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(Circulation. 2002;105:2404.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Red Wine Polyphenols Inhibit Vascular Smooth Muscle Cell Migration Through Two Distinct Signaling Pathways

Katsuya Iijima, MD, PhD; Masao Yoshizumi, MD, PhD; Masayoshi Hashimoto, MD, PhD; Masahiro Akishita, MD, PhD; Koichi Kozaki, MD, PhD; Junya Ako, MD; Tokumitsu Watanabe, MD; Yumiko Ohike, MD; Bokyung Son, MD; Jing Yu, MD; Koichi Nakahara, PhD; Yasuyoshi Ouchi, MD, PhD

From the Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, and Institute for Fundamental Research (K.N.), Suntory Research Center, Suntory Ltd, Osaka, Japan.

Correspondence to Yasuyoshi Ouchi, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail youchi-tky{at}umin.ac.jp

	Abstract

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Background— Red wine polyphenols (RWPs) have been shown to have an antiatherogenic activity mainly through antioxidative effects on LDL oxidation. Although vascular smooth muscle cell (SMC) migration is critical to atherosclerosis formation, the effect of RWPs on SMC migration has not been elucidated. In this study, we investigated whether RWPs could affect the migration of cultured SMCs stimulated by growth factors.

Methods and Results— RWP concentration dependently inhibited platelet-derived growth factor (PDGF)-BB–induced and serum-induced SMC migration in wounding assay and Boyden chamber assay. However, these inhibitory effects of RWPs were not seen in serum-stimulated vascular endothelial cell migration in either assay. Moreover, specific inhibitors of phosphatidylinositol-3' kinase (PI3K) and p38 mitogen-activated protein kinase (p38^MAPK), but not of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), reduced PDGF-BB–induced SMC migration. To elucidate the signaling mechanism underlying the RWP effects, we investigated the effects of RWPs on the activity of PI3K and the phosphorylation of MAPK pathways in PDGF-BB–stimulated SMCs. RWPs inhibited the PI3K activity and p38^MAPK phosphorylation, but not ERK1/2 phosphorylation, in a concentration-dependent manner. Moreover, the phosphorylation of MKK3/6, an upstream kinase of p38^MAPK, was also inhibited by RWP treatment in a concentration-dependent manner, suggesting that the inhibitory effect of RWPs on the p38^MAPK pathway works upstream of MKK3/6. The concentration-effect relationship of RWPs necessary for the inhibition of PI3K and p38^MAPK pathways was similar to that of cell migration assays.

Conclusions— RWPs inhibit the SMC migration through the inhibition of 2 distinct signaling pathways and thus exert antiatherogenic actions.

Key Words: muscle, smooth • endothelium • atherosclerosis

	Introduction

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Lower rates of mortality from coronary heart disease have been observed in the French population despite its having a high-cholesterol diet. Red wine has been assumed to account for the phenomenon, which is known as the "French paradox."¹ Recently, it has been demonstrated that red wine polyphenols (RWPs) have various antiatherogenic effects, such as antioxidation of LDL-cholesterol,^2,3 inhibition of adhesion molecule expression in cytokine-stimulated vascular endothelial cells (ECs), ⁴ inhibition of platelet aggregation,⁵ induction of NO release, and vasorelaxation.⁶

It has been shown that development of atherosclerosis is characterized by EC dysfunction, proliferation and migration of vascular smooth muscle cells (SMCs), and increased extracellular matrix deposition.⁷ SMC migration is believed to play a major role in the pathogenesis of many vascular diseases, including progressive intimal thickening after coronary intervention.⁸ It has been previously reported that activation of the phosphatidylinositol 3'-kinase (PI3K) pathway is a rapid response to platelet-derived growth factor (PDGF), one of the most potent chemoattractants for SMCs, and is implicated in cellular motility, such as actin reorganization and membrane ruffling, in several cell types, including SMCs.^9,10 Moreover, it has been demonstrated recently that activation of mitogen-activated protein kinase (MAPK) pathways also plays a role in cell migration. Specifically, it has been reported that p38^MAPK, one of the MAPK pathways, may mediate the migratory action of tracheal SMCs in response to PDGF.¹¹

We have recently shown that RWPs inhibit SMC proliferation and that the downregulation of a cell-cycle regulator, cyclin A, is at least in part responsible for this growth inhibition.¹² However, the effect of RWPs on SMC migration is not known. Thus, in the present study, we investigated whether RWPs could affect PDGF-BB–induced SMC migration and activation of intracellular signaling pathways that are known to mediate cell migration.

	Methods

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Preparation of Red Wine Polyphenolic Compound
RWPs were extracted from pulverized red wine (7600 mL in total; Suntory Co) by adsorption chromatography using a Diaion HP-20 column as described.¹² The total RWP fraction (24 g total) was obtained by eluting the column with 100% ethanol. This compound was dissolved in 50% ethanol using ultrasonication. The effects of RWPs on our experiments were compared with those of ethanol at a final concentration of 0.5% as vehicle.

Cell Culture
Rat aortic SMCs (RASMCs) were isolated from Sprague-Dawley rats, and human aortic SMCs (HASMCs) were purchased (BioWhittaker, Inc). In cell migration assays, migration of SMCs was compared with that of serum-stimulated ECs, bovine carotid ECs (BCECs), or human umbilical vascular endothelial cells (HUVECs). All cells were subcultured as described.¹²

Cell Migration Assay
Two migration assays were performed. For the monolayer-wounding cell migration assay (wounding assay), SMCs that had been grown to confluence in 6-well culture plates were subjected to wounding as previously described.¹³ The cells were preincubated with serum-free medium for 24 hours and with RWPs at final concentrations of 1 to 100 µg/mL for 10 hours. Cell layers were scraped with a sterile single-edged razor blade and reincubated with RWPs at each concentration and then stimulated with PDGF-BB (10 ng/mL; human recombinant, Sigma) or 10% serum. After 30 hours of incubation at 37°C, the cells were fixed and stained with Giemsa stain. The fixed cells were microscoped, and the number of cells migrated across the regions of the wound edge was counted. In addition, to investigate the effect of RWPs on EC migration, the migration of 10% serum-stimulated BCECs or HUVECs was examined.

In the second assay, migration of SMCs or ECs briefly exposed to chemoattractant and RWPs was assayed using a 96-well modified Boyden chamber housing a collagen-precoated polycarbonate filter with 8.0-µm pores, as previously described.¹⁴ Lower chambers were filled with each chemoattractant, PDGF-BB (0.1 to 20 ng/mL) or 10% serum, and RWPs (1100 µg/mL) in serum-free medium. Under this condition, the duration of RWP treatment was the same as that of the chemoattractant. An equal number of cells (5x10⁵ cells/mL) in 225 µL was loaded into the upper chamber. After incubation for 6 hours, migrating cells on the lower surface of the filter were determined by counting 4 high-power (x200) fields of constant area per well. Moreover, the effect of RWPs on cell migration induced by PDGF-BB in both the upper and lower chambers, which represents chemokinesis, was also investigated. To examine whether the signaling pathway of PI3K or MAPK plays a role in SMC migration, the cells were pretreated with specific inhibitors, wortmannin (PI3K inhibitor; Sigma), SB203580 (p38^MAPK inhibitor; Sigma), or PD98059 (MEK1 inhibitor; New England BioLabs) for 30 minutes, and the inhibitors were added again in the upper and lower chambers using the Boyden chamber assay.

PI3K Activity
The PI3K activity was assayed by the method described with slight modifications.¹⁵ After serum-starved SMCs were pretreated with RWPs for 10 hours, the cells were stimulated by PDGF-BB for 10 minutes, and then cell lysates were incubated with anti-phosphotyrosine antibody (PY-20) for 2 hours and coupled with protein A-Sepharose (Amersham Pharmacia Biotech AB) for 1 hour at 4°C. The immunoprecipitate was washed and resuspended in 50 µL PI3K buffer (10 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, 1 mmol/L EDTA, and 100 µmol/L Na₃VO₄), and 10 µg of phosphatidylinositol was added. The reaction was started by the addition of [-³²P] ATP, and the samples were incubated at 30°C for 15 minutes. The reaction was stopped by the addition of 20 µL of 8N HCl and 160 µL chloroform methanol (1:1). The lower phase containing phospholipids was recovered and spotted on silica gel thin-layer chromatography plates (Gel-60, Merck), impregnated with 1% (wt/vol) potassium oxalate and allowed to dry before sample application, and developed in a mixture of chloroform, methanol, 28% NH₃, and water (120:94:4:23.2 mL). The radioactivity on the dried plate was visualized and quantified by a Phospho-imaging analyzer.

MAPK Phosphorylation
After serum-starved HASMCs were pretreated with RWPs for 10 hours, the cells were stimulated by PDGF-BB for 10 minutes. Total protein extracts (20 µg/lane) were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter. To assess the activation of 3 distinct MAPK pathways, ERK1/2, SAPK-1/JNK, and p38^MAPK, the total and phosphorylated proteins were detected with each specific MAPK antibody (New England BioLabs). Moreover, to examine the activation upstream of the p38^MAPK pathway, antiphosphorylated MKK3/6 antibody was used. Bound proteins were visualized with enhanced chemiluminescence reagents (Amersham).

Cell Proliferation and DNA Synthesis
Serum-starved subconfluent HASMCs plated in 24-well plates (2 cm²/well) were pretreated with RWPs for 10 hours. For cell proliferation assay, the cells were stimulated with PDGF-BB (10 ng/mL) for 60 hours, and then cell number was measured. For DNA synthesis assay, the cells were stimulated with PDGF-BB (10 ng/mL) for 22 hours and then were labeled with [methyl-³H]-thymidine (Du-Pont NEN) at 1 µCi/mL for 3 hours. After the cells had been labeled, incorporated [³H]-thymidine in the cells was extracted in 0.2N NaOH and measured.

Statistical Analysis
When statistically significant, effects were found by ANOVA analysis (Newman-Keuls test). A value of P<0.05 was considered significant. All data in the text and figures are expressed as mean±SEM.

	Results

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PDGF-BB as well as serum-stimulated migration of RASMCs in the wounding assay and pretreatment with RWPs showed potent inhibition of migration (Figure 1, top). The results of quantitative assessment in this assay demonstrated that RWPs inhibited PDGF-BB–induced or serum-induced migration of RASMCs in a concentration-dependent manner (Figure 1, bottom). In contrast, RWPs did not affect the migration of BCECs stimulated with 10% serum. RWPs also inhibited the migration of HASMCs stimulated with serum in a similar concentration-dependent manner but did not inhibit migration of serum-stimulated HUVECs (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 1. Effects of RWPs on cell migration in wounding assay. Serum-starved RASMCs or BCECs that had been grown to confluence were pretreated with RWPs (1 to 100 µg/mL) for 10 hours. Cell layers were scraped with a razor blade, and the cells were reincubated with RWPs and growth stimulator, PDGF-BB (10 ng/mL) or 10% serum. After a 30-hour incubation, the number of migrated cells was counted. Results of quantitative assessment are shown as mean±SEM from 4 independent wells in the lower panel. *P<0.01 vs control treated with PDGF-BB or serum and without RWPs.

Cell migration can be generally characterized by the following 3 forms: (1) random motion, which occurs in the absence of any stimulus; (2) chemokinesis, which is a random motion that is influenced by a stimulus; and (3) chemotaxis, which is directed motion toward a gradient of a stimulus.¹⁶ Growth factor–induced migratory action consists of chemokinesis and chemotaxis. Because the incubation time in the wounding assay was 30 hours, newly divided cells may result in the increase in migrated cells. We additionally performed a Boyden chamber assay to examine the effect of RWPs on cell migration for 6 hours. As shown in Figure 2A, stimulation of RASMCs with PDGF-BB in the lower chamber showed a concentration-dependent induction of cell migration, which presents the chemotactic effect, and RWPs exhibited potent inhibitory effects on the PDGF-BB–induced chemotaxis of RASMCs (58%, 15%, and 4% of the control with 10 ng/mL PDGF-BB at RWP concentrations of 10, 30, and 100 µg/mL, respectively). These findings suggest that RWPs exert antimigratory effects on SMCs even with a short exposure. Moreover, RWPs concentration-dependently inhibited PDGF-BB–induced chemotaxis of HASMCs (Figure 2B). This result suggests that RWPs exert an inhibitory effect on SMC chemotaxis regardless of the species. In contrast, RWPs did not inhibit the chemotaxis of BCECs stimulated with 10% serum, even at the highest concentration (100 µg/mL) of RWPs (Figure 2C).

View larger version (43K): [in this window] [in a new window]   Figure 2. Effects of RWPs on cell migration in Boyden chamber assay. A, Effects of brief exposure (6 hours) to RWPs on migration of RASMCs. RASMCs resuspended in serum-free medium were plated in the upper chamber. Chemoattractant solution containing PDGF-BB with RWPs was placed in the lower chamber. After a 6-hour incubation, cells that had migrated to the lower surface were stained and counted. Results are shown as mean±SEM from 4 independent wells. *P<0.05 vs control treated with PDGF-BB at each concentration and without RWPs. B and C, Effects of RWPs on the migration of PDGF-BB (10 ng/mL)–stimulated HASMCs or 10% serum-stimulated BCECs were examined using the same method. *P<0.05 vs control treated with chemoattractant and without RWPs.

To elucidate whether each intracellular signaling pathway, PI3K or MAPK, mediates PDGF-BB–stimulated SMC migration, we investigated the effect of a specific inhibitor of each pathway. Treatment of RASMCs with wortmannin or SB203580 significantly decreased the migration (chemotaxis) in a concentration-dependent manner (Figure 3A). In contrast, PD98059 did not show any significant effects on the SMC migration. Moreover, simultaneous treatment with wortmannin and SB203580 showed an additional inhibition of SMC migration in our experimental conditions (Figure 3B). These findings suggest that the pathways of both PI3K and p38^MAPK, but not of ERK1/2, are positively implicated in PDGF-BB–induced SMC migration.

View larger version (26K): [in this window] [in a new window]   Figure 3. A, Effects of signaling inhibitors on PDGF-BB–stimulated SMC migration. After resuspended HASMCs in serum-free medium were preincubated with each inhibitor, wortmannin (WN), SB203580 (SB), or PD98059 (PD), for 30 minutes, PDGF-BB (10 ng/mL)–stimulated SMC migration was examined using Boyden chamber assay. B, Additional effects of each inhibitor on PDGF-BB–stimulated SMC migration. HASMCs were preincubated with WN (50 µmol/L), SB (25 µmol/L), or PD (25 µmol/L) for 30 minutes. *P<0.05 vs control treated with PDGF-BB and without inhibitors. P<0.05 vs control treated with PDGF-BB and each inhibitor, WN or SB. C, Effects of RWPs (30 µg/mL) on SMC migration stimulated with PDGF-BB (10 ng/mL) in the lower chamber only (chemotaxis) or in both the upper and lower chambers (chemokinesis) were examined using a Boyden chamber method. *P<0.05 vs control treated with PDGF-BB in the lower chamber. P<0.05 vs control treated with PDGF-BB in both the upper and lower chambers.

Potent SMC chemotaxis induced by PDGF-BB in the lower chamber was inhibited by RWPs (30 µg/mL) in the lower chamber, and this inhibition was enhanced by addition of RWPs in the upper chamber. RWPs also inhibited the random motion (chemokinesis) induced by PDGF-BB in both the upper and lower chambers (Figure 3C).

The effect of RWPs on the lipid kinase activity of PI3K in PDGF-BB–stimulated VSMCs was examined (Figure 4A). Stimulation of RASMCs or HASMCs with PDGF-BB significantly increased PI3K activity, and RWP pretreatment inhibited PI3K activity in a concentration-dependent manner. The effects of RWPs on the phosphorylation of each MAPK pathway, ERK1/2, SAPK-1/JNK, and p38^MAPK, were examined (Figure 4B). RWP pretreatment inhibited the p38^MAPK activation to 28%, 5%, and 3% of the control at concentrations of 10, 30, and 100 µg/mL, respectively. Surprisingly, the phosphorylation of other pathways, ERK1/2 and SAPK1/JNK, was not inhibited by RWPs except at the highest concentration (100 µg/mL). These results suggest that the inhibitory effect of RWPs on the activation of MAPK pathways could be specific for the p38^MAPK pathway. To clarify the mechanism of the inhibition of p38^MAPK phosphorylation by RWPs, we examined the effect of RWPs on the phosphorylation of MKK3/6, an upstream kinase of p38^MAPK. The phosphorylation of MKK3/6 was also inhibited by RWPs in a concentration-dependent manner, suggesting that the inhibitory effect of RWPs on the p38^MAPK pathway works upstream of MKK3/6. It is noteworthy that the concentrations of RWPs necessary for the inhibition of PI3K and p38^MAPK pathways were almost the same as those of RWPs for the inhibition of SMC migration.

View larger version (37K): [in this window] [in a new window]   Figure 4. A, Effects of RWPs on PI3K activity in SMCs. After serum-starved RASMCs or HASMCs were pretreated with RWPs for 10 hours, the cells were stimulated with PDGF-BB (10 ng/mL) for 10 minutes. Immunoprecipitated cell lysates by anti-phosphotyrosine antibody (PY20) were assayed for PI3K activity. Radiolabeled phosphatidylinositol 3' phosphate (PI[3]P), the enzymatic product of PI3K, was separated by thin-layer chromatography. Results of measured radioactivity are shown in the bottom panel. One representative result of 3 separate experiments is shown. B, Effects of RWPs on MAPK phosphorylation in SMCs. After pretreatment of serum-starved HASMCs with RWPs for 10 hours, the cells were stimulated with PDGF-BB (10 ng/mL) for 10 minutes. Western immunoblotting was performed using specific antibodies for each MAPK pathway, either in total or in phosphorylated form (p-). Moreover, effects of RWPs on MKK3/6 phosphorylation (p-MKK3/6) were determined using a specific antibody.

Activation of each signaling in PDGF-BB–stimulated SMCs was concentration-dependently inhibited by pretreatment with each inhibitor, whereas phosphorylation of ERK1/2 and p38^MAPK was slightly inhibited by wortmannin only at the maximum concentration (Figure 5). In our experiments, each inhibitor did not interfere with other pathways.

View larger version (19K): [in this window] [in a new window]   Figure 5. Effects of signaling inhibitors on PI3K activity and MAPK phosphorylation in SMCs. After pretreatment of serum-starved HASMCs with each inhibitor, WN, SB, or PD, for 30 minutes, the cells were stimulated with PDGF-BB (10 ng/mL) for 10 minutes. The effects of each inhibitor on PI3K activity using radioactivity of PI(3)P and MAPK phosphorylation using specific antibodies for phosphorylated ERK1/2 or p38MAPK were examined.

We have previously reported that RWPs inhibited the serum-stimulated SMC proliferation.¹² It is well known that the ERK1/2 pathway is important in growth factor–induced proliferation.¹⁷ However, PDGF-BB–induced activation of ERK1/2 in SMCs was not significantly inhibited by RWPs, as shown in Figure 4B. To investigate whether RWPs could inhibit PDGF-BB–stimulated SMC proliferation, the effect of RWPs on cell proliferation and DNA synthesis in SMCs was examined. RWPs significantly inhibited the cell number and [³H]-thymidine uptake of HASMCs stimulated by PDGF-BB in a concentration-dependent manner (Figure 6). These data suggest that the antiproliferative effect of RWPs on SMCs might be independent of the classic MAPK pathway ERK1/2.

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of RWPs on PDGF-BB–stimulated SMC proliferation. In cell proliferation assay, after pretreatment of serum-starved HASMCs with RWPs for 10 hours, the effects of RWPs on the number of HASMCs stimulated with PDGF-BB (10 ng/mL) for 60 hours were examined. In DNA synthesis assay, the effects of RWPs on thymidine incorporation into HASMCs stimulated with PDGF-BB for 22 hours were examined. The effects of RWPs were expressed as a percentage of control treated with PDGF-BB and without RWPs. *P<0.05 versus control. Results are shown as mean±SEM of 6 wells.

	Discussion

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In the present study, we demonstrated that RWPs potently inhibited PDGF-BB and serum-stimulated SMC migration but not serum-stimulated EC migration. Moreover, RWPs significantly inhibited the activities of 2 signaling pathways, PI3K and p38^MAPK, but not other MAPK pathways. The concentrations of RWPs necessary for the inhibition of these pathways were similar to those for the inhibition of SMC migration. Despite many studies investigating the mechanisms of intimal development using injured vessels of an animal model, there is little evidence that this model is valid either for the spontaneous intimal formation (intimal thickening) or for the accelerated intimal process characteristic of atherosclerosis in human.¹⁸ Ikari et al¹⁹ have previously demonstrated that human coronary intimal cells appearing in the perinatal period may arise by migration after replication of medial SMCs. They speculate that this process may be the first event in a clonal expansion, accounting for the monoclonality of advanced lesions.²⁰ Restenosis, including in-stent restenosis, after successful angioplasty and stenting remains a major limitation to the long-term success of the procedure. It is known that restenosis involves the SMC migration across the basement membrane to the intima, where SMCs proliferate.⁸ If the SMC migration is an early key step in restenosis formation, regular consumption of red wine might result in the inhibition and delay of its development.

Several laboratories have previously reported that PI3K is indispensable for cell migration induced not only by PDGF-BB but also by other growth factors in several cell types.^9,10 One study showed that replacement of 2 tyrosine residues within the PI3K-binding sites of the PDGF ß-receptor causes a loss of chemotactic response to PDGF-BB²¹; however, another study demonstrated that PI3K inhibitors did not inhibit chemotaxis in SMCs and Swiss 3T3 cells.²² In the present study, the inhibition of PI3K by wortmannin partially reduced PDGF-BB–induced SMC migration. The underlying mechanism of the discrepant observations about the functional role of PI3K in PDGF-induced SMC migration remains to be elucidated. It also has been demonstrated that cytoskeletal organization changes dynamically when cells increase their motility, and PI3K activity associates with an increase of cell motility.²³ Small G proteins downstream of the PI3K pathway, Rac and Rho, are known to be involved in PDGF-stimulated cellular responses, such as Rac-mediated lamellipodia and Rho-mediated stress fiber and focal adhesion formation.²⁴ The potent inhibitory effect of RWPs on SMC migration might be associated with, at least in part, the downregulation of these signaling pathways downstream of PI3K.

It has been reported that Akt, which is downstream of PI3K, is a major mediator for growth factors in cell survival.²⁵ Under our experimental conditions, phosphorylation of Akt might not be important for cell survival, because RWPs at the highest concentration (100 µg/mL) significantly downregulated the Akt phosphorylation; however, RWPs did not induce apoptosis in PDGF-BB–stimulated or serum-stimulated SMCs (data not shown).

Recently, several reports have shown that p38^MAPK activation is important for the cytoskeletal formation, including actin reorganization, and phosphorylation of heat shock protein 27 (hsp27), which is downstream of p38^MAPK and has been shown to play a role in membrane ruffling.²⁶ In the present study, we showed that treatment of PDGF-BB–stimulated SMCs with RWPs resulted in inhibition of p38^MAPK phosphorylation but did not affect other MAPK pathways. We also demonstrated that p38^MAPK inhibition by SB203580 showed a potent inhibition on SMC migration, but ERK1/2 inhibition by PD98059 did not show the significant inhibition, in good agreement with previous studies.^11,27 These results suggest that RWPs would exert the antimigratory effect on SMCs via the inhibition of cytoskeletal formation in part.

Matsumoto et al²⁷ have reported that expression of dominant-negative Ras inhibited PDGF-BB–induced migration in ECs expressing wild-type PDGF ß-receptor. They demonstrated that dominant-negative Ras inhibited PDGF-BB–induced activation of p38^MAPK as well as that of ERK1/2 and inhibition of p38^MAPK by SB203580 significantly decreased EC migration, but inhibition of ERK1/2 by PD98059 did not. In the present study, because RWPs (3 to 30 µg/mL) did not inhibit PDGF-BB–induced ERK1/2 phosphorylation, it is very likely that RWPs did not interfere with the pathway from the PDGF receptor, via Ras, to ERK1/2. These findings suggest that the site of RWP action may be between Ras and p38^MAPK. The phosphorylation of MKK3/6, upstream of p38^MAPK, was also inhibited by RWPs, suggesting that RWPs work upstream of p38^MAPK. With regard to the inhibitory effect of RWPs on the p38^MAPK pathway, additional investigation will be necessary to identify the exact point where RWPs work.

In our previous study, RWPs downregulated expression of cyclin A gene, one of the cell-cycle regulators, through downregulation of transcription factors.¹² In the present study, RWPs strongly inhibited the proliferation and DNA synthesis of PDGF-BB–stimulated SMCs; however, the ERK1/2 phosphorylation, which is known to relate to cell growth,¹⁷ was not inhibited by RWPs (up to 30 µg/mL). These results suggest that the inhibitory effects of RWPs on cell-cycle progression in SMCs do not depend on the ERK1/2 pathway. On the other hand, it has been recently reported that PI3K is associated with cell-cycle regulation, indicating that PI3K inhibitors induce G₁ arrest of several cells through upregulation of p27^Kip1, one of the cyclin-dependent kinase inhibitors.^28,29 Moreover, a key role of p27^Kip1 in SMC migration using SMCs from p27^Kip1-/- mice has been demonstrated recently.³⁰ To clarify the mechanism of the antiproliferative and antimigratory effects of RWPs on SMCs, we investigated the effect of RWPs on the p27^Kip1 expression in SMCs. Serum stimulation suppressed the induction of p27^Kip1 in serum-starved SMCs, and treatment of serum-stimulated SMCs with PI3K inhibitor, wortmannin or LY294002, significantly induced the p27^Kip1 expression. It is of interest that RWPs (30 µg/mL) also upregulated the p27^Kip1 expression in serum-stimulated RASMCs (data not shown). This result suggests that RWPs suppress cell-cycle progression not only through the downregulation of cyclin A expression but also through the upregulation of p27^Kip1 and that RWPs may inhibit intracellular signaling cascades activated by stimulators of cell proliferation and migration at an upstream site.

The concentration of RWPs in blood after red wine intake has not been determined, because RWPs consist of polymers as well as monomers, and for now it is impossible to measure the concentration of polymers in blood. Several researchers have shown that the absorption efficiency is 1% to 5% for monomers.³¹ If the absorption efficiency of polymers is similar to this value, the calculated final concentration of RWPs in blood could be 1 to 10 µg/mL after intake of 30 to 300 mL red wine. Furthermore, if this concentration of RWPs in blood is maintained over the long term by the regular consumption of red wine, it might be possible that RWPs would exert antimigratory effects on SMCs in vivo.

When detachment of endothelial monolayer is induced by several injuries, EC migration is mainly responsible for the wound repair of damaged vessels. We previously showed that RWPs had no inhibitory effect on proliferation, DNA synthesis, and cyclin A gene expression in serum-stimulated ECs.¹² In the present study, RWPs did not inhibit serum-induced EC migration in either migration assay. Although RWPs had no significant effects on proliferation and migration of ECs, the mechanism of these phenomena is unclear. However, we found that RWPs inhibited expression of vascular cellular adhesion molecule-1 in tumor necrosis factor-–stimulated HUVECs in a concentration-dependent manner (data not shown). Moreover, it has been recently reported that gallates, a component of RWPs, inhibited cytokine-induced adhesion molecule expression in ECs through a reduction of nuclear factor-B activity.⁴ These beneficial effects of RWPs on ECs suggest that RWPs may play a role not only in the maintenance of vascular structural integrity but also in the functional restoration of damaged ECs. In the future, the effects of RWPs on EC function, including membrane sensitivity and intracellular signaling, need to be elucidated.

In summary, RWPs have potent inhibitory effects on SMC migration and proliferation in the context of atherogenesis. Our findings on the intracellular signaling pathways modulating the proliferation and migration of SMCs may provide a new insight into the mechanism of the French paradox and may enable us to innovate a therapeutic approach for the prevention of atherosclerotic progression.

	Acknowledgments

 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, a Grant-in-Aid from Takeda Medical Research Foundation, and Funds for Comprehensive Research on Aging and Health from the Japan Foundation for Aging and Health.

Received February 4, 2002; revision received March 12, 2002; accepted March 12, 2002.

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