Germany

France

Red wine, cardiovascular

health

& endothelialnitric oxide synthase

Red wine increases the expression of human

endothelial NO synthase (eNOS):

a mechanism that may contribute to its

beneficial cardiovascular effects

Department of Pharmacology, Johannes Gutenberg University, D-55101 Mainz, Germany

Thomas Wallerath, PhD Daniela Poleo

Huige Li MD, PhD Ulrich Förstermann, MD, PhD

AbstractObjectives: In the present study, we tested the effect of red wine on eNOS expression and eNOS activity in human endothelial cells.Background: Endothelial-type NO synthase (eNOS) exerts vasoprotective effects. Moderate alcohol consumption has been associated with a reduction of cardiovascular disease, and red wine seems to offer more benefits than any other kind of drink. However, the molecular basis of this protective effect is unclear.Methods: Human endothelial cells were treated with red wine and eNOS mRNA expression was measured by RNase protection assay, eNOS protein expression by Western blotting, and eNOS activity by RFL-6 reporter cell assay. eNOS promoter activity was analyzed in transfected endothelial cells; binding activities of relevant transcription factors were determined by electrophoretic mobility shift assay.Results: Incubation of endothelial cells with red wines from France upregulated eNOS mRNA and protein expression. In contrast, red wines from Germany showed little or no effect on eNOS expression. No significant difference in eNOS mRNA expression could be detected between ”en barrique” (matured in oak barrels) and ”non barrique” (matured in steel tanks) produced French red wines. Endothelial cells treated with French red wines produced up to three times more bioactive NO than control cells. French red wines increased the activity of the eNOS promoter, with the essential trans-stimulated sequence being located in the proximal 326 bp of the promoter sequence. eNOS mRNA stability was also increased by red wine. Conclusions: The increase in eNOS expression and activity brought about by red wines from France (and probably other locations) may contribute to the beneficial effects of this beverage on the cardiovascular system.

(J Am Coll Cardiol 2003; 41: 471-478)

Aggregatingplatelets

Endothelial cells

Smooth muscle cells GTP cGMPVasodilatation

Ca2+ CaML-Arginine+O2 L-Citrulline

R R

Ca2+

ADPSerotonin

NO

NOReceptoragonists

PDGFFGF

Proliferation

Leukocytes

Blood vessel

CD11/CD18expression

++ -

--

NO is produced by vascular endothelium under basal conditions, and its production can be further stimulated by a variety of receptor agonists. NO dilates all types of blood vessels by stimulating soluble guanylyl cyclase (sGC) and increasing cyclic cGMP in smooth muscle cells. In addition to its vasodilator properties, NO can convey vasoprotection in several ways. NO released towards the vascular lumen is a potent inhibitor of platelet aggregation and adhesion to the vascular wall. Besides protection from thrombosis, this also prevents the release of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) that stimulate smooth muscle proliferation and its production of matrix molecules. Endothelial NO also controls the expression of genes involved in atherogenesis. NO decreases the expression of the chemoattractant protein MCP-1, and of surface adhesion molecules such as CD11/CD18, P-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) thereby preventing leukocyte adhesion to vascular endothelium and leukocyte migration into the vascular wall. This offers protection against an early phase of atherogenesis. Furthermore, NO has been shown to inhibit DNA synthesis, mitogenesis, and proliferation of vascular smooth muscle cells as well as smooth muscle cell migration, thereby protecting against a later phase of atherogenesis. Based on the combination of those effects, NO produced in endothelial cells can be considered an anti-atherosclerotic principle.

Vasoprotective role of NO

eNOS

sGC

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eNO

S m

RN

A le

vel (

% o

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German red wines

Bordeaux

BurgundyRhone French red wines

Effect of red wines on eNOS mRNA expression in human endothelial

EA.hy 926 cells. Cells were left untreated (control, Co) or exposed for 24

h to ethanol (EtOH, 1.25%, v/v), three different German red wines

("Dornfelder 1997“ (1), "Kriegsheimer Rosengarten 1997“ (2) and

"Spätburgunder 1999“ (3)) or six different French red wines from

Bordeaux ("Chateau Bonnet reserve 1997“ (4), "Chateau Bellegrave 1997“

(5) and "Chateau de la Marechaude 1997“ (6)), Rhone ("Chateau Cabieres

1998“ (7)), and Burgundy ("Chateau Rousseau 1998“ (8), "Les Chevaliers

de la Reine 1998“ (9)). In this experiment, the red wines were diluted in

the cell culture medium to a final concentration of 10% (v/v). RNase

protection analyses were performed using antisense RNA probes to

human eNOS and GAPDH (for standardization).

Co EtOH 1 2 3 4 5 6 7 8 9

Effect of different red wines on eNOS mRNA expression in human endothelial cells

Effect of French red wines on eNOS mRNA expression in human endothelial cells

eNOS

GAPDH

EtOH MT N GLes Chev.10%

EtOH

***

Les Chev.10%

0

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eNO

S m

RN

A le

vel (

%of

co

ntro

l)

118

140151

200

249

311417

100

nt

Upregulation of eNOS mRNA by the French red wine "Les Chevaliers de la

Reine 1998" in primary human umbilical vein endothelial cells (HUVEC).

eNOS mRNA expression was quantified with RNase protection assay. RNA

was prepared from HUVEC either incubated for 24 h with ethanol (EtOH,

1.25%, v/v) or with the red wine "Les Chevaliers de la Reine 1998" (Les Chev.,

10% v/v).

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EtOH Chateau Bonnet`non-barrique`

*****

Chateau Bonnet`en -barrique`

eNO

S m

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% o

f con

trol

)

n. s.

Upregulation of eNOS mRNA in human endothelial cells by the French red

wines "Chateau Bonnet 1997“ (“non-barrique“, matured in steel tanks) and

"Chateau Bonnet reserve 1997“ (“en-barrique“, matured in oak barrels).

eNOS mRNA expression was quantified with RNase protection assay. RNAs

were prepared from EA.hy 926 cells either incubated for 24 h with ethanol

(EtOH, 1.25%, v/v) or with the red wines (10% v/v).

B

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0 12 24 36 48 60Incubation time (h)

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*

10%3%1%

Ch.Bonnet

10 d

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*

eNO

S m

RN

A le

vel (

% o

f con

trol

)A

**

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Dornfelder Ch.Bonnet L.Chevaliers

1% 3% 10% 1% 1% 3% 3%10% 10%EtOH

100

150

200

250

0

Concentration- and time-dependent upregulation of eNOS mRNA expression

by French red wines. eNOS mRNA expression was quantified with RNase

protection assay. Panel A: EA.hy 926 cells were incubated for 24 h with

ethanol (EtOH, 1.25%, v/v,), a German red wine ("Dornfelder 1997") and two

French red wines ("Les Chevaliers de la Reine 1998" and "Chateau Bonnet

reserve 1997") diluted to final concentrations of 1%, 3% and 10% (v/v). Panel

B: Cells were exposed to cell culture medium containing 1%, 3% and 10%

(v/v) of " Chateau Bonnet reserve 1997" for different periods of time.

Effect of different red wines on eNOS mRNA expression in human endothelial cells

eNO

S m

RN

A le

vel (

% o

f con

trol

)

Effect of French red wines on eNOS protein expression and NO production in human endothelial cells

eNOS

Les Chevaliers de la Reine

-Tub.

Chateau Bonnet

EtOHEtOH 1% 1% 3%3% 10% 10%

A

NO

pro

duct

ion

(% o

f con

trol

)

050

100150200250

300350400

450

Chateau Bonnet

EtOH 10%1%

Les Chevaliersde la Reine

3% 1% 3% 10%

B

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*

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Effect of French red wines on eNOS protein expression and eNOS activity in endothelial EA.hy 926 cells. Panel A: Western blot analyses using a polyclonal anti-eNOS- and a monoclonal anti-tubulin-antibody (for normalization). Cells were incubated for 48 h with ethanol (EtOH, 1.25 %, v/v) or two different French red wines ("Chateau Bonnet reserve 1997" and "Les Chevaliers de la Reine 1998", 1%, 3% and 10%, v/v). The blots represent four independent experiments each. Panel B shows the effect of a 48 h incubation with "L.Chevaliers" and "Ch.Bonnet reserve" on the NO production in EA.hy 926 cells stimulated for 2 min with the calcium ionophore A23187. Conditioned medium from EA.hy 926 cells was transferred to RFL-6 reporter cells and incubated for 2 min. Thereafter, cGMP content in RFL-6 cells was determined by radioimmunoassay .

Effect of French red wines on eNOS promoter activity in human endothelial cells

eNO

S pr

omot

er a

ctiv

ity

(% o

f con

trol

)

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**

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Chateau Bonnet Les Chevaliersde la Reine

EtOH 1% 3% 10%

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1% 3% 10%

A

0 100 200 300

eNOS promotor activity (% of each control)

pGl3-basic

+1-1600

Luc

+1-1111

Luc

+1-633

Luc

+1-326

Luc

B150 25050 350

EtOHLes Chevaliers 10%

Enhancement of eNOS promoter activity in EA.hy 926 cells after incubation with French red wines. Panel A: EA.hy 926 cells were stably transfected with p-eNOS-3500-Hu-Luc-neo. Stable cells were incubated for 24 h with ethanol (EtOH, 1.25%, v/v), or various concentrations of two French red wines ("Les Chevaliersde la Reine 1998" and "Chateau Bonnet reserve 1997"). The luciferase activity (normalized by protein content) was taken as a measure of eNOS promoter activity. Panel B: EA.hy 926 cells were transiently transfected with pGl3-Basic (containing a promoter-less luciferase gene) or different eNOS promoter luciferase constructs (p-eNOS-1600-Hu-Luc, p-eNOS-1111-Hu-Luc, p-eNOS-633-Hu-Luc and p-eNOS-326-Hu-Luc, containing 1.6-kb to 0.33-kb of the eNOS promoter cloned before a luciferase reporter gene). The different transfected cells were either exposed 24 h to ethanol (EtOH, 1.25%, v/v) or to the red wine "Les Chevaliers de la Reine 1998" (10%, v/v). The relative luciferase activity (corrected with renilla-luciferase activity) was taken as a measure of eNOS promoter activity.

Effect of a French red wines on eNOS mRNA stability

eNO

S m

RN

A le

vel (

% o

f con

trol

) *****

***

0 12 24 36Time (h) after actinomycin D treatment

EtOH

Ch.Bonnet 10%

0

40

60

80

100

120

20

Effect of French red wine on eNOS mRNA stability. EA.hy 926 cells were pre-incubated with ethanol (EtOH, 1.25%, v/v) or with or the red wine "Chateau Bonnet reserve 1997" (10%, v/v) for 24 h. To inhibit transcription, actinomycin D (20 µg/mL) was added to the culture medium. RNA was prepared 0, 12, 24 and 36 h thereafter. The eNOS mRNA expression was determined by RNase protection assay. The eNOS mRNA levels in both groups at the time of addition of actinomycin D (0 h) were set at 100%.

Red wine

eNOS mRNAexpression

eNOS proteinexpression

eNOS promoteractivity

eNOS mRNAstability

bioactive NO

Summary:

vasoprotection ?!