This Article Abstract Full Text (PDF) All Versions of this Article: 23/6/1001    most recent 01.ATV.0000070101.70534.38v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Oak, M.-H. Articles by Schini-Kerth, V. B. Search for Related Content PubMed PubMed Citation Articles by Oak, M.-H. Articles by Schini-Kerth, V. B. Related Collections Other Ethics and Policy Coumarins CV surgery: other

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1001.)
© 2003 American Heart Association, Inc.

Vascular Biology

Red Wine Polyphenolic Compounds Inhibit Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells by Preventing the Activation of the p38 Mitogen-Activated Protein Kinase Pathway

Min-Ho Oak; Marta Chataigneau; Thérèse Keravis; Thierry Chataigneau; Alain Beretz; Ramaroson Andriantsitohaina; Jean-Claude Stoclet; Soon-Jae Chang; Valérie B. Schini-Kerth

From Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034 (M.-H.O., M.C., T.K., T.C., A.B., R.A., J.-C.S., V.B.S.-K.), Université Louis Pasteur de Strasbourg, France, and Research and Development Center, Yangji Chemicals (S.-J.C.), An-San, South Korea.

Correspondence to Valérie B. Schini-Kerth, PhD, UMR CNRS 7034, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, 74, route du Rhin, B.P. 24, F-67401 Illkirch, France. E-mail schini{at}aspirine.u-strasbg.fr

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— Moderate consumption of red wine has a beneficial effect on the cardiovascular system. This study examines whether red wine polyphenolic compounds (RWPCs) affect vascular endothelial growth factor (VEGF) expression, a major angiogenic and proatherosclerotic factor in vascular smooth muscle cells (VSMCs).

Methods and Results— VEGF mRNA expression was assessed by Northern blot analysis and the release of VEGF by immunoassay in cultured VSMCs. Short-term and long-term exposure of VSMCs to RWPCs inhibited VEGF mRNA expression and release of VEGF in response to platelet-derived growth factor AB (PDGF_AB), transforming growth factor-ß₁, or thrombin. The PDGF_AB-induced expression of VEGF was markedly reduced by SB203580 (inhibitor of p38 mitogen-activated protein kinase [MAPK]), antioxidants, and diphenylene iodonium (inhibitor of flavin-dependent enzymes), slightly reduced by PD98059 (inhibitor of MEK), and not significantly affected by wortmannin (inhibitor of PI-3-kinase) and L-JNKI (inhibitor of JNK). Short-term and long-term treatment of VSMCs with RWPCs markedly reduced PDGF_AB-induced production of reactive oxygen species and phosphorylation of p38 MAPK.

Conclusions— These data indicate that RWPCs strongly inhibit growth factor–induced VEGF expression in VSMCs by preventing the redox-sensitive activation of the p38 MAPK pathway. The potential antiangiogenic and antiatherosclerotic properties of RWPCs are likely to contribute to cardiovascular protection by preventing the development of atherosclerotic lesions.

Key Words: red wine polyphenolic compounds • vascular endothelial growth factor • vascular smooth muscle cells • atherosclerosis • angiogenesis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Epidemiological studies have suggested that light to moderate consumption of alcoholic beverages, particularly red wine, is associated with a reduction in overall mortality, and this effect is attributable primarily to a reduced risk of coronary heart disease.^1,2 Although the exact nature of the protective effect of red wine on coronary diseases is unclear, it might be attributable, in part, to its ability to reduce 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. This is consistent with the findings that the consumption of red wine reduced the progression of lesions in experimental models of atherosclerosis.^3–5 The protective effect of red wine seems to be attributable, at least in part, to polyphenols, because nonalcoholic wine products and the red wine polyphenolic compounds (RWPCs) quercetin and catechin also prevented the progression of atherosclerotic lesions.^3–5 The beneficial effect of RWPCs might be related to their ability to prevent oxidation of LDL,⁶ activation of platelets,⁷ and expression of tissue factor and monocyte chemoattractant protein-1.^8,9

Recent findings have indicated that vascular endothelial growth factor (VEGF) is strongly expressed in human atherosclerotic plaques.^10,11 The cellular sources of VEGF in plaques are predominantly vascular smooth muscle cells (VSMCs) and to some extent foamy macrophages.^10,11 Besides being the major inducer of angiogenesis and vasculogenesis, VEGF is also known to induce vascular permeability and to function as a powerful endothelial mitogen and chemoattractant.¹² In addition, VEGF stimulates gene expression of several endothelial proteins involved in prothrombotic and proatherosclerotic responses, including tissue factor,¹³ adhesion molecules,¹⁴ and monocyte chemoattractant protein-1.¹⁵ Moreover, VEGF induces monocyte procoagulant activity and promotes monocytes chemotaxis.¹³ Thus, VEGF is likely to play an important role in the formation of new blood vessels and the expression of proinflammatory and prothrombotic molecules in atherosclerotic plaques. Therefore, the purpose of the present study was to determine whether RWPCs inhibit VEGF expression in VSMCs and, if so, to elucidate the underlying mechanism.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Preparation of RWPC
RWPCs dry powder from red French wine (Corbières A.O.C.) was 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, France). The procedures used to prepare and analyze RWPCs have been described previously.^16,17 In brief, phenolic compounds were adsorbed on a preparative column, and then alcohol was desorbed; the alcoholic-eluent was gently evaporated; the concentrated residue was lyophilized and finely sprayed to obtain RWPCs dry powder. One liter of red wine produced 2.7 g of RWPCs, which contained 471 mg/g of total phenolic compounds expressed as gallic acid. The extract contained 8.6 mg/g catechin, 8.7 mg/g epicatechin, anthocyanins (malvidin-3-glucoside, 11.7 mg/g; peonidin-3-glucoside, 0.66 mg/g; and cyanidin-3-glucoside, 0.06 mg/g) and phenolic acids (gallic acid, 5.0 mg/g; caffeic acid, 2.5 mg/g; and caftaric acid, 12.5 mg/g).

Cell Culture
Rat aortic VSMCs were cultured in MEM containing 10% FCS and antibiotics. 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.

Northern Blot Analysis
The cellular RNA from VSMCs was prepared by isothiocyanate and phenol extraction. VEGF mRNA level was assessed by Northern blot analysis as described.¹⁸ A 350-bp-long restriction fragment obtained from the cloned rat VEGF cDNA (provided by Dr C. Frelin, Université de Nice, France) was labeled with ³²P--dCTP using the Random Primer labeling kit from Stratagene. Autoradiographs were analyzed by scanning densitometry. VEGF mRNA levels were normalized to their respective 18S ribosomal RNA levels and expressed in arbitrary units as a fold increase of the signal obtained with untreated cells.

Western Blot Analysis
Total proteins (20 µg) were subjected to SDS PAGE (12%) and blotted on PVDF membrane. Immunodetection was carried out using antibodies directed against phosphorylated p38 MAPK, JNK, ERK1/2, and Akt (New England Biolabs). The immunoreactive band was detected by enhanced chemiluminescence (Amersham).

Determination of VEGF
A commercially available human VEGF immunoassay (R&D system) was used for the determination of VEGF content in human VSMC-conditioned medium (24 hours).

Determination of Reactive Oxygen Species Formation
The medium of treated and untreated VSMCs grown in 96-well plates was replaced by Hanks’ balanced salt solution (HBSS), and the cells were loaded with dichlorofluorescein (DCF) diacetate (10 µmol/L; Molecular Probes) dissolved in HBSS for 30 minutes at 37°C. The extracellular dye was removed, and HBSS containing PDGF_AB (30 ng/mL) or solvent was added. DCF fluorescence was measured in a Wallac Victor 1420 fluorescence plate reader (EG&G Wallac) at 37°C at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

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

	Results

Top Abstract Introduction Methods Results Discussion References

 
RWPCs Inhibit VEGF Expression
Exposure of rat VSMCs either to PDGF_AB, transforming growth factor (TGF)-ß₁, or -thrombin for 1 hour markedly increased the low basal steady state-level of VEGF mRNA (Figure 1A). The stimulatory effect of the 3 growth factors was markedly reduced by exposure of VSMCs to RWPCs 15 minutes before addition of a growth factor (Figure 1A). RWPCs alone also slightly but significantly reduced the basal expression of VEGF mRNA (Figure 1A). Similar findings were obtained with human VSMCs (data not shown). To characterize the inhibitory effect of RWPCs on VEGF expression, all subsequent experiments were performed with PDGF_AB, because this growth factor induced the strongest expression of VEGF mRNA (Figure 1A). RWPCs inhibited PDGF_AB-induced VEGF mRNA expression in a concentration- dependent manner with a statistically significant inhibition at concentrations of ≥10 µg/mL (Figure 1B). In addition to the 15-minute pretreatment period, exposure of VSMCs to RWPCs for either 24 or 18 hours followed by washout and an additional incubation period with serum-free medium for 6 hours also significantly reduced both the basal and PDGF_AB-induced VEGF mRNA expressions (Figures 2A and 2B). RWPCs treatment did not affect cell viability as assessed by CellTiter 96 MDSU aqueous one solution cell proliferation assay (Promega; the values were 93.5±0.8% and 95.5±1.9% in control [0.1% ethanol] and RWPCs [30 µg/mL]-treated cells after a 24-hour incubation period, respectively, n=6).

View larger version (47K): [in this window] [in a new window]   Figure 1. RWPCs inhibit growth factor-induced VEGF mRNA expression in VSMCs. A, Rat VSMCs were exposed to either solvent (0.1% ethanol) or RWPC for 15 minutes before the addition of a growth factor for 1 hour. Thereafter, VEGF mRNA levels were assessed by Northern blot analysis. B, Concentration-dependent inhibitory effect of RWPC on PDGFAB (1 hour)-induced VEGF mRNA expression in rat VSMCs. Depicted are representative Northern blots showing VEGF mRNA (top), 18S ribosomal RNA (center), and cumulative data (bottom). Results are shown as mean±SEM of 4 different experiments. #Treatment vs control; *Treatment vs the respective growth factor alone.

View larger version (34K): [in this window] [in a new window]   Figure 2. Long-term inhibitory effect of RWPCs on PDGFAB-induced VEGF mRNA expression in VSMCs. Rat VSMCs were exposed to either solvent (0.1% ethanol) or RWPCs for 24 hours (A) or 18 hours followed by washout and an additional 6-hour incubation period with serum-free culture (B) before the addition of PDGFAB (1 hour). Thereafter, VEGF mRNA levels were assessed by Northern blot analysis. Depicted are representative Northern blots showing VEGF mRNA (top), 18S ribosomal RNA (center), and cumulative data (bottom). Results are shown as mean±SEM of 4 different experiments. #Treatment vs control; *Treatment vs PDGFAB treatment alone.

To demonstrate that the inhibitory effect of RWPCs on VEGF mRNA expression was followed by a reduction of the secretion of VEGF protein, human VSMCs were exposed to either vehicle or PDGF_AB for 24 hours, and thereafter the amount of VEGF in conditioned medium was determined by immunoassay. PDGF_AB markedly increased the modest basal release of VEGF, and this response was significantly inhibited by pretreatment of VSMCs with RWPCs for 15 minutes before the addition of PDGF_AB (Figure 3A). The inhibitory effect of RWPCs was concentration-dependent and was statistically significant at concentrations of ≥3 µg/mL (Figure 3A). The PDGF_AB-stimulated release of VEGF was also reduced by pretreatment of VSMCs with RWPCs for either 24 or 18 hours followed by washout and a subsequent 6-hour incubation period with serum-free culture medium (Figure 3B). In addition, exposure of VSMCs to RWPCs alone either for 24 or 18 hours followed by washout slightly but significantly reduced the basal release of VEGF, whereas the 15-minute pretreatment period was without effect (Figures 3A and 3B).

View larger version (41K): [in this window] [in a new window]   Figure 3. RWPCs inhibit PDGFAB-induced release of VEGF protein into conditioned medium of VSMCs. Human VSMCs were exposed to either solvent (0.1% ethanol) or RWPCs for 15 minutes (A), 18 hours followed by washout and an additional 6-hour incubation period in serum-free culture medium (B), or 24 hours (B) before the addition of PDGFAB for 24 hours. Thereafter, the amount of VEGF protein into conditioned medium was determined by immunoassay. A, Results are shown as mean±SEM of 3 different experiments performed in triplicate. #Treatment vs control; *Treatment vs PDGFAB treatment alone.

Putative Role of Reactive Oxygen Species and Protein Kinases
The interaction of PDGF_AB with PDGF receptors in vascular cells activates several intracellular signal transduction pathways, which result in the formation of reactive oxygen species (ROS) and activation of p38 MAPK, ERK1/2, c-Jun NH₂-terminal kinase (JNK), and phosphoinositide 3-kinase/Akt.^19–22 Therefore, the role of these signal transduction pathways in the PDGF_AB-induced VEGF expression was examined. The results shown in Figure 4A indicate that PDGF_AB caused the formation of ROS as assessed by DCF fluorescence; this response was abolished by N-acetylcysteine. In addition, N-acetylcysteine and vitamin C (two antioxidants) and diphenylene iodonium (an inhibitor of flavin-dependent enzymes such as the NAD(P)H oxidase) significantly reduced the PDGF_AB-induced expression of VEGF mRNA, indicating the involvement of a redox-sensitive event (Figure 4B). In the absence of PDGF_AB, N-acetylcysteine, vitamin C, and diphenylene iodonium alone affected the basal expression of VEGF mRNA only minimally (Figure 4B). Immunoblot analysis indicated that PDGF_AB caused a transient phosphorylation of p38 MAPK, ERK1/2, JNK, and Akt with a maximal response occurring within 10 minutes (Figures 5A and 5B). Next, the involvement of these protein kinase–dependent pathways in the PDGF_AB-induced expression of VEGF was assessed using SB203580, an inhibitor of p38 MAPK; PD98059, an inhibitor of MEK; L-JNKI, an inhibitor of JNK; and wortmannin, an inhibitor of phosphoinositide 3-kinase. The stimulatory effect of PDGF_AB was abolished by inhibition of the p38 MAPK pathway, significantly reduced by inhibition of the ERK1/2 kinase pathway, and minimally affected by inhibition of the JNK and phosphoinositide 3-kinase pathways (Figure 6). Neither SB203580 nor PD98059 and L-JNKI alone affected significantly the basal release of VEGF, whereas a slight but significant increase was obtained in response to wortmannin (Figure 6). Because phosphorylation of p38 MAPK and ERK1/2 in VSMCs is markedly induced by H₂O₂²³ (Figure 5C), the possibility that ROS mediate the PDGF_AB-induced phosphorylation of p38 MAPK and ERK1/2 was evaluated. N-acetylcysteine and diphenylene iodonium significantly attenuated the PDGF_AB-induced phosphorylation of p38 MAPK and had only minor effects on that of ERK1/2 (Figure 5C). In addition, the stimulatory effect of PDGF_AB on p38 MAPK was also reduced by the combination PEG-superoxide dismutase (500 U/mL) and PEG-catalase (500 U/mL, data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 4. A, RWPCs and N-acetylcysteine (NAC) inhibit the PDGFAB-induced formation of ROS in VSMCs. Human VSMCs were exposed to NAC (30 minutes), RWPCs, or solvent (0.1% ethanol) for 15 minutes or 24 hours before the addition of PDGFAB. After a 1-hour incubation period, the generation of intracellular ROS was assessed using DCF and expressed in arbitrary units as a fold increase of the signal obtained with control cells. B, Effect of NAC and vitamin C (two antioxidants) and diphenylene iodonium (DPI, an inhibitor of flavin-dependent enzymes such as NAD(P)H oxidase) on PDGFAB-induced expression of VEGF mRNA in VSMCs. Rat VSMCs were exposed either to solvent, NAC, vitamin C, or DPI for 30 minutes before addition of PDGFAB for 1 hour. Thereafter, VEGF mRNA levels were assessed by Northern blot analysis. Depicted are representative Northern blots showing VEGF mRNA (top), 18S ribosomal RNA (center), and cumulative data (bottom). A, Results are shown as mean±SEM of 3 different experiments performed in triplicate. B, Results are shown as mean±SEM of 4 different experiments. #Treatment vs control; *Treatment vs PDGFAB treatment alone.

View larger version (37K): [in this window] [in a new window]   Figure 5. A, Effect of short-term and long-term exposure of VSMCs to RWPCs on the PDGFAB-induced phosphorylation of p38 MAPK, ERK1/2, JNK, and Akt in VSMCs. VSMCs were exposed to either solvent or RWPCs before the addition of PDGFAB. Thereafter, the phosphorylation level of the different protein kinases was assessed by Western blot analysis. Depicted in A are representative Western blots; B, cumulative data. Results are shown as mean±SEM of 3 different experiments. C, Effect of N-acetylcysteine (NAC) and diphenylene iodonium (DPI) on PDGFAB-induced phosphorylation of p38 MAPK and ERK1/2. VSMCs were exposed either to solvent, NAC, or DPI for 30 minutes before addition of PDGFAB for 10 minutes. The effect of H2O2 (10 minutes) on the phosphorylation of p38 MAPK and ERK1/2 is also shown. Similar observations were obtained in 2 additional experiments. *Treatment vs PDGFAB treatment alone.

View larger version (38K): [in this window] [in a new window]   Figure 6. Effect of SB203580 (an inhibitor of p38 MAPK), PD98059 (an inhibitor of ERK1/2 kinase kinase), L-JNKI (an inhibitor of JNK), and wortmannin (an inhibitor of phosphoinositide-3-kinase) on PDGFAB-induced release of VEGF protein into the incubation medium. VSMCs were exposed to either solvent or an inhibitor for 30 minutes before the addition of PDGFAB for 24 hours. Thereafter, the amount of VEGF protein into conditioned medium was determined by immunoassay. Results are shown as mean±SEM of 3 different experiments performed in triplicate. #Treatment vs control; *Treatment vs PDGFAB treatment alone.

Next, the possibility that RWPCs inhibit the PDGF_AB-induced expression of VEGF by preventing the generation of ROS or subsequent activation of the p38 MAPK pathway was determined. Exposure of VSMCs to RWPCs for either 15 minutes or 24 hours significantly blunted the PDGF_AB-induced generation of ROS (Figure 4A) and phosphorylation of p38 MAPK (Figures 5A and 5B). In contrast to p38 MAPK, RWPCs did not significantly affect the phosphorylation of ERK1/2, JNK, and Akt (Figure 5A). In the absence of PDGF_AB, RWPCs alone affected the basal generation of ROS and phosphorylation of p38 MAPK and ERK1/2 only minimally (Figures 4A, 5A, and 5B). RWPCs (30 µg/mL) also reduced the H₂O₂ (100 µmol/L)-stimulated release of VEGF from 304±18% to 239±8% (n=3) after a 24-hour incubation period.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present findings indicate that RWPCs strongly inhibit VEGF expression in VSMCs in response to PDGF_AB, TGF-ß₁, and -thrombin. These 3 growth factors are all potential endogenous stimuli of VEGF expression in atherosclerotic lesions, because an increased expression of PDGF-B and TGF-ß has been found in human atherosclerotic lesions, and procoagulant and prothrombotic responses have been observed at the surface of plaques.^24,25 The inhibitory effect of RWPCs is detected at concentrations of ≥3 µg/mL. Although the concentration of RWPCs in blood after intake of red wine remains unknown, a previous study has indicated that intake of 100 mL of red wine by healthy volunteers caused an increase in plasma concentration of polyphenolic monomers of 2.5 µg/mL (gallic acid equivalents).²⁶ Thus, the inhibitory effect of RWPCs on VEGF expression observed in the present study occurs at concentrations that are likely to be achieved in blood after moderate consumption of red wine. The present findings also indicate that RWPCs caused a sustained inhibition of VEGF expression in VSMCs that lasts several hours after their removal from the incubation medium. Such a long-lasting effect of RWPCs might reflect their association with VSMCs or the possibility that RWPCs induce the production of one or several peptides/proteins, which, in turn, contribute to prevent the expression of VEGF.

In addition to growth factors, VEGF expression can also be strongly stimulated by ROS such as H₂O₂ in most types of cells, including VSMCs.²⁷ More recent findings have suggested that ROS can also act as key signaling molecules controlling VEGF expression in response to growth factors. This is supported by the fact that exposure of VSMCs to thrombin or PDGF_AB caused the generation of substantial amounts of ROS via activation of a p22phox-containing NAD(P)H oxidase.^19,28 Moreover, prevention of the generation of ROS by either antioxidant treatment or diphenylene iodonium, a nonselective inhibitor of NAD(P)H oxidase, attenuated the expression of VEGF in response to thrombin and PDGF_AB.^19,28,29 ROS seem to control the expression of VEGF by activating the heterodimeric transcription factor hypoxia-inducible factor-1.²⁸ RWPCs are known to have antioxidant properties most likely attributable to their ability to scavenge ROS, such as hydroxyl radical and superoxide anion.^30–33 Altogether, these previous findings suggest that the inhibitory effect of RWPCs on the expression of VEGF involves their antioxidant properties. Consistent with such an idea, both short- and long-term exposure of VSMCs to RWPCs prevented the PDGF_AB-induced generation of ROS.

To further characterize the signaling pathways involved in the PDGF_AB-induced expression of VEGF, the activation of several protein kinases, including members of the mitogen-activated protein kinases and Akt, has been assessed by immunoblot analysis. Consistent with previous findings,^22,34,35 PDGF_AB caused a transient phosphorylation of ERK1/2, p38 MAPK, JNK, and Akt. Because activation of these protein kinase pathways has been involved in the upregulation of VEGF expression in several cell types,^36–38 their role in the PDGF_AB-induced expression of VEGF was determined using specific pharmacological inhibitors. The findings indicate that the stimulatory effect of PDGF_AB is critically dependent on the activation of the p38 MAPK pathway and also, to some extent, ERK1/2 but not JNK and phosphoinositide-3-kinase/Akt. ROS seem to mediate the phosphorylation of p38 MAPK in response to PDGF_AB, because this effect is attenuated by antioxidant treatments and by diphenylene iodonium. Moreover, H₂O₂ markedly increased the phosphorylation level of p38 MAPK. The present findings also indicate that RWPCs selectively prevent the PDGF_AB-induced phosphorylation of the p38 MAPK without affecting those of ERK1/2, JNK, and Akt. These findings are in good agreement with previous ones showing that red wine polyphenols inhibited PDGF_BB-induced activation of p38 MAPK whereas higher concentrations were also able to inhibit the activity of phosphoinositide-3-kinase in VSMCs.³⁵ Although red wine has been shown to inhibit PDGF_BB binding to PDGF ß receptor and PDGF ß receptor phosphorylation,³⁹ such explanations are unlikely to account for the present findings, because PDGF_AB-induced phosphorylation of ERK1/2, JNK, and Akt was unaffected by RWPCs. Altogether, these findings suggest that RWPCs inhibit growth factor–induced VEGF expression by preventing the redox-sensitive activation of the p38 MAPK, which in turn upregulates VEGF gene expression.

Previous studies have indicated that resveratrol, a polyphenolic compound found in wine, suppresses the growth of new blood vessels in several in vivo models of angiogenesis⁴⁰ and inhibits tumor growth and tumor-induced neovascularization in vivo.^41,42 Although the molecular mechanism of the in vivo antiangiogenic activity of wine-derived polyphenolic compounds remains unknown, it could be attributable to their direct inhibitory effect on endothelial cell growth^40,43 and migration⁴³ and, as suggested by the present findings, inhibition of the endogenous production of VEGF.

In conclusion, RWPCs strongly inhibit growth factor-induced VEGF expression by preventing the redox-sensitive activation of the p38 MAPK pathway in VSMCs. These effects are observed at concentrations that are likely to be achieved in blood after moderate wine consumption. Therefore, the antiangiogenic and antiatherosclerotic properties of RWPCs could contribute to explain the reduced risk of coronary heart disease and cancer mortality after moderate consumption of red wine for long-term periods.

	Acknowledgments

 
This study was supported in part by Yangji Chemicals (South Korea) and the Institut Européen Vin et Santé des Régions Viticoles (France). The authors thank Evelyne Lacofrette for technical help.

Received January 19, 2003; accepted March 20, 2003.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Rimm EB, Williams P, Fosher K, Criqui M, Stampfer MJ. Moderate alcohol intake and lower risk of coronary heart disease: meta-analysis of effects on lipids and haemostatic factors. BMJ. 1999; 319: 1523–1528.[Abstract/Free Full Text]

2. Renaud S, Guéguen R, Schenker J, d’Houtaud A. Alcohol and mortality in middle-aged men from eastern France. Epidemiology. 1998; 9: 184–188.[CrossRef][Medline] [Order article via Infotrieve]

3. Hayek T, Fuhrman B, Vaya J, Rosenblat M, Belinky P, Coleman R, Elis A, Aviram M. Reduced progression of atherosclerosis in apolipoprotein E-deficient mice following consumption of red wine, or its polyphenols quercetin or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler Thromb Vasc Biol. 1997; 17: 2744–2752.[Abstract/Free Full Text]

4. da Luz PL, Serrano Junior CV, Chacra AP, Monteiro HP, Yoshida VM, Furtado M, Ferreira S, Gutierrez P, Pileggi F. The effect of red wine on experimental atherosclerosis: lipid-independent protection. Exp Mol Pathol. 1999; 65: 150–159.[CrossRef][Medline] [Order article via Infotrieve]

5. Vinson JA, Teufel K, Wu N. Red wine, dealcoholized red wine, and especially grape juice, inhibit atherosclerosis in a hamster model. Atherosclerosis. 2001; 156: 67–72.[CrossRef][Medline] [Order article via Infotrieve]

6. Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet. 1993; 341: 454–457.[CrossRef][Medline] [Order article via Infotrieve]

7. Wang Z, Huang Y, Zou J, Cao K, Xu Y, Wu JM. Effects of red wine and wine polyphenol resveratrol on platelet aggregation in vivo and in vitro. Int J Mol Med. 2002; 9: 77–79.[Medline] [Order article via Infotrieve]

8. Feng AN, Chen YL, Chen YT, Ding YZ, Lin SJ. Red wine inhibits monocyte chemotactic protein-1 expression and modestly reduces neointimal hyperplasia after balloon injury in cholesterol-fed rabbits. Circulation. 1999; 100: 2254–2259.[Abstract/Free Full Text]

9. Pendurthi UR, Williams JT, Rao LV. Resveratrol, a polyphenolic compound found in wine, inhibits tissue factor expression in vascular cells: a possible mechanism for the cardiovascular benefits associated with moderate consumption of wine. Arterioscler Thromb Vasc Biol. 1999; 19: 419–426.[Abstract/Free Full Text]

10. Chen YX, Nakashima Y, Tanaka K, Shiraishi S, Nakagawa K, Sueishi K. Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerotic intimas of human coronary arteries. Arterioscler Thromb Vasc Biol. 1999; 19: 131–139.[Abstract/Free Full Text]

11. Couffinhal T, Kearney M, Witzenbichler B, Chen D, Murohara T, Losordo DW, Symes J, Isner JM. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol. 1997; 150: 1673–1685.[Abstract]

12. Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med. 1999; 77: 527–543.[CrossRef][Medline] [Order article via Infotrieve]

13. Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, Pan YC, Olander JV, Connolly DT, Stern D. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med. 1990; 172: 1535–1545.[Abstract/Free Full Text]

14. Melder RJ, Koenig GC, Witwer BP, Safabakhsh N, Munn LL, Jain RK. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat Med. 1996; 2: 992–997.[CrossRef][Medline] [Order article via Infotrieve]

15. Marumo T, Schini-Kerth VB, Busse R. Vascular endothelial growth factor activates nuclear factor-B and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells. Diabetes. 1999; 48: 1131–1137.[Abstract]

16. Andriambeloson E, Magnier C, Haan-Archipoff G, Lobstein A, Anton R, Beretz A, Stoclet JC, Andriansitohaina R. Natural dietary polyphenolic compounds cause endothelium-dependent vasorelaxation in rat thoracic aorta. J Nutr. 1998; 128: 2324–2333.[Abstract/Free Full Text]

17. Landrault N, Poucheret P, Ravel P, Gasc F, Cros G, Teissedre PL. Antioxidant capacities and phenolics levels of French wines from different varieties and vintages. J Agric Food Chem. 2001; 49: 3341–3348.[CrossRef][Medline] [Order article via Infotrieve]

18. Kronemann N, Bouloumie A, Bassus S, Kirchmaier CM, Busse R, Schini-Kerth VB. Aggregating human platelets stimulate expression of vascular endothelial growth factor in cultured vascular smooth muscle cells through a synergistic effect of transforming growth factor-beta1 and platelet-derived growth factor AB. Circulation. 1999; 100: 855–860.[Abstract/Free Full Text]

19. Görlach A, Brandes RP, Bassus S, Kronemann N, Kirchmaier CM, Busse R, Schini-Kerth VB. Oxidative stress and expression of p22phox are involved in the up-regulation of tissue factor in vascular smooth muscle cells in response to activated platelets. FASEB J. 2000; 14: 1518–1528.[Abstract/Free Full Text]

20. Yu J, Deuel TF, Kim HR. Platelet-derived growth factor (PDGF) receptor-alpha activates c-Jun NH2-terminal kinase-1 and antagonizes PDGF receptor-beta-induced phenotypic transformation. J Biol Chem. 2000; 275: 19076–19082.[Abstract/Free Full Text]

21. Fu M, Zhu X, Wang Q, Song Q, Zheng H, Ogawa W, Du J, Chen YE. Platelet-derived growth factor promotes the expression of peroxisome proliferator-activated receptor gamma in vascular smooth muscle cells by a phosphatidylinositol 3-kinase/Akt signaling pathway. Circ Res. 2001; 89: 1058–1064.[Abstract/Free Full Text]

22. Yamaguchi H, Igarashi M, Hirata A, Susa S, Ohnuma H, Tominaga M, Daimon M, Kato T. Platelet-derived growth factor BB-induced p38 mitogen-activated protein kinase activation causes cell growth, but not apoptosis, in vascular smooth muscle cells. Endocr J. 2001; 48: 433–442.[Medline] [Order article via Infotrieve]

23. Brandes RP, Viedt C, Nguyen K, Beer S, Kreuzer J, Busse R, Gorlach A. Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells. Thromb Haemost. 2001; 85: 1104–1110.[Medline] [Order article via Infotrieve]

24. Barrett TB, Benditt EP. Sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery. Proc Natl Acad Sci U S A. 1987; 84: 1099–1103.[Abstract/Free Full Text]

25. Bobik A, Agrotis A, Kanellakis P, Dilley R, Krushinsky A, Smirnov V, Tararak E, Condron M, Kostolias G. Distinct patterns of transforming growth factor-ß isoform and receptor expression in human atherosclerotic lesions: colocalization implicates TGF-ß in fibrofatty lesion development. Circulation. 1999; 99: 2883–2891.[Abstract/Free Full Text]

26. Duthie GG, Pedersen MW, Gardner PT, Morrice PC, Jenkinson AM, McPhail DB, Steele GM. The effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers. Eur J Nutr. 1998; 52: 733–736.

27. Ruef J, Hu ZY, Yin LY, Wu Y, Hanson SR, Kelly AB, Harker LA, Rao GN, Runge MS, Patterson C. Induction of vascular endothelial growth factor in balloon-injured baboon arteries: a novel role for reactive oxygen species in atherosclerosis. Circ Res. 1997; 81: 24–33.[Abstract/Free Full Text]

28. Görlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, Kietzmann T, Busse R. Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22(phox)-containing NADPH oxidase. Circ Res. 2001; 89: 47–54.[Abstract/Free Full Text]

29. Bassus S, Herkert O, Kronemann N, Gorlach A, Bremerich D, Kirchmaier CM, Busse R, Schini-Kerth VB. Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species. Arterioscler Thromb Vasc Biol. 2001; 21: 1550–1555.[Abstract/Free Full Text]

30. Miyagi Y, Miwa K, Inoue H. Inhibition of human low-density lipoprotein oxidation by flavonoids in red wine and grape juice. Am J Cardiol. 1997; 80: 1627–1631.[CrossRef][Medline] [Order article via Infotrieve]

31. Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet. 1993; 341: 454–457.

32. Hanasaki Y, Ogawa S, Fukui S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radic Biol Med. 1994; 16: 845–850.[CrossRef][Medline] [Order article via Infotrieve]

33. Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochem Pharmacol. 1988; 37: 837–841.[CrossRef][Medline] [Order article via Infotrieve]

34. Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, Law RE. Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells. Hypertension. 1997; 29: 334–339.[Abstract/Free Full Text]

35. Iijima K, Yoshizumi M, Hashimoto M, Akishita M, Kozaki K, Ako J, Watanabe T, Ohike Y, Son B, Yu J, Nakahara K, Ouchi Y. Red wine polyphenols inhibit vascular smooth muscle cell migration through two distinct signaling pathways. Circulation. 2002; 105: 2404–2410.[Abstract/Free Full Text]

36. Jiang B-H, Zheng JZ, Aoki M, Vogt PK. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci U S A. 2000; 97: 1749–1753.[Abstract/Free Full Text]

37. Tanaka T, Kanai H, Sekiguchi K, Aihara Y, Yokoyama T, Arai M, Kanda T, Nagai R, Kurabayashi M. Induction of VEGF gene transcription by IL-1 beta is mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes. J Mol Cell Cardiol. 2000; 32: 1955–1967.[CrossRef][Medline] [Order article via Infotrieve]

38. Milanini J, Vinals F, Pouysségur J, Pages G. p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts. J Biol Chem. 1998; 273: 18165–18172.[Abstract/Free Full Text]

39. Rosenkranz S, Knirel D, Dietrich H, Flesch M, Erdmann E, Böhm M. Inhibition of the PDGF receptor by red wine flavonoids provides a molecular explanation for the "French paradox." FASEB J. 2002; 16: 1958–1960.[Abstract/Free Full Text]

40. Bråckenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB J. 2001; 15: 1798–1800.[Free Full Text]

41. Kimura Y, Okuda H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice. J Nutr. 2001; 131: 1844–1849.[Abstract/Free Full Text]

42. Brakenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB J. 2001; 15: 1798–1800.

43. Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 2001; 171: 11–16.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				A. Walter, N. Etienne-Selloum, D. Brasse, R. Schleiffer, V. Bekaert, P. M. Vanhoutte, A. Beretz, and V. B. Schini-Kerth Red Wine Polyphenols Prevent Acceleration of Neovascularization by Angiotensin II in the Ischemic Rat Hindlimb J. Pharmacol. Exp. Ther., May 1, 2009; 329(2): 699 - 707. [Abstract] [Full Text] [PDF]

				J. Lu, K. Zhang, S. Chen, and W. Wen Grape seed extract inhibits VEGF expression via reducing HIF-1{alpha} protein expression Carcinogenesis, April 1, 2009; 30(4): 636 - 644. [Abstract] [Full Text] [PDF]

				K. W. Lee, N. J. Kang, Y.-S. Heo, E. A. Rogozin, A. Pugliese, M. K. Hwang, G. T. Bowden, A. M. Bode, H. J. Lee, and Z. Dong Raf and MEK Protein Kinases Are Direct Molecular Targets for the Chemopreventive Effect of Quercetin, a Major Flavonol in Red Wine Cancer Res., February 1, 2008; 68(3): 946 - 955. [Abstract] [Full Text] [PDF]

				C. Baron-Menguy, A. Bocquet, A.-L. Guihot, D. Chappard, M.-J. Amiot, R. Andriantsitohaina, L. Loufrani, and D. Henrion Effects of red wine polyphenols on postischemic neovascularization model in rats: low doses are proangiogenic, high doses anti-angiogenic FASEB J, November 1, 2007; 21(13): 3511 - 3521. [Abstract] [Full Text] [PDF]

				E. Anselm, M. Chataigneau, M. Ndiaye, T. Chataigneau, and V. B. Schini-Kerth Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src- and Akt-dependent activation of eNOS Cardiovasc Res, January 15, 2007; 73(2): 404 - 413. [Abstract] [Full Text] [PDF]

				S. Li, H. Tanaka, H. H. Wang, S. Yoshiyama, H. Kumagai, A. Nakamura, D. L. Brown, S. E. Thatcher, G. L. Wright, and K. Kohama Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1262 - H1272. [Abstract] [Full Text] [PDF]

				M. Sarr, M. Chataigneau, S. Martins, C. Schott, J. El Bedoui, M.-H. Oak, B. Muller, T. Chataigneau, and V. B. Schini-Kerth Red wine polyphenols prevent angiotensin II-induced hypertension and endothelial dysfunction in rats: Role of NADPH oxidase Cardiovasc Res, September 1, 2006; 71(4): 794 - 802. [Abstract] [Full Text] [PDF]

				M. Dell'Agli, A. Busciala, and E. Bosisio Vascular effects of wine polyphenols Cardiovasc Res, September 1, 2004; 63(4): 593 - 602. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 23/6/1001    most recent 01.ATV.0000070101.70534.38v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Oak, M.-H. Articles by Schini-Kerth, V. B. Search for Related Content PubMed PubMed Citation Articles by Oak, M.-H. Articles by Schini-Kerth, V. B. Related Collections Other Ethics and Policy Coumarins CV surgery: other