of thrombin is unclear, MMP-2 activation is strictly dependent on its proteolytic activity by a mechanism distinct from PAR activation (Lafleur et al,¹² Zucker et al,¹⁸ and present findings). Previous studies have suggested that reactive oxygen species activate MMP-2 and that thrombin stimulates NADPH oxidase–dependent formation of reactive oxygen species in vascular cells.^25–29 However, in our experimental model of VSMCs, the stimulatory effect of thrombin remains unaffected by antioxidants such as N-acetylcysteine and vitamin C. Moreover, MMP-2 activation is not induced by other potent activators of NADPH oxidase, including angiotensin II and platelet-derived growth factor_AB, or by hydrogen peroxide (data not shown). Therefore, the involvement of a redox-sensitive mechanism in the activation of MMP-2 observed in the present study is very unlikely.

The major novel finding of the present study is that RWPCs strongly reduce the formation of active MMP-2 in response to thrombin in VSMCs. The inhibitory effect of RWPCs cannot be explained by their antioxidant properties or by a direct inhibition of thrombin because its proteolytic activity remains unchanged in the presence of RWPCs. This inhibitory effect is reversible and is detected at concentrations >3 µg/mL. Although the concentration of RWPCs in blood after red wine intake remains unknown, intake of 100 mL of red wine by healthy volunteers caused an increase in the plasma concentration of polyphenolic monomers of 2.5 µg/mL (gallic acid equivalents).³⁰ The inhibitory effect of RWPCs on pro-MMP-2 activation observed in the present study occurs at concentrations that are therefore likely to be reached in blood after moderate consumption of red wine. In addition to the reduced activation of MMP-2, RWPCs decreased the expression of MMP-2 mRNA and that of its cell-associated protein.

The thrombin-induced activation of pro-MMP-2 is markedly accelerated in the presence of vascular cells, and this effect has been attributed to MT-MMPs (Lafleur et al¹² and present findings). Although several MT-MMPs can activate pro-MMP-2, MT1-MMP appears to be its major physiological activator.³¹ MT1-MMP is expressed in VSMCs of normal arteries and balloon-injured arteries and in human atherosclerotic plaques.^32,33 Thus, VSMC-associated MT1-MMP is likely to play a key role in pro-MMP-2 activation in the arterial wall and, in turn, in vascular remodeling in normal and atherosclerotic arteries. The present data indicate that although thrombin did not affect the expression level of MT1-MMP mRNA and protein, it increased markedly its cell-associated activity in VSMCs. The stimulatory effect of thrombin on MT1-MMP activity required the proteolytic activity of thrombin and was independent of PAR-1 activation. In contrast to thrombin, increased MT1-MMP mRNA and protein level were observed in VSMCs in response to several proinflammatory mediators including interleukin-1, tumor necrosis factor-, oxidized LDL, and serum.^31,33

The present findings indicate that exposure of VSMCs to RWPCs decreased the expression of MT1-MMP mRNA. However, this effect was not associated with a reduced MT1-MMP protein level, possibly as a result of its stability and its high basal expression level in VSMCs (Shofuda et al,³¹ Rajavashisth et al,³³ and present findings). In addition, RWPCs directly and effectively inhibited the activity of MT1-MMP, similar to the MMP inhibitor GM6001. Therefore, the prevention of MMP-2 activation by RWPCs is likely to be mediated by the direct inhibition of MT1-MMP activity. Because these effects are reversible, the continuous presence of RWPCs is required to prevent efficiently the degradation of extracellular matrix components. Finally, and more importantly, RWPCs prevented thrombin-induced VSMC invasion as efficiently as a broad-spectrum MMP inhibitor. On the basis of the role of MMPs in cell migration and invasion, this striking effect further supports the potential role of RWPCs in the control of these enzymes.

In conclusion, the present findings indicate that thrombin stimulates matrix invasion of VSMCs, at least in part, by enhancing the activity of cell-associated MT1-MMP, thereby promoting the conversion of pro-MMP-2 into its catalytically active form. Furthermore, RWPCs strongly inhibit VSMC invasion, and this effect is associated with a direct inhibition of MT1-MMP activity and a reduced expression and activation of pro-MMP-2. RWPCs might therefore prevent excessive vascular remodeling and thus contribute to the beneficial effect of RWPCs on the cardiovascular system.

	Acknowledgments

 
This study was supported in part by ONIVINS (French Ministry of Agriculture) and Institut Européen Vin et Santé des Régions Viticoles (France).

	Footnotes

 
*The first 2 authors contributed equally to this work.

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