Differential effects of red and white wines on inhibition ...

Cardiovascular Research (2009) 81, 758–770 doi:10.1093/cvr/cvn340

Differential effects of red and white wines on inhibition of the platelet-derived growth factor receptor: impact of the mash fermentation Jan Sparwel1,2, Marius Vantler1,2, Evren Caglayan1,2, Kai Kappert1, Jochen W.U. Fries3, ¨hm5, Erland Erdmann1, and Stephan Rosenkranz1,2* Helmut Dietrich4, Michael Bo ¨t zu Ko ¨ln, Germany; 2Center for Molecular Medicine of the University of ¨r Innere Medizin, Universita Klinik III fu 3 ¨t zu Ko ¨ln, Germany; 4Fachgebiet Weinanalytik ¨r Pathologie, Universita Cologne (CMMC), Cologne, Germany; Institut fu 5 ¨nkeforschung, Geisenheim, Germany; and Medizinische Klinik III, Universita ¨tskliniken des Saarlandes, und Getra Homburg/Saar, Germany Received 1 May 2008; revised 1 December 2008; accepted 3 December 2008; online publish-ahead-of-print 11 December 2008 Time for primary review: 25 days

KEYWORDS Atherosclerosis; Platelet-derived growth factor; Tyrosine kinases; Wine; French paradox

Aims Moderate wine consumption is associated with a significant reduction of cardiovascular mortality. The molecular basis of this phenomenon remains unknown. Platelet-derived growth factor (PDGF) is an important contributor to atherogenesis. We investigated the effects of selected red and white wines on PDGF receptor (PDGFR) signalling in rat and human vascular smooth muscle cells (VSMCs). Methods and results All red wines concentration dependently inhibited the ligand-induced tyrosine phosphorylation of the PDGFR, downstream signalling events such as mitogen activated protein (MAP) kinase activation (Erk 1/2) and induction of immediate early genes (Egr-1, c-fos), and PDGF-induced cellular responses, whereas all white wines had no effect. At concentrations achieved after wine consumption in humans, all red wines completely abolished PDGF-dependent VSMC proliferation and migration. Red wines also inhibited PDGFR phosphorylation in vascular tissue, and in human coronary smooth muscle cells. Quantitative analyses of all tested wines and of samples collected at various time points (Days 0–16) of the ‘mash fermentation’, which is only performed for red wine, revealed that flavonoids of the catechin family, which potently inhibit PDGFR signalling, are extracted from grape seeds and skins during this process and therefore accumulate specifically in red wine. The accumulation of flavonoids correlated with the inhibitory potency of red wines on PDGFR signalling. Furthermore, this procedure could be imitated by incubation of wines with shredded grape seeds, and flavonoid-enriched white wine inhibited the PDGFR as potently as red wines. Conclusion Only red wines abrogate a critical pathogenic mechanism during atherogenesis, PDGFR signalling, in VSMCs. This effect is mediated by non-alcoholic constituents, which accumulate during the mash fermentation. Our findings offer a molecular explanation for the vasoprotective effects particularly of red wine. Therefore, future epidemiological studies should consider differential protective effects of red and white wine in vivo.

1. Introduction Moderate alcohol consumption is associated with a significant reduction of cardiovascular mortality in humans.1 Some studies indicate that wine may be more protective than other liquors such as beer or spirits.2,3 A recent meta-analysis of 26 studies on more than 200 000 individuals revealed that when compared with abstainers, cardiovascular risk was reduced by 22% in beer drinkers, and by 32% in wine drinkers.4 This and other studies suggest that wine may contain non-alcoholic compounds which—in addition

* Corresponding author. Tel: þ49 221 478 32402; fax: þ49 221 478 32400. E-mail address: [email protected]

to alcohol—protect against atherosclerotic vascular disease. Consistent with this idea the relatively high wine intake in France is thought to account for the lower mortality from coronary heart disease (CHD), which is reduced by 50% compared with other European countries and the USA, despite similar intakes of saturated fats.5,6 However, the molecular basis of this phenomenon, commonly known as the ‘French paradox’, remains unknown. Furthermore, it is not clear whether red wine confers any advantage over white wine. Atherogenesis is recognized as a complex inflammatory process involving several cell types such as macrophages, Tlymphocytes, platelets, endothelial cells, and vascular smooth muscle cells (VSMCs).7 These cells secrete numerous

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1

Red wine inhibits PDGFR signalling

2. Methods 2.1 Cell culture Vascular smooth muscle cells were isolated from rat thoracic aorta (male Wistar Kyoto; 6–10-week-old; Charles River Wega, Sulzfeld, Germany) by enzymatic dispersion and maintained as described.22 Human coronary smooth muscle cells (hcSMC) were purchased from Lonza Biosciences (Verviers, Belgium) and maintained in SmGM-2 according to the manufacturer’s instructions. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as described.23 Cells were grown in a 5% CO2 atmosphere at 378C. Experiments were performed with cells from passages 5–10.

2.2 Immunoprecipitation and western blot analysis Quiescent VSMCs were left resting or stimulated with 50 ng/mL PDGF-BB for 5 min in the presence or absence of wines as indicated. The bPDGFR was immunoprecipitated as previously described.19,24 Immunoprecipitates were resolved by SDS–PAGE and subjected to western blot analysis. To monitor the association of signalling molecules with the activated bPDGFR, immunoprecipitates were subjected to western blot analysis using antisera against PLCg, RasGAP, p85, and SHP-2.

2.3 Erk phosphorylation and induction of immediate early genes Quiescent VSMCs were left resting or stimulated with PDGF-BB (10 ng/mL) for times indicated in the presence or absence of red or white wines. The cells were harvested, the lysates were resolved by SDS–PAGE, and subjected to western blotting using antisera against phospho-Erk 1/2, Egr-1, or c-fos.

2.4 DNA synthesis assay DNA synthesis was measured by a 5-bromodeoxyuridine (BrdU)-incorporation assay according to the manufacturer’s specifications (Roche) as described.19

2.5 Chemotaxis assay Platelet-derived growth factor-dependent chemotaxis was assayed utilizing a 48-well modified Boyden chemotaxis chamber (NeuroProbe Inc., Baltimore, MD, USA) and collagen-coated PVP-free polycarbonate filters (8 mm pore size) (Poretics Corp., Livermore, CA, USA) as described previously.19,24

2.6 PDGF receptor phosphorylation in rat aorta Rat aortas were isolated from 12-week-old male rats, perfused with buffer containing 1% v/v of red wine (Chateauneuf-du-Pape) or white wine (Riesling) for 1 h at 378C, and stimulated with PDGF-BB (50 ng/mL) for 15 min. For immunohistochemical analyses, tissue was paraffin embedded, 5 mm aortic cross sections were prepared, and counterstained with haematoxylin and an antibody that recognizes phosphorylated bPDGFR (Tyr1021) as described.25 In addition, aortic tissue was homogenized, and western blot analyses were performed using antibodies that recognize the bPDGFR, phosphorylated bPDGFR (Tyr1021), and phospho-Erk1/2. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.7 Wines Red wines used were: Chianti Classico (Laborel, 1995), Barolo (Oddero, 1995), Bordeaux (Cha ˆteau Lanessan, 1985), red Rioja (Campo Viejo, 1998), Spa ¨tburgunder (Recher Herrenberg, 1999), Cha ˆteauneuf-du-Pape (Domaine Duclaux, 2002), and Avignonesi (Rosso di Toscana, 2000). White wines used were: Pinot Grigio (San Simone, 2000), Chardonnay (Hahnmu ¨hle, 1998), white Cha ˆteauneuf-du-Pape (Domaine du Vieux Lazaret, 2000), white Rioja (Conde de Valdemar, 2000), Mu ¨ller-Thurgau (Konstanzer, 1999), and Riesling Kabinett (1997er Weisenheim/Sd. Rosenbu ¨hl, Pfalz). Grape juices used were: Wu ¨rttemberger red grape juice (2000) and white grape juice (Weingut H.J. Fries, 2001).

2.8 Analysis of wine components Quantitative analysis was carried out by high performance liquid chromatography (HPLC) as described.26 In brief, a Merck-Hitachi HPLC system with a L-6200 pump and a diode array detector L-7450, equipped with a Fluofix 120E column (2504,6 mm; 5 mm; NEOS Company, Kobe, Japan) was used for analytical separation (flow rate 1.0 mL/min at 258C). The detection wavelength was 280 nm for flavonoids, 320 nm for phenol carbonic acids, and 520 nm for anthocyanins. The gradient elution was performed with water/O-phosphoric acid, and acetonitrile/water/phosphoric acid. Quantification was carried out using an external standardization.

2.9 Mash fermentation The mash fermentation of the 2003 Cha ˆteauneuf-du-Pape (Domaine Duclaux) was carried out for 16 days at 248C for the first three days,


cytokines and growth factors which act in an auto-/paracrine manner and promote the development and progression of atherosclerotic plaques. One such growth factor, which is critically involved in all phases of atherogenesis, is plateletderived growth factor (PDGF).7 PDGF and its receptors are significantly upregulated and activated in human atherosclerotic plaques,7–9 and inhibition of PDGF receptor (PDGFR) signalling by neutralizing antibodies or tyrosine kinase inhibitors potently inhibited neointima formation in various models.10–13 PDGF exerts its biological effects via activation of two subtypes of highly specific, transmembrane receptor tyrosine kinases (RTKs), termed a- and bPDGFR.14,15 Recent studies demonstrated that of the two receptor subtypes, bPDGFR-mediated signals are particularly important for vascular development and neointima formation following vascular injury,11 mainly by promoting the migration and proliferation of VSMCs in neointimal lesions. In fact, PDGF is the most potent mitogenic and chemotactic agent for VSMCs at the site of lesion formation.16 The prominent role of PDGF for de novo atherogenesis was recently underlined by a study on LDLR2/2 /smLRP-12/2 mice, in which upregulation and activation of the bPDGFR was associated with a dramatic acceleration of atherosclerotic lesion formation, and this response was prevented by a PDGFR-specific tyrosine kinase inhibitor.17 Thus, numerous studies indicate that PDGFR signalling is highly relevant for vascular disease. We and others have recently demonstrated that flavonoids known to be present in wine and green tea interfere with signal relay by the bPDGFR.18,19 In addition, the amount of flavonoid intake was shown to inversely correlate with CHD mortality in humans.20,21 Therefore, we now investigated the effects of selected red and white wines on PDGFR activation and PDGF-induced atherogenic responses in VSMCs. Furthermore, we sought to relate the inhibitory effects of some wines on PDGFR signalling to their flavonoid content and to specific procedures performed during the production of wine, e.g. fermentation and maturation.

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Table 1 Quantitative analysis of polyphenols in various red wines Red wines Chianti Classico

Bordeaux

Rioja (red)

Spa ¨tburgunder

Oddero, 1995 France Steel 12.8 2939

Cha ˆteau Lanessan, 1985 France Oak 12.5 2190

Campo Viejo, 1998 Spain Oak 12.6 1599

Recher Herrenberg, 1999 Germany Oak 12.6 2749

4.3 3.0 33.8 61.8 69.3 34.8 n.d. n.d. 2.2 0.4 9.9 2.0 0 3.6 0 0 0 0 0

11.8 n.d. 59.3 26.5 23.1 0 0 0 1.6 11.2 9.4 n.d. 0 12.3 0 0 0 0 0

22.7 4.8 28.1 34.6 19.2 8.5 35.9 8.5 3.3 2.7 n.d. 15.5 0 4.1 0 0 0 1 0

9.3 3.3 18.6 88.3 221.9 119.5 n.d. n.d. 0.4 0.3 16.6 2.8 0 12.7 0 0 0 0 0

Table 2 Quantitative analysis of polyphenols in various white wines White wines Pinot Grigio

Chardonnay

Cha ˆteauneuf-du-Pape (white)

Rioja (white)

Mu ¨ller-Thurgau

San Simone, 2000 Country Italy Maturation Steel Alcohol (% v/v) 12.5 Total phenols (Folin) 211 Phenolic components (mg/L) Caftaric acid 15.2 Protocatechueic 0 acid Tyrosol 0 Gallic acid 0 (þ)-Catechin 0 (2)-Epicatechin 0 Procyanidin B1 0 Procyanidin B2 0 GRP 1 p-CGT 1.8 Syringic acid 0 Coutaric acid 5.3 Fertaric acid 0 p-Coumaric acid 1.2 Quercetin 0 trans-piceid 0 cis-piceid 0 trans-resveratrol 0 cis-resveratrol 0

Hahnmu ¨hle, 1998 Germany Oak 12.4 314

Domaine du Vieux Lazaret, 2000 France Oak 13.9 213

Conde de Valdemar, 1998 Spain Oak 12.8 395

Konstanzer, 1999 Germany Steel 11.9 149

1.0 3.4

23.8 3.8

17.8 5

4.1 0

11.5 4.0 0 1.1 0 0 3.5 0 0 0.3 1.1 2.1 0 0 0 0 0

20.7 2.6 0 0 0 0 2.4 2.0 0 5.5 2.8 0.7 0 0.17 0.15 0 0

18 7.2 1.1 23.8 0 0 3 4.5 0 9.9 0 0.9 0 0 0.25 0 0

11.3 0 0 0 0 0 1.6 0.5 0 2.3 0 2.1 0 0 0 0 0

Wine


Wine Laborel, 1995 Country Italy Maturation Steel Alcohol (% v/v) 12.4 Total phenols (Folin) 2399 Phenolic components (mg/L) Caftaric acid 24.7 Protocatechueic acid 5.2 Tyrosol 42.0 Gallic acid 76.3 (þ)-Catechin 24.0 (2)-Epicatechin 21.0 Procyanidin B1 n.d. Procyanidin B2 n.d. GRP 2.4 p-CGT 1.3 Syringic acid 13.1 Coutaric acid 9.7 Fertaric acid 1.8 p-Coumaric acid 3.5 Quercetin 0 trans-piceid 0 cis-piceid 0 trans-resveratrol 0 cis-resveratrol 0

Barolo

Red wine inhibits PDGFR signalling and at 30–328C for the remaining 13 days. The temperature was controlled three times per day. The initial grape blend contained the following grape types: approximately 60% Grenache, 30% Mourve `dre, and 10% Syrah. Samples were collected and frozen at –208C until HPLC analyses or experimental procedures were performed.

2.10 Grape seeds incubations

2.11 Materials and antibodies PDGF-BB and VEGF-A were purchased from Promo Cell. The antiphosphotyrosine antibodies were from Santa Cruz (PY20) and Upstate Biotechnology (4G10). The antibody against SHP-2 was obtained from Transduction Labs (dPTP1D), the PLCg-antibody was from Upstate Biotechnology. The antibodies against the bPDGFR (97A), RasGAP (69.3), and the p85 subunit of PI3K were a kind gift from Andrius Kazlauskas and Alex Toker (Harvard Medical School, Boston, USA). The phospho-bPDGFR (Tyr1021) antibody was obtained from Abcam. Egr-1-, c-myc-, and c-fos-antibodies were purchased from Santa Cruz, and the phospho-Erk1/2, VEGFR-2, and phospho-VEGFR-2 (Tyr1175) antibodies were from Cell Signaling. Rat tail Collagen type I was obtained from BD Biosciences.

2.12 Statistical analysis All data are expressed as mean + SEM. Statistical significance was evaluated by non-parametric analysis. P , 0.05 was considered significant.

Figure 1 Differential effects of various red and white wines on bPDGFR activation and platelet-derived growth factor-dependent mitogen activated protein kinase activation and induction of immediate early genes. (A) Quiescent vascular smooth muscle cells were stimulated with PDGF-BB (50 ng/mL) in the presence of increasing concentrations of red or white wines as indicated. The bPDGFR was immunoprecipitated, and the immunoprecipitates were subjected to western blotting using antisera against the bPDGFR (top) or phosphotyrosine (P-Y; bottom). (B and C ) Quiescent vascular smooth muscle cells were stimulated with PDGF-BB (10 ng/mL) for the indicated times in the presence of 1% v/v of red (B) or white wines (C). Cell lysates were subjected to western blotting using antisera against RasGAP (lysate control), phospho-Erk1/2, Egr-1, or c-fos.


Italian red wine (Avignionesi) and German white wine (Riesling Kabinett) were incubated with various concentrations (10, 20, 30%) of shredded grape seeds for 92 h at room temperature under continuous stirring. The grape seed flour contained 2.97 mg/g catechin, 1.62 mg/g epicatechin, 3.94 mg/g procyanidin B1, and 4.94 mg/g procyanidin B2. After the incubation procedure, all samples were frozen at –208C until HPLC analyses or experimental procedures were performed.

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3. Results 3.1 Selected red and white wines differentially inhibit the bPDGFR and atherogenic responses of vascular smooth muscle cells

and the immunoprecipitates were subjected to western blot analysis. As demonstrated in Figure 1A, all red wines concentration dependently inhibited the ligand-induced phosphorylation of the bPDGFR, whereas all white wines had no effect (Figure 1A). Importantly, a complete inhibition of PDGFR activation by red wines in vitro occurred at concentrations (1.0% v/v of wine in the culture medium) that correspond to the consumption of two to three glasses of red wine. As a consequence of reduced receptor activation, red wines also inhibited the recruitment of bPDGFR-associated signalling molecules such as RasGAP, PLCg, PI3K, and SHP-2, to the activated receptor (not shown). Furthermore, all red wines abolished the PDGF-dependent activation of critical signalling events downstream from the receptor such as mitogen-activated protein (MAP) kinase activation (Erk 1/2), and the induction of immediate early genes including Egr-1 and c-fos (Figure 1B). In contrast, all tested white wines had no effect on PDGF-dependent downstream signalling (Figure 1C). We next investigated the effects of each wine on PDGF-dependent cellular responses. DNA synthesis was measured in a BrdU-incorporation assay. Stimulation of

Figure 2 Effects of red and white wines on platelet-derived growth factor-dependent cellular responses. (A and B) DNA synthesis. Quiescent vascular smooth muscle cells were stimulated with PDGF-BB (50 ng/mL) in the presence of various concentrations of red (A) or white wines (B) as indicated, and a BrdU incorporation assay was performed. (C and D) Chemotaxis. Quiescent vascular smooth muscle cells were stimulated with PDGF-BB (10 ng/mL) in the presence of red or white wines as indicated, and chemotaxis was measured by utilizing modified Boyden chambers. (C ) Representative stainings of filters showing migrated vascular smooth muscle cells. (D) Quantification of chemotaxis was performed by counting of migrated cells at 200, using a micrometer grid. All data are expressed as the percentage of the platelet-derived growth factor response and represent means + SEM from at least three independent experiments. (*P , 0.05; #P , 0.01 vs. PDGF).


To investigate specific effects of various red and white wines on PDGFR signalling, selected wines were tested for their ability to inhibit the ligand-induced activation of the bPDGFR in VSMCs. Since differences between red and white wines on the modulation of gene expression and vascular responses have been reported (reviewed by Opie and Lecour27), and some studies even suggested that only wines fermented in oak barrels (‘en barrique’) exert protective effects,28 we chose wines representing various countries and varieties, grape blends, fermentation and maturation procedures, and vintages. These included red and white wines matured in steel tanks or oak barrels (see Tables 1 and 2). In order to measure the ligand-induced tyrosine phosphorylation of the bPDGFR in the presence of each wine, VSMCs were pretreated with increasing concentrations of red or white wines and subsequently stimulated with PDGF-BB (50 ng/mL). The bPDGFR was immunoprecipitated,

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3.2 Red and white wines constitute significant differences regarding their content of polyphenolic compounds

Figure 3 Effects of red and white wine on bPDGFR activation in rat aorta. Rat aortas were isolated and perfused with buffer containing 1% v/v of red wine (Cha ˆteauneuf-du-Pape) or white wine (Riesling) for 1 h at 378C, and then stimulated with PDGF-BB (50 ng/mL) for 15 min. (A) Immunohistochemical analysis of aortic cross sections; (B) western blot analyses of tissue homogenates using antibodies that recognize the phosphorylated bPDGFR (Tyr1021) or Erk1/2. Shown are representative stainings and western blots from three independent experiments.

quiescent VSMCs with PDGF-BB led to a concentrationdependent increase of BrdU uptake to maximally 3-fold at 50 ng/mL (not shown). When the cells were stimulated with 50 ng/mL PDGF-BB in the presence of wine, all red wines inhibited the PDGF response at concentrations similar to those inhibiting bPDGFR activation (Figure 2A), whereas all white wines had no effect (Figure 2B). PDGF-dependent migration of VSMCs was measured using a modified Boyden chemotaxis chamber. Figure 2C demonstrates VSMCs that have migrated towards PDGF-BB through collagen-coated polycarbonate filters. PDGF-BB (10 ng/mL) led to an 8-fold increase of VSMC migration (P , 0.01) (Figure 2C and D). All red wines (1.0% v/v) completely inhibited PDGF-dependent chemotaxis (all P , 0.01 vs. PDGF-BB alone), whereas the pre-incubation with either white wine did not significantly affect the migratory response (Figure 2C and D), with the exception of chardonnay which constituted a slight inhibitory effect (P , 0.05). Importantly, inhibition of the PDGFR by red wine was also observed in vascular tissue. In isolated rat aortas that were perfused with buffer containing 1% v/v of red wine, the ligand-induced phosphorylation of the bPDGFR was completely inhibited, whereas it was not affected by the same concentration of white wine (Figure 3A and B).

The differential effects of red and white wines on PDGFR signalling raise the question about the nature of these profound differences. Consistent with other investigators,18 we have previously shown that polyphenolic flavonoids known to be present in wine and green tea inhibit signal relay by PDGFRs.19 Therefore, all tested wines were quantitatively analysed for specific polyphenolic compounds by HPLC. As expected, the total amount of polyphenols —which constitute the major part of ‘wine tannins’ was far greater in red wines as compared with white wines (Tables 1 and 2). The most abundant differences were found for gallic acid and flavonoids of the catechin family including (þ)-catechin, (2)-epicatechin, and procyanidin B2, which only appeared in red wines at relevant concentrations. Interestingly, we found no significant differences between wines produced ‘en barrique’ compared with those produced in steel tanks (see Tables 1 and 2). Only minimal amounts of monomeric anthocyanins, quercetin, or resveratrol were detected in all of the wines. These data show a clear correlation between the flavonoid content of wines and their potency to attenuate PDGFR signalling in VSMCs.

3.3 The accumulation of flavonoids and the inhibitory effects on the platelet-derived growth factor receptor depend on the process of ‘mash fermentation’ In contrast to white wines, red wines are typically fermented in the presence of grape seeds, skins, and stalks (‘mash fermentation’), which contain the majority of flavonoids present in grapes. Therefore, we hypothesized that flavonoids accumulate during this process and related the flavonoid contents at various time points of the mash fermentation to the inhibitory effects on PDGFR signalling. To this end, wine samples were collected at the time points 0 (grape juice), day 1, day 7, and day 16 (final) of the mash fermentation of a 2003 Cha ˆteauneuf-du-Pape (Domaine Duclaux). The samples were analysed by HPLC and tested for their ability to inhibit PDGFR activation and PDGF-dependent cellular responses. As shown in Table 3, the flavonoid levels in red grape juice were rather low and comparable to white grape juice, however, their content significantly rose during the 16 days of mash


In order to investigate whether the observed effects of red wine may be relevant in humans, key experiments were performed in hcSMC. As demonstrated in Figure 4A– C, all red wines but not white wines inhibited the ligand-induced tyrosine phosphorylation of the bPDGFR, downstream signalling events such as phosphorylation of Akt and Erk1/2, and PDGF-dependent proliferation and migration of hcSMC. Interestingly, an inhibitory effect was observed at even lower concentrations of red wine (0.3% v/v) as compared with rat VSMCs (1.0% v/v). Consistent with previous reports on catechins and vascular endothelial growth factor receptor (VEGFR) signalling,29 red wine but not white wine also inhibited the phosphorylation of the VEGFR in human endothelial cells (Figure 4D). In contrast, other RTKs such as the insulin and epidermal growth factor receptors were not affected by red wine (not shown).

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fermentation similarly to the alcohol concentration and ultimately reached the levels previously observed in red wines. When the mash samples were investigated for their effects on PDGFR signalling, the increase of flavonoid concentrations correlated with the inhibitory potency on bPDGFR

phosphorylation (Figure 5A). Consequently, the duration of the mash fermentation was also associated with the capability to inhibit PDGF-dependent DNA synthesis and chemotaxis, as only samples collected at days 7 and 16 effectively attenuated these cellular responses (Figure 5B and C).


Figure 4 Effects of red and white wines on bPDGFR activation and cellular responses in human coronary smooth muscle cells. Quiescent human coronary smooth muscle cells were stimulated with 50 ng/mL PDGF-BB in the presence of 0.3% v/v of red or white wines. (A) Platelet-derived growth factor-dependent tyrosine phosphorylation of the bPDGFR, and Akt and Erk phosphorylation. (B) PDGF-dependent DNA synthesis. (C ) PDGF-dependent migration. All data are expressed as the percentage of the platelet-derived growth factor response and represent means + SEM from at least three independent experiments (*P , 0.05; #P , 0.01 vs. PDGF). (D) Effects of red and white wine on vascular endothelial growth factor receptor activation in human endothelial cells. Quiescent human umbilical vein endothelial cells were stimulated with VEGF-A (50 ng/mL) in the presence of various concentrations of red (Cha ˆteauneuf-du-Pape) or white wine (Riesling) as indicated. The cells were harvested, and cell lysates were subjected to western blotting using antisera that recognize VEGFR-2 or phosphorylated VEGFR-2 (Tyr1175).


765

Table 3 Quantitative analysis of polyphenols in grape juice and wine samples collected at different time points of the mash fermentation of a 2003 Cha ˆteauneuf-du-Pape as indicated Mash fermentation Grape Juice (white)

0

0

30.8 2.3 0 0 0.8 0 0 0 0 2.9 2.5 0 5.6 0 1.6 0 0 0 0 0 0.4 0 0

57.4 0 0 0 5.2 8.0 0 0 0 7.1 3.2 0 18.4 0 0 0 0 0 0 0 1.2 0 0

3.4 Enrichment of flavonoids in red and white wine enhances the inhibitory effects on bPDGFR signalling The above findings indicate that developing red wines require a rather long contact time with grape seeds and skins in order to accumulate significant amounts of flavonoids. Hence, it should be possible to mimic this scenario and thereby enhance the flavonoid content of wine. Therefore, we sought to imitate the mash fermentation by incubating German white wine (Riesling Kabinett) and Italian red wine (Avignonesi) with various concentrations of shredded grape seeds. This procedure led to an accumulation of polyphenols, particularly (þ)-catechin, (2)-epicatechin, and gallic acid in white wine, and further increased the content of these polyphenolic compounds in red wine (Table 4). As expected, ‘polyphenol-enriched’ white wine potently inhibited bPDGFR phosphorylation (Figure 6A), and PDGF-induced DNA synthesis and chemotaxis to the same extent as red wines (Figure 6B and C). Moreover, even the inhibitory effects of red wine (Avignonesi) on PDGFR signalling were further enhanced by increasing the level of flavonoids (Figure 6A and C).

4. Discussion The present study demonstrates that only red wines abrogate a specific pathogenic mechanism involved in the complex process of atherogenesis, namely PDGFR signalling.

Cha ˆteauneuf-du-Pape (Domaine Duclaux, 2003) Day 1 0 36.6 0 0 0 3.5 4.0 4.8 5.3 0 4 0.1 0 6.9 0 2.9 0 0 0 0 0 2.4 0.7 0

Day 7 15.4

Day 16 14.6

24.2 4 12.1 0 3.6 21.7 3.8 4.8 0 1.4 2.9 0 9.1 0 0 0 0 0 0 0 3.4 1.3 0

22.3 2 9.8 0 13 33.5 7.3 11.3 0 0.8 2.3 0 7.8 0 1.4 0 0 0 0 0 3.1 0.7 0

This effect correlates with their content of non-alcoholic polyphenlic compounds, flavonoids of the catechin family, which attenuate bPDGFR activation. We demonstrate that flavonoids accumulate during the mash fermentation and therefore appear specifically in red wine. Furthermore, the increase of the flavonoid content during this process correlated with the inhibitory potency on PDGFR signalling. The inhibition of the PDGFR by red wine in the vessel wall implicates that our observations are relevant in vivo and may contribute to protective cellular events against atherosclerosis in humans. PDGF-dependent proliferation and migration of VSMCs are crucial events for de novo atherogenesis and restenosis formation following coronary angioplasty and stenting.7 In atherosclerotic plaques, PDGFRs are highly upregulated and appear to be in a phoshorylated, thus activated state during disease progression.7–9 Here we show that red wines, but not white wines, potently inhibit PDGFR activation and PDGF-dependent proliferation and migration of VSMCs in vitro. Although we investigated only one specific cellular mechanism involved in the complex pathogenic process of plaque formation, and it is difficult to dissect the role of a single receptor tyrosine kinase and its signalling pathways for disease progression, numerous studies have collectively shown that inhibition particularly of the bPDGFR subtype is sufficient to attenuate neointima formation in vivo.10–13 The present study reveals that red wines potently inhibit PDGFR signalling and PDGF-dependent atherogenic


Duration Alcohol (% v/v) Phenolic components (mg/L) Caftaric acid Protocatechueic acid Tyrosol 3-OH-Benzoic acid Gallic acid (þ)-Catechin (2)-Epicatechin Procyanidin B1 Procyanidin B2 GRP p-CGT Syringic acid Coutaric acid Sinapic acid Fertaric acid p-Coumaric acid Ferulic acid Ellic acid Quercetin trans-piceid cis-piceid trans-resveratrol cis-resveratrol

Grape Juice (red)

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Table 4 Quantitative analysis of polyphenols in red and white wine at baseline and after incubation with various concentrations of shredded grape seeds Grape seed incubations White wine (Riesling Kabinett) 0 350 37.9 2.1 0 0 0.9 5.2 1.6 0 3.8 5.2 0.0 16.7 0 0 5.9 6.8 0 0 0 0 0 0 0

10 295

20 2110

30 2708

40.0 6.7 0 0 4.9 13.2 6.5

25.0 6.2 0 0 10.9 52.6 32.9

40.2 2.8 14.0 0 14.1 97.8 61.6

7.3 4.6 0.0 5.8 13.4 0 6.1 4.9 0 0 0 0

5.7 4.2 0.0 4.5 9.2 0 4.4 4.3 0 0 0 0

4.2 1.0 0.4 3.2 9.7 0 3.8 1.3 0 0 0 0

0

0

0

responses in VSMCs. These effects are most likely mediated by flavonoids of the catechin family. Our findings are consistent with previous observations demonstrating that catechin–flavonoids attenuate signal relay by the bPDGFR.18,19 Inhibition of the ligand-induced receptor activation may be achieved either by blunting the receptors’ intrinsic tyrosine kinase activity or by preventing ligand binding to the receptor. Consistent with our previous observation that flavonoids inhibit binding of PDGF-BB to its receptor, a recent study demonstrated that another member of the catechin family, epigallocatechin gallate (EGCG), is incorporated into cell surface membranes and leads to irreversible binding of PDGF-BB to non-receptor sites and thus prevents its binding to the bPDGFR.30 Since gallic acid and catechins tend to form oligomers (procyanidins) and polymers, it is possible that currently undetectable flavonoid polymers also contribute to bPDGFR inhibition. Importantly, red wines also attenuated PDGF signalling in hcSMCs, which were even more sensitive to the inhibitory effect of red wine than rat VSMCs. The relevance of VEGFR inhibition by red wine flavonoids, as shown in Figure 4D and by other investigators,29 for the process of atherogenesis is difficult to interpret. While diminished VEGF signalling may compromise the regenerative capacity of the endothelium, it may also lead to reduced angiogenesis within atherosclerotic lesions and thereby prevent from plaque destabilization. Flavonoids are rather hydrophobic compounds which require a certain alcohol content to be solubilized sufficiently. The combination of increasing alcohol content and

0 2144 24.7 4.4 40.0 0 55.4 24.3 15.1 16.7 2.1 0 0 3.1 13.2 0 0 3.1 0 0 0 0 0.9 2.1 0

10 2152

20 4321

30 5651

26.4 3.8 42.7 0 50.7 18.6 13.5

11.6 5.8 13.3 0 31.3 46.5 31.0

20.3 7.7 38.5 0 66.4 125.6 110.7

2.4 2.1 7.5 4.0 9.2 0 1.2 2.8 0 0 0 0

3.7 4.2 7.2 4.1 6.7 0 0 4.6 0 0 0 0

4.9 4.9 0 5.4 9.7 0 0 5.7 0 0 0 0

0

0

0

long contact time with grape seeds and skins during the mash fermentation may therefore reflect the special merit of red wines as compared with white wines, grape juices, or other nutritional sources. This is supported by the fact that the amount of polyphenols, particularly flavonoids, was increased in both red and white wine by incubation with shredded grape seeds. Our data demonstrate that polyphenol-enriched white wine (Riesling) exerts inhibitory effects on PDGFR signalling that are similar to the effects induced by red wines. In agreement with these findings, a polyphenol-enriched white wine (Chardonnay) was recently shown to restore the impaired antioxidant capacity in a model of streptozotocin-induced diabetes in rats.31 Furthermore, red grape skin and grape seed extract supplementation significantly reduced atherosclerosis in male Wantanabe hyperlipidemic rabbits.32 These data indicate that white wines, when enriched in polyphenols/flavonoids, or grape seed extract supplementation may induce ethanol-independent effects in vitro and in vivo, that are comparable to the protective effects of red wines. The physiological relevance of our findings for inhibition of atherogenesis in vivo depends on the bioavailability of flavonoids after wine consumption, and on the concentrations required for sufficient inhibition of PDGFR signalling. Several recent studies indicated that flavonoids and their metabolites appear in the serum after wine intake.33–35 In our experiments, the concentrations of (þ)-catechin in the tissue culture medium after preincubation with 1.0% v/v of wine (which corresponds to an alcohol concentration of 1.0‰) ranged between 200 and 750 mg/L for most red


% Grape seeds Total phenols (Folin) Phenolic components (mg/L) Caftaric acid Protocatechueic acid Tyrosol 3-OH-Benzoic acid Gallic acid (þ)-Catechin (2)-Epicatechin Procyanidin B2 GRP p-CGT Syringic acid Caffeic acid Coutaric acid Sinapic acid Fertaric acid p-Coumaric acid Ferulic acid Ellic acid Quercetin trans-piceid cis-piceid trans-resveratrol cis-resveratrol

Red wine (Avignonesi)


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wines compared with 0–10 mg/L for white wines. In humans, the catechin serum levels after consumption of 200 mL red wine are approximately 600 mg/L.33 Hence, the concentrations of flavonoids used in vitro correlate with the serum levels achieved after moderate red wine intake in humans. Animal studies and epidemiological data in humans also support the relevance of red wine/flavonoids for the prevention of atherosclerosis.20,21,36–40 Apolipoprotein E-deficient mice, which were fed with either red wine or catechin showed a reduction of atherosclerotic lesion areas of 48 and 39%, respectively,37 and oral ingestion of a mixture of various catechins led to a decrease of atheromatous areas by 23%.38 EGCG particularly inhibited the development of atherosclerotic plaques in apo E-null mice,39 and local delivery of catechins potently inhibited neointima formation after experimental vascular injury in the rat.40 In humans, an inverse association between flavonoid intake and cardiovascular risk has also been demonstrated. A prospective cohort study including 806 men revealed that the age-adjusted risk ratios of myocardial infarction and CHD mortality were 0.54 and 0.48 in subjects with high catechin intake compared with those with low catechin intake, respectively.20 These data were confirmed in a case–control study, in which a daily intake of 21 mg of flavonoids was associated with a reduction of cardiovascular risk by 24%.21 Our data indicate that the protective effects of wine against atherosclerosis, that have been proposed in numerous studies, may be exclusively mediated by red wines.

Previous in vitro studies have shown differential effects of red and white wines on cellular responses including the expression of genes (MCP 1, cyclin A, MMP-2, eNOS, ET-1, monocyte tissue factor), thrombosis, proliferation of VSMCs, reduction of neointima formation, and vasorelaxation of coronary arteries (reviewed by Opie and Lecour27). Here, we relate the inhibitory effects of red wines on PDGFR signalling to their content of specific flavonoids of the catechin family, which were previously shown to inhibit bPDGFR activation. Based on a quantitative analysis of French wines and a reported average wine consumption of 180 mL/day/ person in France, the average daily intake of major wine catechins [(þ)-catechin, (2)-epicatechin, procyanidin dimers B1, B2, B3, and B4] for the French population was estimated as 5 mg/resident/day in white wine drinkers and 32 mg/resident/day in red wine drinkers.41 A recent case– control study revealed that flavonoids present in wine and tea are particularly protective against CHD, and that an average intake of 21 mg per day corresponds to a 24% decrease of cardiovascular risk.24 Hence, the high catechin intake with red wine and other nutritional sources such as fruits, vegetables, and tea, which also contain small amounts of flavonoids, may partly explain the protective effects of the ‘mediterranian diet’ against CHD.33,42,43 This may also explain the results of the PRIME study in which an inverse relation between alcohol consumption and cardiovascular risk was observed in France, whereas no significant relationship was found in Northern Ireland, where wine consumption is low.44


Figure 5 Impact of the mash fermentation on the inhibitory potency of red wine on platelet-derived growth factor receptor activation and platelet-derived growth factor-dependent cellular responses. Wine samples were collected at the time points 0 (grape juice), day 1, day 7, and day 16 (final) of the mash fermentation of a 2003 Cha ˆteauneuf-du-Pape (Domaine Duclaux). (A) Platelet-derived growth factor-dependent tyrosine phosphorylation of the bPDGFR. (B) PDGF-dependent DNA synthesis. (C ) PDGF-dependent migration (*P , 0.05; #P , 0.01 vs. PDGF).

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Figure 6 Imitation of the mash fermentation by wine incubation with shredded grape seeds enhances the inhibitory effects of red and white wine on bPDGFR signalling. An Italian red wine (Avignionesi) and a German white wine (Riesling Kabinett) were incubated with various concentrations (10, 20, 30%) of shredded grape seeds for 92 h as indicated. (A) Platelet-derived growth factor-dependent tyrosine phosphorylation of the bPDGFR. (B) PDGF-dependent DNA synthesis. (C ) PDGF-dependent migration (*P , 0.05; #P , 0.01 vs. PDGF).

In summary, we demonstrate that only red wines exert inhibitory effects on PDGFR signalling that are mediated by non-alcoholic constituents and are likely to be relevant in vivo. In addition to other effects of red wine such as modulation of the autonomic nervous system, endocrine system, and lipid and glucose metabolism,27,45 inhibition of PDGFR signalling

provides a molecular explanation for the lower CHD mortality in France (‘French paradox’). Future epidemiological studies should consider differential protective effects of red and white wine in vivo. Furthermore, the use of isolated flavonoids—either applied orally or in stent-based approaches— may be sufficient to prevent atherogenesis and restenosis.


Acknowledgements We thank Andrius Kazlauskas and Alex Toker (Harvard Medical School) for generously supplying antibodies against the bPDGFR (97A), RasGAP (69.3), and p85. We thank M. Je ´ro ˆme R. Quiot (Cha ˆteauneuf-du-Pape, France) for kindly collecting wine samples from the mash fermentation, Martin Pour Nikfardjam (Geisenheim) for HPLC analysis, and Norbert Knauth (Roche Pharma) and Peter Eckert (University of Bonn) for wine/grape seeds incubations. The technical assistance by Manuela Uebel, Jessica Schnitker and Tanja Roth is greatly acknowledged. This manuscript contains parts of a doctoral thesis by J.S.

Conflict of interest: none declared.

This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) (Ro 1306/2-1 and 2-2), and by the ‘Ernst und Berta Grimmke-Stiftung’ (Du ¨sseldorf, Germany).

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