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Inhibition of the PDGF receptor by red wine flavonoids provides a molecular explanation for the "French paradox" ¹

STEPHAN ROSENKRANZ², DENIS KNIREL, HELMUT DIETRICH^*, MARKUS FLESCH, ERLAND ERDMANN and MICHAEL BÖHM

Klinik III für Innere Medizin, Universität zu Köln, Germany;
^* Fachgebiet Weinanalytik und Getränkeforschung, Forschungsanstalt Geisenheim, Germany; and
Innere Medizin III, Universitätsklinken des Saarlandes, Homburg/Saar, Germany

²Correspondence: Klinik III für Innere Medizin, Universität zu Köln, Joseph-Stelzmann-Str. 9, 50924 Köln (Lindenthal), Germany. E-mail: Stephan.Rosenkranz{at}medizin.uni-koeln.de

SPECIFIC AIMS

Because 1) the mortality rate from coronary artery disease (CAD) in France is only 50% compared with other European countries and the U.S. despite similar intakes of animal fats (a phenomenon termed "French paradox"), 2) moderate wine consumption is inversely correlated with CAD mortality, and 3) cellular signals initiated by the platelet-derived growth factor ß receptor (ßPDGFR) play a critical role in the pathogenesis of atherosclerosis, we hypothesized that specific constituents of red and/or white wine may inhibit ßPDGFR signaling and PDGF-dependent cellular responses in vascular smooth muscle cells (VSMC). We sought to relate the quantitative amount of constituents and differential effects of red and white wine to specific procedures performed during wine production.

PRINCIPAL FINDINGS

1. Red, but not white, wine inhibits the ligand-induced activation of the ßPDGFR
Preincubation of VSMC with red wine (Chateauneuf-du-Pape), but not white wine (Riesling Kabinett) or ethanol, dose-dependently inhibits PDGF-induced tyrosine phosphorylation of the ßPDGFR. A statistically significant effect is observed at a concentration of 0.1% v/v (Fig. 1 A, B). Radioligand binding studies with [¹²⁵I]-PDGF-BB revealed that red, but not white, wine inhibits binding of PDGF-BB to its receptor in a concentration-dependent manner. Thus, red wine specifically inhibits PDGF ligand binding and subsequent tyrosine phosphorylation of the ßPDGFR.

View larger version (40K): [in this window] [in a new window]   Figure 1. Red wine dose-dependently inhibits ßPDGFR tyrosine phosphorylation and binding of receptor-associated signaling molecules. A) Quiescent VSMC were left resting (-) or stimulated with 50 ng/mL PDGF-BB (+) for 5 min in the presence of increasing concentrations of red wine. Immunoprecipitation of the ßPDGFR and immunoblotting were performed. B) Western blot signals from panel A were quantitated by densitometry and the P-Y signal was normalized by the ßPDGFR signal. Data are expressed as % of the maximal PDGF response and represent mean values ± SE of 3 independent experiments. *P < 0.05; **P < 0.01. C) Western blot analysis of ßPDGFR immunoprecipitates was performed using antisera against PLC, RasGAP, p85, and SHP-2 to detect coimmunoprecipitation of these signaling molecules with the activated ßPDGFR.

2. Red wine abrogates the ligand-induced recruitment of ßPDGFR-associated signaling molecules and the activation of PDGF-dependent downstream events
As a result of reduced receptor activation, red wine abrogates the ligand-induced recruitment of ßPDGFR-associated signaling molecules including RasGAP, PI-3 kinase, SHP-2, and PLC (Fig. 1C ). Red wine inhibits activation of PDGF-dependent downstream events such as Erk activation (p42/44^MAPK) and the induction of immediate early genes including Egr-1 and c-fos. In contrast, red wine does not affect serum- (FCS) or EGF-dependent Erk activation or IEG induction, indicating it specifically inhibits PDGF and not all mitogens.

3. Red wine potently inhibits PDGF-dependent proliferation and migration of VSMC
PDGF-dependent cell cycle progression was measured by BrdU incorporation. PDGF-BB leads to a concentration-dependent increase in BrdU uptake in VSMC (Fig. 2 A). Preincubation with red wine potently inhibits the maximal PDGF response at concentrations similar to those inhibiting PDGFR activation whereas white wine has no effect (Fig. 2B ). Moreover, red wine only slightly inhibits serum (FCS) -induced mitogenesis, further indicating the specificity of the inhibitory effect for PDGF. PDGF-BB leads to a dramatic increase in cell migration to eightfold of the basal level. When the chemoattractant is administered in the presence of red wine, PDGF-dependent chemotaxis is almost completely inhibited; coadministration with white wine does not affect the migratory response toward PDGF-BB (Fig. 2C, D ).

View larger version (43K): [in this window] [in a new window]   Figure 2. Differential effects of red wine and white wine on PDGF-BB-dependent cellular responses of VSMC. A) PDGF-dependent DNA synthesis was measured by BrdU incorporation. Shown is a concentration-response curve of PDGF-BB in VSMC. Data are expressed as fold increase over buffer and represent mean values ± SE of 3 independent experiments. B) Effects of red (RW) and white wine (WW) on DNA synthesis in response to PDGF-BB or 10% fetal calf serum (FCS). Data are expressed as % of the maximal response to PDGF or FCS. The experiment shown is representative of 3 independent experiments; each experimental condition was performed in triplicate. C) PDGF-dependent chemotaxis of VSMC was measured using a modified Boyden chamber. Shown are VSMC migrating through 8 µm pores of collagen-coated polycarbonate filters in the presence of buffer, PDGF-BB, PDGF-BB + red wine, and PDGF-BB + white wine. D) Quantification of chemotaxis was performed at x200 using a micrometer grid. Data are expressed as a fold increase over buffer and represent mean values ± SE from 3 independent experiments (*P<0.01 vs. buffer; #P<0.01 vs. PDGF-BB).

4. Quantitative analysis of polyphenols in red and white wine by HPLC
Only minimal amounts of anthocyanins, quercetin, and resveratrol were detected in both red and white wine. The total amount of polyphenols, which constitute the major part of "wine tannins", is far greater in the Chateauneuf-du-Pape (2113 mg/L) than the Riesling Kabinett (350 mg/L). The most dramatic differences were found for gallic acid (44.9 vs. 0.9 mg/L) and flavonoids of the catechin family including (+)-catechin (37.3 vs. 5.2 mg/L), (-)-epicatechin (12.0 vs. 1.6 mg/L), and procyanidin B2 (9.7 vs. 0.0 mg/L).

5. Flavonoids present in red wine inhibit the ßPDGFR
All flavonoids of the catechin family found in red wine potently inhibit the PDGF-dependent tyrosine phosphorylation of the ßPDGFR at concentrations present in wine, whereas gallic acid alone does not mediate a significant effect. Although the inhibitory effects of each catechin are less potent than red wine, they appear to be additive. As a result of their inhibitory effects on ßPDGFR activation, catechins dose-dependently inhibit PDGF-dependent S phase entry and VSMC migration.

6. Mimicking the "mash fermentation": enrichment of flavonoids in white wine leads to an inhibitory effect on ßPDGFR activation
To mimic the long contact time of wine with grape skins and seeds (which are the major sources of flavonoids) during mash fermentation (which is specific for red wine), the Riesling Kabinett was incubated with various concentrations of shredded grape seeds for 96 h. This procedure led to a significant concentration-dependent increase in the amount of polyphenols including catechins. The "flavonoid-enriched" white wine potently inhibits ßPDGFR signaling and PDGF-dependent DNA synthesis in a dose-dependent manner, and these inhibitory effects are similar to the ones induced by the Chateauneuf-du-Pape.

CONCLUSIONS AND SIGNIFICANCE

The data summarized provide evidence that nonalcoholic constituents of red wine that accumulate during mash fermentation act as potent inhibitors of ßPDGFR signaling and PDGF-dependent cellular responses in VSMC. Signals initiated by the ßPDGFR play an important role in vascular development and the pathogenesis of atherosclerosis. PDGF-dependent migration and proliferation of VSMC are critical steps during atherogenesis. Thus, inhibition of ßPDGFR signaling by red wine flavonoids offers a molecular explanation for the phenomenon that moderate wine consumption protects from atherosclerosis.

Some epidemiological studies have demonstrated that moderate wine consumption is inversely related to cardiovascular mortality. The higher wine intake of the French population is believed to account for the lower mortality rate from CAD (50%) in France compared with other European countries and the U.S. However, these studies do not distinguish between red and white wine, and the molecular mechanisms underlying this "wine CAD hypothesis" have remained elusive. Here we describe differential effects of red and white wine on receptor tyrosine kinase inhibition such that red wine—but not white wine—specifically inhibits ßPDGFR signaling, PDGF-dependent proliferation, and migration of VSMC. Wine analysis revealed that red wine contains significantly more polyphenols/flavonoids, particularly catechin and its derivatives such as epicatechin and procyanidin B2. These data are consistent with the scenario schematically outlined in Fig. 3 , whereby the long contact time of the originating wine with grape skins and seeds during the mash fermentation leads to an accumulation of flavonoids in red but not white wine. Consumption of red wine results in high serum levels of catechins, which on the cellular level inhibit activation of the ßPDGFR, recruitment of ßPDGFR-associated signaling molecules, and PDGF-dependent responses of VSMC. The inhibition of PDGF-induced proliferation and migration of VSMC and possibly inflammatory cells by red wine flavonoids disrupts the atherogenic process and thus protects the vessel wall from atherosclerosis.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proposed role of nonalcoholic constituents of red wine for inhibition of atherosclerosis. Extraction of polyphenols from grape skins and seeds during mash fermentation leads to an accumulation of flavonoids (catechins) in red wine. In contrast, white wine is produced by the "must fermentation" performed in the absence of grape skins and seeds. Consumption of red wine results in high serum levels of flavonoids, which inhibit PDGF ligand binding and subsequent tyrosine phosphorylation of the ßPDGFR on the cellular level. As a result, ßPDGFR-associated signaling molecules including Src, PI3K, RasGAP, SHP-2, and PLC are not recruited to the receptor and PDGF-induced cellular responses are blunted. Inhibition of PDGF-induced proliferation and migration of VSMC by red wine flavonoids disrupts the atherogenic process and protects the vessel wall from atherosclerosis.

The physiological relevance of the in vitro findings depends on the bioavailability of catechins in humans and on the efficacy of PDGFR inhibition for prevention of atherosclerosis. Recent studies have demonstrated that catechins are largely bioavailable after red wine intake. Concentrations of red wine/catechins shown to inhibit the ßPDGFR in vitro (400 µg/L) correlate with the serum levels of catechin after red wine consumption in humans (up to 600 µg/L). The idea that the catechins found in red wine are indeed responsible for the inhibitory effects of red wine on ßPDGFR activation is proved by the fact that each substance that was isolated mediated inhibitory effects on ßPDGFR signaling. The effects of different flavonoids of the catechin family appeared to be additive. In apolipoprotein E-deficient mice, which constitute an established animal model for atherosclerosis, oral ingestion of red wine, catechin, or a mixture of various catechins led to a reduction of atherosclerotic lesion areas of 48%, 39%, and 23%, respectively. Like catechin, functional blockade of the ßPDGFR by injection of a monoclonal antibody into apolipoprotein E-deficient mice led to a reduction in aortic atherosclerotic lesion size and the number of intimal VSMC by 67% and 80%, respectively. Finally, the "Zutphen Elderly Study" recently demonstrated an inverse association between catechin intake and ischemic heart disease mortality in humans.

In conjunction with the importance of ßPDGFR signaling for atherogenesis and the bioavailability of catechins in humans reported by others, the data above highlight the crucial role of red wine flavonoids for inhibition of receptor tyrosine kinases such as the ßPDGFR in VSMC, and thus identify one important mechanism in how red wine protects from atherosclerosis. Therefore, our study offers a molecular explanation for the "French paradox".

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0207fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0207fje

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