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Dealcoholized red wine reverse vascular remodeling in an experimental modelof metabolic syndrome: role of NAD(P)H oxidase and eNOS activity

Marcela Alejandra Vazquez-Prieto,ab Nicol�as Federico Renna,ab Carina Lembo,ab Emiliano Ra�ul Diezab

and Roberto Miguel Miatello*ab

Received 15th July 2010, Accepted 13th August 2010

DOI: 10.1039/c0fo00077a

The present study examines the effect of chronic administration of dealcoholized red wine Malbec

(DRW) on vascular remodeling and NAD(P)H oxidase and endothelial nitric oxide synthase activity

(eNOS) in an experimental model of metabolic syndrome induced by fructose administration. Thirty-

day old male Wistar rats were fed a normal rat diet (control) or the same diet plus 10% fructose in

drinking water (FFR). During the last 4 weeks of a 10-week period of the corresponding diet,

a subgroup of control and FFR (n ¼ 8 each) received DRW in their drinking water. Systolic blood

pressure (SBP), a homeostasis model assessment of insulin resistance (HOMA-IR), aortic NAD(P)H

oxidase and eNOS activity in the heart and vascular tissue were evaluated. Vascular remodeling was

evaluated in the left carotid artery (CA) and interlobar, arcuate and interlobular renal arteries (RA)

through lumen to media (L/M) ratio determination. At the end of the study FFR increased the SBP (p <

0.001), HOMA-IR (p < 0.001), and aortic NAD(P)H oxidase activity (p < 0,05) but reduced cardiac

and vascular eNOS activity (p < 0.01), L/M ratio in CA (p < 0.001) and RA (p < 0.01) compared with

the C group. DRW reduced SBP (p < 0.05), aortic NAD(P)H oxidase (p < 0.05), and recovered eNOS

activity (p < 0.001) and L/M in CA (p < 0.001) and RA (p < 0.001) compared with FFR. This study

provides new data about the beneficial effect of DRW on oxidative stress and vascular remodeling in

the experimental model of metabolic syndrome. Data suggest the participation of mechanisms

involving oxidative stress in FFR alterations and the usefulness of natural antioxidant substances

present in red wine in the reversion of these changes.

Introduction

Metabolic syndrome (MS), characterized by insulin resistance,

dyslipidemia and hypertension, is an important risk factor for

cardiovascular diseases.1 Rats chronically receiving fructose

(FFR) provide a useful experimental model for the study of the

interaction between factors clustered in MS.2 Endothelial

dysfunction is associated with this experimental model.3 We

previously reported a decrease of the endothelial isoform of nitric

oxide synthase activity (eNOS) at cardiovascular level and an

increase in vascular smooth muscle cell proliferation in primary

culture, showing also evidence involving the renin angiotensin

system (RAS) in the pathophysiology of these injuries.4,5

Furthermore, it has been demonstrated that angiotensin, acting

on AT1 receptors, could induce oxidative stress, through acti-

vation of nicotinamide adenine dinucleotide phosphate

(NAD(P)H) oxidase, the most important source of intracellular

reactive oxygen species (ROS) in vascular cells.6

ROS play a physiological role in the vessel wall and participate

as second messengers in endothelium dependent function, in

smooth muscle cell and endothelial cell growth and survival, and

in remodeling of the vessel wall.7,8 The major vascular ROS is

superoxide anion ($O2�), which inactivates nitric oxide (NOc),

aInstitute of Experimental Medicine and Biology of Cuyo (IMBECU),National Council of Research (CONICET), Mendoza, ArgentinabDepartment of Pathology, School of Medicine, National University ofCuyo, Av. Libertador 80, 5500 Mendoza, Argentina. E-mail: rmiatell@fcm.uncu.edu.ar; Fax: +54 261 4135242; Tel: +54 261 41305000 ext 2697

124 | Food Funct., 2010, 1, 124–129

the main vascular relaxing factor.9 The relationship between

oxidative stress and vascular remodeling had been previously

reported in human and animal experimental studies,7 including

fructose-fed rats.10,11

The study of the beneficial effect on human health of

consumption of natural antioxidants, present in vegetables,

fruits and beverages such as red wine, has recently increased in

significance. Epidemiological studies suggest that moderate red

wine consumption could decrease the risk of cardiovascular

mortality,12 mainly attributable to the polyphenol content but

also attributable to the alcohol content.13 Polyphenols could

favor endothelium-dependent vasodilatation in aorta and

human coronary arteries, inhibit vascular smooth muscle cell

proliferation.14–16 We previously reported that resveratrol was

able to increase the eNOS activity and reduce the systolic blood

pressure (SBP) in this model of MS.17 In order to establish the

beneficial effects of non-alcoholic constituents of red wine on

vascular remodeling, the aim of this study was to determine the

effect of chronic administration of dealcoholized red wine

(DRW) in fructose-fed rats upon the possible participation of

changes in ROS and NOc generation in the development of

structural and functional alterations at cardiovascular and

metabolic levels. Specifically, ROS production by the

NAD(P)H oxidase system, and NOc generation by eNOS were

examined in order to establish whether these systems are

involved as pathogenic mechanisms in metabolic and structural

cardiovascular changes associated with this experimental

model.

This journal is ª The Royal Society of Chemistry 2010

Fig. 1 NAD(P)H oxidase aorta activity from C, C + DRW, FFR, and F

+ DRW rats. Values are mean � SEM (n ¼ 8). Bars without a common

letter differ, P < 0.05.

Fig. 2 eNOS activity in mesenteric vascular bed homogenates (A) and

heart tissue homogenates from the left ventricle (B), from C, C + DRW,

FFR, and F + DRW rats. Values are mean � SEM (n ¼ 4). Bars without

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Results

No differences were observed in food and drink intake between

groups throughout the experimental period. Table 1 shows body

weight, metabolic variables and SBP. The body weight did not

vary among groups. At the end of the study fructose-fed rats

developed insulin resistance, increased significantly the triglyc-

eride levels and reduced the HDL cholesterol compared with

control groups. Chronic administration of DRW significantly

reduced the insulin resistance state and increased the HDL

cholesterol. Systolic blood pressure was gradually increasing

throughout the experimental period in FFR and reached

a significant difference compared to controls. DRW adminis-

tration to FFR during the last four weeks was able to reduce SBP

in a slight but significantly way, without effect on control rats.

The NAD(P)H oxidase activity in aortic tissue was higher in

FFR, compared with the C group. Administration of DRW

significantly reduced the NAD(P)H oxidase activity (Fig. 1).

Fig. 2 shows eNOS activity levels, measured in a mesenteric

vascular bed (panel A) and heart tissue from left ventricle

homogenates (panel B). The eNOS activity was significantly

diminished in the FFR group, compared to control rats. DRW

chronic administration to FFR was able to return NOc

production to control levels in both mesenteric vascular and

heart tissue, while DRW to control rats increased significantly

the eNOS activity in mesenteric vascular tissue.

Arterial wall modifications were detected by structural anal-

ysis performed by histological methods, which allow us to

observe changes in arteries from different localizations and

calibers. Fig. 3 shows lumen : media (L/M) ratios and repre-

sentative microphotographs observed in arteries from different

localizations: left carotid (A), renal interlobar (B), renal arcuate

(C), and renal interlobular (D) arteries in each group. The

carotid lumen to media ratio in the FFR group was significantly

Table 1 Body weight, SBP, and metabolic parameters from C, C +DRW, FFR, and F + DRW rats.

Groups

C C + DRW FFRFFR +DRW

Body weight/g 340 � 7 324 � 9 349 � 7 325 � 7Plasma glycemia/

mmol L�1

4.0 � 0.2b 4.0 � 0.2b 6.3 � 0.4a 5.6 � 0.2a

Plasma insulin(pmol/L)

72 � 6c 75 � 8c 152 � 7a 118 � 6b

HOMA-IR 1.8 � 0.4c 1.9 � 0.5c 6.1 � 0.8a 4.2 � 0.6b

Plasmatriglycerides/mmol L�1

0.81 � 0.02b 0.72 � 0.07b 1.23 � 0.08a 1.08 � 0.08a

Plasma HDL/mmol L�1

0.92 � 0.04a 0.95 � 0.04a 0.80 � 0.02b 0.92 � 0.01a

Plasma totalcholesterol/mmol L�1

1.48 � 0.04 1.40 � 0.06 1.46 � 0.08 1.41 � 0.08

SBP (mmHg)1

Baseline 100 � 1 102 � 1 103 � 1 100 � 1Week 6 106 � 1b 107 � 1b 130 � 1a 129 � 1a

Week 10 115 � 1c 116 � 1c 136 � 1a 125 � 1b

Values are expressed as mean � SEM, n ¼ 8; means without a commonletter differ, P < 0.05.

a common letter differ, P < 0.05.

This journal is ª The Royal Society of Chemistry 2010

reduced, compared to control rats. Chronic administration of

DRW to FFR increased the L/M ratio to control levels. A similar

structural pattern was found in interlobar renal arteries (caliber

between 120 to 180 mm), in smaller caliber arteries (50 a 120 mm)

such as arcuate renal arteries and in very small arteries (10 a

50 mm) such as interlobular renal arteries.

Discussion

In the present study we demonstrate that DRW was able to

reverse vascular remodeling in fructose-fed rats, an experimental

model of MS, associated with an increased eNOS activity and

a reduced aortic NAD(P)H oxidase activity. These results

suggest that non-alcoholic constituents of red wine reverse the

structural and functional changes by mechanisms related to

oxidative stress enhanced in this model.

We have previously demonstrated the development of endo-

thelial dysfunction in this experimental model, supported by

a diminished NOc generation capability and changes in vascular

smooth muscle cell proliferative behavior in primary culture.4

These changes could be attributed to a significant increase in

ROS production, evaluated through an increased activity of

NAD(P)H oxidase, the most quantitatively important source of

Food Funct., 2010, 1, 124–129 | 125

Fig. 3 Lumen to media ratio observed in arteries from different localizations: left carotid (A), renal interlobar (B), renal arcuate (C), renal interlobular

(D) arteries described by representative microphotographs and analyzed in the bar graph from C, C + DRW, FFR, and F + DRW rats. Values are mean

� SEM (n ¼ 8). Bars without a common letter differ, P < 0.05. Arrows indicate the location of the arteries.

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superoxide at vascular level.10,11,18 ROS produced by vascular

wall cells can directly inactivate other biologically active free

radicals, thereby disturbing vascular homeostasis. One of the

main targets of ROS, particularly superoxide anion is NOc,

decreasing its bioavailability and favoring the formation of

peroxynitrite, a potent vasoconstrictor.19 In this study, we found

a decreased eNOS activity in both heart and mesenteric vascular

tissue in FFR. DRW administered to FFR was able to restore the

activity of this enzyme, suggesting that red wine polyphenols

could be responsible for these beneficial effects. The effects of

some polyphenols in these variables had been achieved. Quer-

cetin increased the activity of eNOS and downregulates the

activity and expression of NAD(P)H oxidase in an experimental

model of hypertension.20 Others studies have shown that

metabolites of flavonoids inhibit the activity of NAD(P)H

oxidase.21,22

In this study, DRW administered to FFR induced a slight but

significant decrease in systolic blood pressure, reduced the index

of insulin resistance and increased HDL cholesterol. Further-

more, chronic administration of DRW was able to revert the

vascular remodeling in FFR in both distribution and resistance

arteries. The structural changes in FFR could be associated to

vascular smooth muscle cell proliferative behavior previously

observed in vitro in this model.4 Red wine administration could

protect the NOc inactivation process by ROS and also increased

the NOc generating system activity. The final result could be the

inhibition of vascular remodeling associated with this experi-

mental model.

The beneficial effects of moderate red wine consumption have

been demonstrated in several studies. Some mechanisms involved

in those effects have pointed to the action of antioxidant prop-

erties of different polyphenols present in red wine.23 These

substances could induce endothelium-dependent vasodilatation

in human aorta and coronary arteries, inhibit vascular

smooth muscle cell proliferation and protect ischemic

126 | Food Funct., 2010, 1, 124–129

myocardium.15,16,24,25 In vivo, flavonoids such as quercetin

prevent endothelial dysfunction and reduce blood pressure,

oxidative stress and end-organ damage in hypertensive animals.26

Quercetin and theaflavin significantly attenuated the athero-

sclerotic lesion size in aorta arteries in ApoE deficient mice by

alleviating inflammation, improving NOc bioavailability, and

inducing heme oxygenase-1.27 The prevention of angiotensin

II-induced hypertension and endothelial dysfunction by red wine

polyphenol extract administration, with a normalization of

vascular superoxide anion production and NAD(P)H oxidase

expression, has also been described.28 It is important to note that

the administration of DRW to the control rats had no positive

effects on almost of all variables studied, suggesting that under

normal conditions DRW adds no further benefit.

Our results are in agreement with epidemiological and exper-

imental evidence demonstrating the beneficial effects of moder-

ated red wine consumption on cardiovascular pathology and

contribute to support the hypothesis that the non-alcoholic

fraction of wine, represented mainly by phenolic compounds

with antioxidant properties, may be the primary factor respon-

sible for this protective effect. Further studies are needed

to clarify the molecular mechanism of DRW on vascular

alterations.

Experimental

Animals and experimental design

All procedures were performed according to institutional

guidelines for animal experimentation and were approved by the

Technical and Science Secretary from the School of Medicine of

National University of Cuyo, Mendoza, Argentina. Thirty-day-

old male Wistar rats, weighting 90–130 g were housed during the

experimental period of 10 weeks in a room under conditions

of controlled temperature (21 � 2 �C), humidity and a 12 h

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light/dark cycle. At the beginning of the study, 32 rats were

randomly distributed into two groups: one control group (C)

(n ¼ 16) and one experimental group (FFR) (n ¼ 16). After six

weeks of treatment, the half of C and experimental groups were

assigned to receive 10 mL/Kg daily of DRW for four more weeks.

The names of each group were assigned as follows: Control (C);

C + DRW; FFR: 10% (w/v) fructose solution administration in

the drinking water during all the experimental protocol; and F +

DRW. All groups were fed the same standard rat diet (Gepsa-

Feeds, Buenos Aires, Argentina) and tap water ad libitum.

Administration of 10% fructose (Saporiti Labs., Buenos Aires,

Argentina) solution in drinking water was used to achieve the

pathological model.

The red wine (Malbec grape variety) was provided by the

School of Agricultural Sciences, National University of Cuyo.

The phenolic characterization of RW Malbec was evaluated by

high performance liquid chromatography as previously

described.29 One litre of red wine contained 2.9 g of total phenols

expressed as gallic acid. The main phenolic content was

(expressed as mg L�1): non-flavonoids: 18.2 gallic acid; 2 caffeic

acid; 4.2 cis-caftaric acid, trans-resveratrol: 1.1, flavonoids: 24.1

catechin; 14.2 epicatechin; procyanidin (11.3 B1; 3.1 B3), flavo-

nols: 4.9 quercetin, and anthocyanins (344 malvidin-3-glucoside;

16.2 peonidin-3-glucoside; 60.3 delphinidin-3-glucoside).

Red wine was dealcoholized by rotary evaporation at low

pressure and temperature, and then the volume of alcohol

evaporated was reconstituted with water, in order to conserve

phenolic composition. DRW (10 mL/Kg) was administered in

drinking water.

The weight of each animal was measured weekly and the

energy intake was recorded twice per week during the experi-

mental period in all groups.

At the end of the experimental period, and after an overnight

fast, the rats were weighed, anesthetized with ketamine (50 mg

kg�1) and acepromazine (1 mg kg�1). Blood was collected from

the abdominal aorta into heparinized tubes. Plasma obtained

after centrifugation was frozen at �70 �C until assayed. Arteries

and organs were excised aseptically for the measurement of

various parameters described below.

Systolic blood pressure (SBP). Systolic blood pressure was

monitored indirectly in conscious, pre-warmed (32 �C) slightly

restrained rats by the tail-cuff method and recorded on a Grass

Model 7 polygraph (Grass Instruments Co., Quincy, MA, USA).

Biochemical determinations

Plasma glucose, triglycerides, HDL-cholesterol and total

cholesterol concentrations were determined using commercial

kits by enzymatic colorimetric methods (Wiener Lab, Rosario,

Argentina). Insulin was measured by RIA (Coat-A-Count,

Siemens, CA, USA), and insulin resistance was assessed using the

homeostasis model assessment (HOMA-IR) described by

Mathew et al.30 HOMA-IR was calculated using the following

formula: HOMA-IR (mmol L�1 � mU/mL) ¼ fasting glucose

(mmol L�1) � fasting insulin (mU/mL)/22.5.

Measurements of eNOS activity. Ca2+/calmodulin-dependent

nitric oxide synthase activity was measured in homogenates from

This journal is ª The Royal Society of Chemistry 2010

mesenteric resistance arteries and left ventricular cardiac tissue

by the conversion of L-[3H]arginine to L-[3H]citrulline, as

previously described.4 Mesenteric vessels were homogenized on

ice for four 15 s intervals with a Politron homogenizer and then

sonicated in a buffer (pH 7.4, 37 �C) containing 50 mmol L�1

Tris, 20 mmol l�1 HEPES, 250 mmol L�1 sucrose, 1 mmol L�1

dithiothreitol, 10 mg mL�1 leupeptin, 10 mg mL�1 soybean trypsin

inhibitor, 5 mg mL�1 aprotinin and 0.1 mmol L�1 phenyl methyl

sulfonyl fluoride. Heart tissue from left ventricle myocardium

was also homogenized on ice for four 15 s intervals with a poly-

tron homogenizer and then sonicated in the same buffer. After

centrifugation of the homogenates (100 g, 5 min, 4 �C) and

determination of the protein content (Bradford method),

aliquots of 50 mL were added to 100 mL of a cocktail reaction

buffer containing 50 mmol L�1 Tris, 20 mmol L�1 HEPES, 1

mmol L�1 dithiothreitol, 1 mmol L�1 NADPH, 0.1 mmol L�1

tetrahydrobiopterin, 50 mmol L�1 FAD, 50 mmol L�1 FMN, and

10 mCi/ml L-[2,3-3H]-arginine (New England Nuclear, Boston

MA), and incubated for 30 min at 37 �C in a shaking bath in the

presence of 10 mg ml�1 calmodulin and 3 mmol l�1 CaCl2 or with

3 mmol L�1 EGTA in the absence of Ca2+/calmodulin. The

reaction was stopped by adding 1 mL cold distilled water and the

mixture applied to an anion-exchange chromatography column

containing Dowex AG 50W-X8 (200–400 Mesh) resin previously

saturated with 50 mL of 100 mmol L�1 L-citrullin and 2 mL of

50 mmol L�1 Tris, 20 mmol L�1 HEPES buffer (pH 7.4) and

eluted with 2 mL of distilled water. Specifically eluted L-[3H]ci-

trulline concentration was determined by liquid scintillation

counting. The calcium-dependent NOS activity was calculated as

the difference between activity in the presence and absence of

Ca2+/calmodulin. Values were corrected to the amount of protein

present in the homogenates and the incubation time (dpm/mg

protein/min). Each rat mesenteric vascular bed and heart tissue

was processed and eNOS activity measured independently.

Measurement of vascular NAD(P)H oxidase activity. The

lucigenin-derived chemiluminescence assay was used to determine

NAD(P)H oxidase activity in the aorta as previously described.18

A 2 cm length segment of thoracic aorta was cut, cleaned, washed,

transferred to a tube with 2 mL of Jude’s Krebs buffer (JKB)

containing (in mmol L�1) 2 HEPES, 11.9 NaCl, 0.46 KCl, 0.1

MgSO4$7H2O, 0.015 Na2HPO4, 0.04 KH2PO4, 0.5 NaHCO3, 1.2

CaCl2, 5.5 glucose; pH 7.40; and equilibrated at 37 �C during

30 min. Then the aortic segment was transferred to a tube con-

taining 1 mL JKB and 5 mmol L�1 lucigenin and left in darkness at

room temperature for 10 min. This concentration of lucigenin

does not appear to be involved in redox cycling and specifically

detects superoxide anion. To assess NAD(P)H oxidase activity,

500 mmol L�1 bNAD(P)H was added and chemiluminescence was

immediately measured in a liquid scintillation counter (LKB

Wallac Model 1219 Rack-Beta Scintillation Counter, Finland) set

in the out-of coincidence mode. Time-adjusted and normalized to

tissue weight scintillation counts were used for calculations.

Measurements were repeated in the absence and presence of

diphenylene iodinium (DPI) (10�6 mol L�1), which inhibits flavin-

containing enzymes, including NAD(P)H oxidase.

Tissue preservation. Tissue samples for histopathology were

processed as previously reported.10 Samples from all rats were

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used in these observations. The kidneys were in vivo perfused

with PBS (pH 7.40, 4 �C) through the renal artery over 5 min.

For histological studies, left kidneys were then perfused with 4%

paraformaldehyde solution for 10 min, then additionally fixed by

immersion in the same solution for 48 h, introduced to a 30%

sucrose solution and kept at �70 �C. Five mm thick tissue slices

were transversely cut through the entire kidney on a cryostat

(Microm HM 505E, Germany) at �26 �C and processed for

histological studies. Common left carotid arteries were fixed and

processed as described above for kidneys.

Histopathology and morphometry. Lumen to media ratio in

kidney arteries transversal slices from common left carotid artery

and left kidney were placed on microscope slides and stained with

Masson’s trichrome solution and examined under a light micro-

scope (Nikon Optiphot-2, Kanagawa, Japan). Images were digi-

talized with a digital camera (GP-KR222 color CCD, Panasonic,

Osaka, Japan) and processed with an analysis system Scion Image

4.01 (Scion, Bethesda, MD, USA). To evaluate the renal arterial

wall thickening, images from three different artery types were

studied in each kidney: interlobar, arcuate and interlobular

arteries. The lumen-to-wall media ratio (the internal diameter to

the medial thickness) was then calculated. Forty slices from each

kidney were processed and 5 to 10 arteries of each type in each slice

were analyzed, in order to obtain an average value for each rat.

The average values were then used for final analysis. Common left

carotid arteries were sectioned transversely. L/M was then

calculated in 10 slices from each artery, in order to obtain an

average value for each rat and then used for final analysis.

Reagents

Unless otherwise noted, reagents were purchased from Sigma

Chemical Co, MO USA. All other chemicals were of molecular

biology or reagent grade.

Statistical and data analysis

Results were expressed as mean and their deviation errors. The

statistical significance was assessed by one-way ANOVA fol-

lowed by Student-Newman-Keuls post-test using GraphPad

Prism version 5.00 for Windows, GraphPad Software, San

Diego, California USA. Differences were considered significant

at p < 0.05. In the figures and tables, data shown without

a common letter differ at a p < 0.05 significance level.

Conclusions

The non-alcoholic constituents of red wine increased the eNOS

activity, reduced the activity of the enzyme NAD(P)H oxidase

and reversed vascular remodeling. The antioxidant properties of

polyphenols could be responsible for the beneficial effects of

DRW.

Acknowledgements

We thank Susana Gonzalez and Cristina Lama for their technical

assistance. This work was supported by grants from Program 06/

P01 SECTyP Universidad Nacional de Cuyo, and PIP-5192

CONICET.

128 | Food Funct., 2010, 1, 124–129

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27 W. M. Loke, J. M. Proudfoot, J. M. Hodgson, A. J. McKinley,N. Hime, M. Magat, R. Stocker and K. D. Croft, Specific dietarypolyphenols attenuate atherosclerosis in apolipoprotein E-knockoutmice by alleviating inflammation and endothelial dysfunction,Arterioscler., Thromb., Vasc. Biol., 2010, 30, 749–757.

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