Ecole Doctorale des Sciences de la Vie et de la Santé

Laboratoire de Biophotonique et Pharmacologie

UMR CNRS 7213 - Faculté de Pharmacie

THÈSE

Présentée pour l’obtention du grade de

DOCTEUR DE L’UNIVERSITE DE STRASBOURG

Discipline : Sciences du Vivant – Aspect Moléculaire et Cellulaire de la Biologie

Spécialité : Pharmacologie

Potentiel des polyphénols à prévenir la dysfonction endothéliale liée au

vieillissement et à la sénescence des cellules endothéliales

Par Noureddine IDRIS KHODJA

Soutenue publiquement le 22 juin 2012 devant le jury composé de :

Rapporteur externe : Docteur Jean Max ROUANET, Montpellier, France

Rapporteur externe : Professeur Paul M VANHOUTTE, Hong Kong, Chine

Rapporteur interne : Professeur Jean-Christophe CASSEL, Strasbourg, France

Membre invité : Docteur Martin BURKART, Karlsruhe, Allemagne

Directrice de thèse : Professeur Valérie SCHINI-KERTH, Strasbourg, France

Remerciements

Je tiens à exprimer mes remerciements les plus sincères au Professeur

Valérie SCHINI-KERTH, ma directrice de thèse, pour m’avoir conseillée,

encouragée et soutenue tout au long de la thèse avec patience et disponibilité, et

pour la confiance qu’elle m’a accordée.

Je remercie très chaleureusement Professeur Paul VANHOUTTE d’avoir

accepté d’être rapporteur de cette thèse. Sa présence dans le jury est un grand

honneur pour moi.

Je tiens également à remercier le Professeur Jean-Christophe CASSEL

et le Docteur Jean Max ROUANET de me faire l’honneur d’être membre de ce

jury. Qu’ils trouvent ici le témoignage de ma profonde reconnaissance.

Je dois aussi un grand merci au Docteur Thierry CHATAIGNEAU, qui fut

mon tuteur de Master, à mon arrivée au laboratoire. C’est lui qui m’a initié à la

recherche ; et tout au long de ces années, il m’a toujours apporté son soutien.

Qu’il retrouve ici le témoignage de ma gratitude.

Je remercie aussi Docteur Cyril AUGER pour sa disponibilité, sa

sympathie et sa transmission de savoir.

Je suis tout particulièrement gré au Docteur Phillipe CHABERT pour sa

relecture et ses conseils toujours très pertinents.

Merci également aux personnels techniques du laboratoire, et plus

particulièrement Brigitte POLLET et Evelyne LACOFFRETTE, pour leurs aides

et gentillesse.

Mes remerciements vont aussi à toutes les personnes du laboratoire de

Valérie et de l’UMR7213

i
 Enfin, je remercie du plus profond de mon cœur toute ma famille. Leur

présence inconditionnelle a fait de moi la personne que je suis devenue. Leur

appui et encouragements m’ont aidé à progresser et à évoluer. Pour ça et pour

tout ce qu’ils ont pu m’offrir, je leur en serai éternellement reconnaissant. Merci.

Je vous aime.

ii
Citation

Longevity is a vascular question, which has been well expressed in the

axiom that man is only as old as his arteries. To a majority of men

death comes primarily or secondarily through this portal. The onset of

what may be called physiological arteriosclerosis depends, in the first

place, on the quality of arterial tissue which the individual has

inherited, and secondarily on the amount of wear and tear to which he

has subjected it.

—Sir William Osler, 1891

iii
Publications et communications scientifiques

Publications originales

Faisant l’objet de ce travail :

1. Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valerie B. Schini-Kerth. Intake of
grape-derived polyphenols causes a persistent improvement of aging-related endothelial
dysfunction in the rat mesenteric artery. PLoS One. 2012;7(2):e32039. Epub 2012 Feb 27.

2. Noureddine Idris Khodja & Valerie B. Schini-Kerth. Thymoquinone improves aging-related

endothelial dysfunction in the rat mesenteric artery. Naunyn Schmiedebergs Arch Pharmacol, sous
presse, 2012.

3. Noureddine Idris Khodja, Cyril Auger, Egon Koch, Valerie B Schini-Kerth. Crataegus special
extract WS®1442 prevents aging-related COX-mediated endothelium-dependent contractions.
Phytomedicine 2012, sous presse.

4. Noureddine Idris Khodja, Marouane Kheloufi, Valerie B. Schini-Kerth. Crataegus special

extract WS®1442 delays replicative endothelial senescence: role of eNOS-derived NO. (en
préparation, 2012).

Ne faisant pas l’objet de ce travail :

1. Jean-Marie Tokoudagba, Cyril Auger, Lise Bréant, Saliou N’Gom, Philippe Chabert, Noureddine
Idris-Khodja, Fernand Gbaguidi, Joachim Gbenou, Mansourou Moudachirou, Annelise Lobstein,
Valerie B. Schini-Kerth. Procyanidin-rich fractions from Parkia biglobosa (Mimosaceae) leaves
cause redox-sensitive endothelium-dependent relaxation involving NO and EDHF in porcine
coronary artery. J Ethnopharmacol. 2010 Oct 28;132(1):246-50. Epub 2010 Aug 18.

2. Noureddine Idris Khodja, Paola Di Marco, Mona Farhat, Thierry Chataigneau, Bernard Geny,
Valerie B. Schini-Kerth. Grape-derived polyphenols prevent the blunted EDHF-mediated
relaxation induced by doxorubicin in the rat mesenteric artery. (en préparation, 2012).

3. Noureddine Idris Khodja & Valerie B. Schini-Kerth. Grape-derived polyphenols don’t

improve aging-related endothelial dysfunction in mesenteric artery of very old rats. (en
préparation, 2012).

iv
4. Hélène Kremer, Jan Jun Zhu, Noureddine Idris Khodja, Julie Boisramé-Helms, Veronique
Kemmel, Eric Epailly, Bernard Geny, Valerie Schini-Kerth, Ferhat Meziani. Black currant
polyphenols decrease cyclosporine-induced hypertension and endothelial dysfunction. (soumis,
2012).

Revue

1. Cyril Auger, Noureddine Idris-Khodja, Valerie B. Schini-Kerth. Endothelial control of vascular

tone by chocolate and other polyphenols. In Chocolate in Health and Nutrition, Watson, Ronald
Ross; Preedy, Victor R.; Zibadi, Sherma (Eds.) Humana Press, 2012.

Communications orales

1. Noureddine Idris Khodja & Valerie B. Schini-Kerth. Thymoquinone improves aging-related

endothelial dysfunction in the rat mesenteric artery. Printemps de la Cardiologie. 12-13 Avril
2012, Bordeaux, France.

2. Sherzad Khorsheed Rashid, Noureddine Idris Khodja, Cyril Auger, Monique Oswald-
Mammosser, Nelly Boehm, Valerie B Schini-Kerth. Probiotics (VSL #3) prevent endothelial
dysfunction in the mesenteric artery of cirrhotic rats with hepatopulmonary syndrome. Journées du
Campus d'Illkirch, 2- 3 Avril 2012, Illkirch, France.

3. Cyril Auger, Noureddine Idris Khodja, Valerie B Schini-Kerth. Prevention of aging-related

endothelial dysfunction by polyphenols: evidence from animal studies. 5th International
Conference on Polyphenols and Health, 17-20 octobre 2011, Sitges, Barcelona, Espagne.

4. Noureddine Idris Khodja, Cyril Auger, Egon Koch, Valerie Schini-Kerth. Crataegus special
extract WS1442® prevents aging-related endothelial dysfunction: Role of ROS and COX-
dependent EDCFs. Journées du Campus d'Illkirch, 3-4 mai 2011, Illkirch, France.

5. Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valerie B Schini-Kerth. Aging-
induced endothelial dysfunction in the rat mesenteric artery is reversed by chronic intake of grape-
derived polyphenols. Winehealth 2010, October 2010, Udine, Italie.

v
 Posters

1. Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valerie B. Schini-Kerth. Grape-
derived polyphenols improved aging-related endothelial dysfunction in the rat mesenteric artery:
Role of oxidative stress and the angiotensin system. Printemps de la Cardiologie. 12-13 Avril
2012, Bordeaux, France.

2. Hélène Kremer, Janjiun Zhu, Noureddine Idris khodja, Anne Laure Lang, Valerie Schini-Kerth,
Bernard Geny, Ferhat Meziani. Beneficial effects of polyphenols (PP) on cyclosporine-induced
endothelial dysfunction. 8ème congrès de Physiologie, Pharmacologie et Thérapeutique, 4-6 avril
2012, Dijon, France.

3. Noureddine Idris Khodja, Cyril Auger, Egon Koch, Valerie Schini-Kerth. Crataegus special
extract WS1442® prevents aging-related endothelial dysfunction: Role of ROS and COX-
dependent EDCFs. 5th International Conference on Polyphenols and Health, 17-20 octobre 2011,
Sitges, Barcelona, Espagne.

4. Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valerie Schini-Kerth. Grape-
derived polyphenols cause a persistent improvement of aging-related endothelial dysfunction in
the rat mesenteric artery: Role of oxidative stress and the angiotensin system. 5th International
Conference on Polyphenols and Health, 17-20 octobre 2011, Sitges, Barcelona, Espagne.

5. Noureddine Idris Khodja, Cyril Auger, Egon Koch, Valerie B Schini-Kerth. Crataegus special
extract WS1442® prevents aging-related COX-mediated endothelium-dependent contractions.
Printemps de la cardiologie. 12-14 mai 2011, Lyon, France.

6. Noureddine Idris Khodja, Paola Di Marco, Mona Farhat, Thierry Chataigneau, Bernard Geny,
Valerie B. Schini-Kerth. Grape-derived polyphenols prevent the blunted EDHF-mediated
relaxation induced by doxorubicin in the rat mesenteric artery. Printemps de la Cardiologie. 12-14
mai 2011, Lyon, France.

7. Noureddine Idris Khodja, Paola Di Marco, Mona Farhat, Thierry Chataigneau, Bernard Geny,
Valerie B. Schini-Kerth. Intake of grape-derived polyphenols prevents doxorubicin-induced
endothelial dysfunction in the rat mesenteric artery. Journées du Campus d'Illkirch, 3-4 mai 2011,
Illkirch, France.

vi
8. Noureddine Idris Khodja, Cyril Auger, Egon Koch, Valerie Schini-Kerth. Crataegus special
extract WS1442 prevents aging-related endothelial dysfunction in the mesenteric artery: Role of
COX-dependent EDCFs. Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie
und Toxikologie. 30 March - 1 April, 2011, Frankfurt am Main, Allemagne.

9. Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valerie.B Schini-Kerth. Chronic
intake of grape-derived polyphenols improves EDHF-mediated relaxation in the mesenteric artery
of old rats: Role of angiotensin II and AT1 receptors. WorldPharma2010. 16th World Congress on
Basic and Clinical Pharmacology, 17-23 July 2010, Copenhagen, Danemark.

10. Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valérie.B Schini-Kerth. Grape-
derived polyphenols improve NO- and EDHF-mediated relaxation in the mesenteric artery of old
rats: role of angiotensin II and oxidative stress. Journées du Campus d'Illkirch, 3-4 mai 2010,
Illkirch, France.

vii
 ABREVIATIONS

AA acide arachidonique
AngII angiotensine II
ANOVA analyse de variance
AMPc adénosine monophosphate cyclique
APA apamine
Ach acétylcholine
AT1 récepteur de l’angiotensine II de type 1
Cav-1 cavéoline 1
COX cyclooxygénases
C.R.E.M.E.A.S. Comité Régional d'Ethique en Matière d'Expérimentation Animale de
Strasbourg
CTX charybdotoxine
CYP cytochrome P450 époxygénases
EDCFs facteurs vasoconstricteurs dérivés de l’endothélium
EDHF facteur hyperpolarisant dérivés de l’endothélium
EDRFs facteurs vasodilatateurs dérivés de l’endothélium
EETS acides époxyeicosatriénoiques
eNOS NO synthase endothéliale
ET-1 endothéline
sGC Guanylate cyclase soluble
GMPc Guanosine monophosphate cyclique
GPx Glutathion peroxydase
H2O2 Peroxyde d’hydrogène
iNOS NO synthase inductible
IKCa canaux potassiques de moyenne conductance, dépendant du calcium
L-NA Nω-nitro-L-arginine
NADPH oxidase nicotinamide adénine dinucléotide phosphate oxydase
NO monoxyde d’azote
O2●- anion superoxide
OH●- ion hydroxyles
ONOO●- peroxynitrite
PAI-1 plasminogen activator inhibitor-1
viii
PGI2 prostacycline
ROS espèces réactives de l’oxygène
SA-β-Gal Senescence Associated β-galactosidase
RWPs composés polyphénoliques du vin rouge
SOD Superoxyde dismutase
SKCa canaux potassiques de faible conductance, dépendant du calcium
SNP nitroprussiate de sodium
TGF-β Transforming Growth Factor-β
TNF-α Facteur de nécrose tumoral α (Tumor Necrosis Factor-α)
TQ thymoquinone
X-Gal 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside

ix
 RÉSUMÉ

L’endothélium qui tapisse la paroi interne des vaisseaux sanguins joue un rôle

primordial dans la régulation de l’homéostasie vasculaire, en partie, par la libération de

facteurs relaxants et contracturants qui contrôlent le tonus vasculaire. Les principaux facteurs

relaxants sont : le monoxyde d’azote (NO), le facteur hyperpolarisant dérivé de l’endothélium

(endothelium-derived hyperpolarizing factor, EDHF) et la prostacycline (PGI2). Les facteurs

vasoconstricteurs sont représentés essentiellement par certains prostanoïdes, l’endothéline

(ET-1) et les anions superoxydes (O2●-).

Le vieillissement se traduit par une altération progressive du système cardiovasculaire

avec notamment l’apparition d’une dysfonction endothéliale caractérisée par une diminution

de la relaxation dépendante de l’endothélium dans différents lits vasculaires. La dysfonction

endothéliale est considérée comme un marqueur, mais aussi comme un événement précoce

contribuant à l’initiation de pathologies cardiovasculaires. Ainsi, la prévention ou le

traitement de la dysfonction endothéliale pourrait réduire leur incidence. Des études récentes

suggèrent que la dysfonction endothéliale liée au vieillissement est associée à une sénescence

des cellules endothéliales, définie par un arrêt irréversible de la division cellulaire avec des

modifications morphologiques et métaboliques et un phénotype pro-inflammatoire et pro-

athérosclérotique.

Des études épidémiologiques ont montré qu’une consommation régulière de

polyphénols contenus dans certains aliments (comme le vin rouge, le thé, les céréales et

certains fruits) est associée à une réduction de la morbidité et de la mortalité cardio-

vasculaires. Les effets protecteurs des composés polyphénoliques pourraient être attribués, au

moins en partie, à leurs propriétés antioxydantes permettant de neutraliser les espèces

réactives de l’oxygène (ROS), à la stimulation d’enzymes antioxydantes endogènes et à

x
l’inhibition d’enzymes pro-oxydantes comme la xanthine oxydase et la NADPH oxydase.

Ainsi, il a été proposé que ces propriétés antioxydantes améliorent la biodisponibilité du NO

en le protégeant de sa dégradation par les anions superoxydes mais aussi en empêchant le

découplage de la eNOS. L’effet protecteur des polyphénols pourrait aussi s’expliquer par leur

capacité à induire des relaxations endothélium-dépendantes des artères en stimulant la

formation endothéliale des facteurs vasoprotecteurs : le NO et l’EDHF.

Le but de ce travail de recherche a été d’évaluer les effets préventifs et curatifs de produits

naturels (extrait du vin rouge, extrait d’aubépine et la thymoquinone) contre la dysfonction

endothéliale liée au vieillissement chez le rat et l’induction d’une sénescence réplicative des

cellules endothéliales en culture.

La première étude a porté sur les effets curatifs ainsi que la cinétique d’induction et de

disparition des effets protecteurs des polyphénols du vin rouge sur la dysfonction endothéliale

liée au vieillissement chez des rats Wistar âgés de 46 semaines en comparaison à des rats

jeunes âgés de 16 semaines. Le vieillissement vasculaire est caractérisé par une diminution

des composantes NO et l’EDHF de relaxation dans l’artère mésentérique et est associé à une

diminution de l’expression de la NO synthase endothéliale (eNOS) et des canaux potassiques

dépendant du calcium (SKCa, IKCa small and intermediate conductance calcium-activated

potassium channels respectivement). Le vieillissement est également associé à un stress

oxydant et une surexpression de l’angiotensine II et des récepteurs AT1 dans la paroi

artérielle. L’ingestion de polyphénols du vin rouge dans l’eau de boisson pendant deux

semaines améliore les composantes NO et l’EDHF de relaxation chez les rats âgés. Cet effet

est associé à une normalisation de l’expression de la eNOS, de SKCa, de IKCa et une

diminution du stress oxydant et de la surexpression de l’angiotensine II et des récepteurs AT1.

Cet effet protecteur est maintenu après deux semaines d’arrêt de la consommation des

polyphénols indiquant que les polyphénols ont un effet curatif soutenu dans le temps.

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 La deuxième étude a évalué l’effet curatif d’une molécule naturelle non

polyphénolique qui est la thymoquinone (TQ). La TQ est le composé le plus actif de l’huile de

graines de Nigella sativa (Cumin noir), présentant des propriétés antioxydantes et

vasodilatatrices. Notre étude indique que le traitement de rats âgés de 46 semaines pendant 2

semaines avec la TQ améliore les composantes NO et l’EDHF de relaxation dans l’artère

mésentérique, grâce en partie, à l'inhibition du stress oxydant et la normalisation de

l’expression de l'angiotensine II et des récepteurs AT1 dans la paroi artérielle.

Dans la troisième étude, nous avons montré qu'un traitement préventif et chronique

avec un extrait d’aubépine (Crataegus spp) pendant 40 semaines empêche l’apparition d’une

dysfonction endothéliale liée au vieillissement chez des rats Wistar âgé de 65

semaines, laquelle est caractérisée par une augmentation des réponses contractiles

dépendantes de l’endothélium. Cet effet protecteur est associé à une diminution du

stress oxydant et de la surexpression des cyclooxygénases dans la paroi artérielle.

Enfin, dans la dernière étude, nous nous sommes intéressés à la sénescence réplicative

des cellules endothéliales des artères coronaires de porc et à l’effet protecteur des

polyphénols. La sénescence réplicative a été induite par des réplications successives d’une

culture primaire de cellules endothéliales d’artères coronaires de porc. La sénescence a été

évaluée par la mesure de l’activité de la β-galactosidase à pH=6, par l’expression de p53

protéine suppresseur de tumeurs et son effecteur p21 protéine inhibitrice du cycle cellulaire,

toutes les deux fortement impliquées dans la sénescence réplicative. Notre étude indique que

les réplications successives à partir d’une culture primaire de cellules endothéliales sont

associées à une augmentation de l’activité de la β-galactosidase, de l’expression de p53 et de

p21. La sénescence réplicative est diminuée par l’inhibiteur de l’activité de p53 (pifithrin).

L'extrait d’aubépine réduit de manière concentration-dépendante l’induction de la sénescence

réplicative des cellules endothéliales en culture évaluée par l’activité de la β-galactosidase.

xii
Cet effet protecteur est associé à une diminution l’expression de p21 sans effet sur

l’expression de p53. Nous avons également trouvé que la sénescence réplicative est associée à

une diminution de l’expression de la eNOS. L’inhibition pharmacologique de la eNOS dans

les cellules endothéliales de passage 1 augmente l’activité de la β-galactosidase. Cette

augmentation de la sénescence est inhibée par un prétraitement avec l’extrait d’aubépine. De

plus, l’effet protecteur d’un traitement avec l’extrait d’aubépine contre la sénescence dans des

cellules endothéliales de passage 3 est partiellement bloqué par l’inhibition pharmacologique

de la eNOS. Le traitement avec l’extrait d’aubépine augmente l’expression de la eNOS dans

les cellules de passage 3 et 4. L’ensemble de ces résultats suggère que l’extrait d’aubépine

diminue la sénescence endothéliale probablement par l’augmentation de l’expression de la

eNOS et de la formation du NO et par diminution de l’activité de p53.

En conclusion, les polyphénols ont des propriétés préventives et curatives vis-à-vis de

la dysfonction endothéliale liée au vieillissement qui se manifestent par une amélioration des

relaxations médiées par le NO et l’EDHF et par une diminution des réponses contractiles

dépendantes de l’endothélium. Ces effets protecteurs s’expliquent vraisemblablement par une

diminution du stress oxydant et une normalisation du système angiotensine II dans la paroi

artérielle. Les effets protecteurs des polyphénols se manifestent également par une diminution

de la sénescence réplicative des cellules endothéliales probablement par augmentation de

formation de NO. Les résultats de ce travail de recherche confirment et expliquent les données

cliniques indiquant que la consommation régulière d’aliments riches en polyphénols diminue

le risque cardiovasculaire et augmente l’espérance de vie.

xiii
 Table de Matière

Remerciements i

Citation iii

Publications et communications scientifiques iv

Abréviations viii

Résumé x

Table de matière xii

1. Introduction 1

1.1. Rappel historique 1

1.2. La fonction endothéliale : rôle clé dans la régulation du tonus vasculaire 3

1.2.1. Les facteurs relaxants dérivés de l’endothélium 3

1.2.1.1. Le monoxyde d’azote 3

1.2.1.2. Le facteur hyperpolarisant dérivé de l’endothélium 10

1.2.1.3. La prostacycline 11

1.2.2. Les facteurs contracturants dérivés de l’endothélium 11

1.2.2.1. Les dérivés de l’acide arachidonique vasoconstricteurs 12

1.2.2.2. Les espèces réactives d’oxygène 12

1.3. La dysfonction endothéliale liée au vieillissement 13

1.3.1. Diminution des facteurs vasodilatateurs dérivés de l’endothélium 14

1.3.1.1. Diminution de la composante NO de relaxation 14

1.3.1.1.1. Diminution de la disponibilité du précurseur L-arginine 14

1.3.1.1.2. Diminution de la disponibilité du cofacteur BH4 15

1.3.1.1.3. Augmentation des inhibiteurs endogène de la eNOS 15

1.3.1.1.4. Diminution de l’activité de la eNOS 16

1.3.2. Diminution de l’EDHF 16

1.3.3. Augmentation de l’EDCF 16

1.3.4. Augmentation du stress oxydant 17

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 1.4. La sénescence endothéliale 18

1.5. Propriétés protectrices des composés polyphénoliques vis-à-vis le système

cardiovasculaire 19

1.5.1. Les flavonoïdes 22

1.5.2. Les non-flavonoïdes 24

1.6. La thymoquinone : rôle dans la protection cardiovasculaire 25

2. Objectifs des études expérimentales 27

3. Publications scientifiques 28

Article 1 "Les polyphénols dérivés du raisin améliorent la dysfonction endothéliale

liée au vieillissement : Rôle des ROS et du système angiotensine II 29

Article 2 "La thymoquinone améliore la dysfonction endothéliale liée au vieillissement" 43

Article 3 "Les polyphénols de l’aubépine préviennent la dysfonction endothéliale liée

au vieillissement : Rôle des COX" 56

Article 4 "Effets protecteurs des polyphénols de l’aubépine contre la sénescence

réplicative des cellules endothéliales : role du NO produit par la eNOS" 68

4. Discussion générale et perspectives 91

5. Bibliographie 103

xv
 1. INTRODUCTION

1. Rappel historique

L’endothélium tapisse la lumière de tous les vaisseaux sanguins et lymphatiques et

joue un rôle primordial dans plusieurs fonctions physiologiques telles que la régulation du

tonus vasculaire, l’hémostase, la perméabilité endothéliale, la prolifération des cellules

musculaires lisses et la réponse immunitaire. Le terme endothélium a été donné pour la

première fois par l’anatomiste suisse Wilhelm His en 1661 pour différencier la paroi interne

des cavités du corps. La définition initiale inclut la paroi interne des vaisseaux sanguins,

lymphatiques, et des cavités mésothéliales. La définition a évolué plus tard pour inclure

seulement la couche intérieure des vaisseaux sanguins et lymphatiques (D'après Aird 2007).

En 1980, Furchgott a montré que l’endothélium est indispensable pour la relaxation

des vaisseaux sanguins induite par l’acétylcholine via la libération d’un facteur relaxant

dérivé de l’endothélium appelé sous l’acronyme EDRF (Endothelium-Derived Relaxing

Factor, Furchgott & Zawadzki 1980). Ensuite, il a été montré que l’EDRF stimule la guanylyl

cyclase soluble dans le muscle lisse vasculaire (Ignarro et al. 1986) et est dégradé par les

anions superoxydes (Gryglewski et al. 1986; Rubanyi & Vanhoutte 1986). Des propriétés

similaires à celles du monoxyde d’azote (NO) connu également pour activer la guanylyl

cyclase soluble et être piégé par les anions superoxydes (Murad et al. 1978). En 1986,

Furchgott et Ignarro rapportaient pour la première fois que le NO et l’EDRF relaxaient le

muscle lisse vasculaire de façon identique et proposaient que l’EDRF soit le NO (Ignarro et

al. 1986; Furchgott et al.,1988). Cette conclusion a été confirmée par les travaux du groupe de

Moncada montrant que les cellules endothéliales produisaient du NO et de la L-citruline à

partir de la L-arginine par une enzyme appelée par suite la NO synthase (NOS) et qui est

inhibée par la NG-monomethyl-L-arginine (Palmer et al. 1988; Palmer & Moncada 1989). Les

1
travaux de recherche de Robert Furchgott, Louis Ignarro et Ferid Murad ont été honorés par

l’attribution de Prix Nobel de Médecine en 1998.

Le NO n’est pas le seul EDRF. Dans certains vaisseaux, la prostacycline (PGI2)

formée par la cyclooxygénase joue également un rôle important dans les relaxations

dépendantes de l’endothélium (De Mey et al. 1982). De plus, Bolton a montré que la

stimulation muscarinique de l’endothélium entraine une hyperpolarisation du muscle

lisse vasculaire via la libération d’un facteur endothélial (Bolton et al. 1984). Ce troisième

EDRF impliquant une hyperpolarisation du muscle lisse vasculaire dépendante de

l’endothélium a été nommé EDHF (Endothelium-derived hyperpolarizing factor, Bolton &

Clapp 1986; Komori & Suzuki 1987; Feletou & Vanhoutte 1988).

L’endothélium peut aussi libérer des facteurs endothéliaux entrainant des réponses

contractiles du muscle lisse vasculaire (De Mey & Vanhoutte 1982), notamment chez les

animaux âgés (Koga et al. 1989), hypertendus (Luscher & Vanhoutte 1986; Yang et al. 2003)

ou diabétiques (Shi et al. 2007a). Ces facteurs contracturants dérivés de l’endothélium sont

regroupés sous l’acronyme EDCFs (Endothelium-Derived Contracting Factors).

2
1.2. La fonction endothéliale : rôle clé dans la régulation du tonus vasculaire

L’endothélium joue un rôle primordial dans la régulation du tonus vasculaire en

libérant des facteurs endothéliaux relaxants et contractiles.

1.2.1. Les facteurs relaxants dérivés de l’endothélium

Il était rapporté dans le rappel historique sur la fonction endothéliale qu’il existe trois

facteurs relaxants endothéliaux qui sont : le monoxyde d’azote, le facteur hyperpolarisant

dérivé de l’endothélium et la prostacycline (Figure 1).

1.2.1.1. Le monoxyde d’azote (NO)

En plus de ses propriétés vasodilatatrices, le NO a des effets anti-inflammatoires,

antiprolifératifs et antiagrégants. Le NO est formé par une enzyme qui s’appelle la NO

synthase (NOS). Il existe trois isoformes de NOS numérotées par ordre de séquençage : la

NOS neuronale (nNOS, NOS 1), la NOS inductible (iNOS, NOS 2) et la NOS endothéliale

(eNOS, NOS 3) qui nous intéressera plus particulièrement dans ce travail. La eNOS comprend

deux domaines structuraux et fonctionnels : un domaine oxygénase du côté N-terminal,

auquel se lient l’arginine, la tétrahydrobioptérine (BH4) et l’hème, relié par le site de fixation

de la calmoduline à un domaine réductase du côté C- terminal, contenant les sites de fixation

du FAD+, du FMN et du NADPH (Rasmusen et al. 2005). La NOS catalyse la synthèse du

radical libre NO, de L-citruline et de NADP à partir de L-arginine, de NADPH et d’oxygène

(Figure 2) (Moncada et al. 1991).

3
 ADP Shear stress Plaquettes
BK
5-HT
-
+
Src NO PGI2
Cellule [Ca2+]i +
endotheliale PI3K
+ +
Ca2+/CaM Akt

+ +
eNOS
L-Arg COX AA
+
K+ K+ PGI2
EDHF NO
Jonction gap IKCa SKCa
myoendothéliale
+ +
sGC GTP AC ATP
Hyperpolarisation

cGMP cAMP

cellules
musculaires lisses
Relaxation

Figure 1: Facteurs vasodilatateurs dérivés de l’endothélium. La eNOS produit du NO à partir de la

L-arginine (L-Arg) après activation par les forces de cisaillement (shear stress) via la voie Src/PI3-
kinase/Akt ou par des agonistes (5-HT, sérotonine; BK, bradykinine; ADP, adénosine diphosphate) via
la voie calcium/calmodulin (Ca2+/CaM). Le NO relache les cellules musculaires lisses vasculaires via
l’activation de la guanylyl cyclase soluble (sGC). Le facteur hyperpolarisant dérivé de l’endothélium
(Endothelium-derived hyperpolarizing factor, EDHF) implique l’activation des canaux potassiques
dépendant du calcium de faible et de moyenne conductance, SKCa et IKCa (small and intermediate
conductance Ca2+-activated K+ channels, respectivement) induisant une hyperpolarisation ( ) de
l’endothélium, qui est ensuite transmise, en partie, aux cellules musculaires lisses vasculaires via les
jonctions gap myo-endothéliales, entrainant à ce niveau, une relaxation. La prostacycline (PGI2) est un
vasodilatateur agissant par activation de l’adénylyl cyclase (AC). Abréviations : AA, acide
arachidonique; ATP, adénosine triphosphate; cAMP, adénosine monophosphate cyclique; cGMP,
guanosine monophosphate cyclique; eNOS, endothelial NO synthase; GTP, guanosine triphosphate;
NO, monoxyde d’azote; PI3K, phosphatidylinositol 3-kinase; Src, Src family kinase.

4
Figure 2: Réprésentation schématique de l’activité catalytique de la NO synthase. La NOS
comprend un domaine oxygénase du côté N-terminal, auquel se lient l’arginine, la
tétrahydrobioptérine (BH4) et l’hème et un domaine réductase du côté C- terminal, contenant les sites
de fixation du FAD+, de FMN et du NADPH. Les deux domaines sont liés par le site de fixation de la
calmoduline. La eNOS catalyse la transformation de L-arginine, de NADPH et d’oxygène en NO et L-
citruline et de NADP. Les électrons sont fournis par le NADPH au niveau du domaine réductase et
transmis via le couple FAD et FMN au fer de l’hème où l’arginine et l’oxygène sont transformés en
NO et L-citruline en présence de BH4. (D'après Alderton et al. 2001)

La forme active de la eNOS est un homodimère stabilisé par l’hème, l’arginine et la BH4.

Quand la disponibilité de la L-arginine ou de la BH4 devient trop faible, la eNOS fonctionne

en mode découplé (Figure 3). Dans ce cas, les électrons ne sont pas transférés à la L-arginine

pour former du NO mais réagissent avec l’oxygène pour former des anions superoxyde (O2●-).

L’anion superoxyde réagit très rapidement avec le NO pour former l’anion peroxynitrite

(ONOO●-). Dans des conditions physiologiques, le peroxynitrite est produit en très faibles

quantités (pico au nanomolaire) et a des propriétés très proches de celles du NO, car il agit

comme un donneur de NO. En revanche, à des concentrations plus fortes, il est cytotoxique.

Dans les réponses inflammatoires, l’expression de la iNOS est souvent stimulée en réponse à

des médiateurs d’inflammation. Elle produit 100 à 1000 fois plus de NO que la eNOS et dans

un environnement riche en espèces oxydantes, ce qui favorise la formation de peroxynitrite et

diminue la disponibilité du NO pour la relaxation artérielle. Le peroxynitrite engendre un

stress oxydant et l’oxydation de la BH4, entraine le découplage des NOS et, comme dans un

5
cercle vicieux, favorise à nouveau la production de l’anion superoxyde (Rasmusen et al.

2005).

Figure 3 : Découplage de la eNOS.A. Mode couplé : dans une cellule endothéliale stimulée, la eNOS
est sous forme d’un homodimère actif. Si l’apport en tétrahydrobioptérine (BH 4) et en arginine est
suffisant, les deux sous-unités oxygénases de l’enzyme fonctionnent ensemble et produisent du NO à
partir d’arginine, grâce au flux d’électrons. B. Mode découplé : en absence de BH4 et/ou d’apport
insuffisant en arginine, les deux sous-unités oxygénases de la eNOS fonctionnent indépendamment et
produisent simultanément du NO et de l’anion superoxyde. Ces deux entités réagissent ensemble pour
●-
former le peroxynitrite (ONOO ), un puissant pro-oxydant (d'après Rasmusen et al. 2005).

Figure 4 : Régulation de l’activité de la eNOS. La eNOS fonctionnelle est une protéine dimérique
localisée dans les cavéoles membranaires et dans l’appareil de Golgi. (1) Au repos, la eNOS est
couplée à la cav-1 (cavéoline-1, une protéine structurale des cavéoles) qui diminue son activité. (2)
eNOS est constitutivement phosphorylée au niveau de la Thr 495 empêchant son activation par la
CaM. (3) La eNOS peut être inhibée en réponse à un stress oxydant par phosphorylation de la tyrosine
par PYK2 (proline-rich tyrosine kinase). (4) La eNOS peut être activée aussi bien par la CaM
(calcium/calmodulin) ou phosphorylation de la Ser1177. La protéine Hsp90 (heat shock protein)
facilite le recrutement de l’akt responsable de la phosphorylation de la eNOS. (5) la dislocation de
l’enzyme eNOS vers le cytoplasme (D'après Fleming 2010)

6
La eNOS peut être activée aussi bien par la voie calcium-dépendante (calcium/calmodulin)

que par phosphorylation par différentes kinases. Au repos, la eNOS est localisée au niveau

des cavéolés relié par la protéine cavéoline-1 qui maintient l’enzyme à l’état inactif limitant la

formation du NO. L’augmentation du calcium intracellulaire en réponse à divers stimuli

(acétylcholine, bradykinine, VEGF…) entraine la fixation de la CaM sur son site de liaison au

niveau de la eNOS et la dislocation de l’enzyme eNOS des cavéoles vers le cytoplasme

facilitant ainsi le transfert des électrons et augmentant la formation du NO (Figures 1 et 4)

(Busse & Mulsch 1990). La protéine Hsp90 (heat shock protein) se fixe par la suite à la eNOS

et facilite le recrutement de l’akt (phospholipase β) qui est responsable de la phosphorylation

de la eNOS. L’activité de la eNOS peut être régulée par phosphorylation de résidus sérine,

thréonine et tyrosine. Il existe de nombreux sites potentiels de phosphorylation, mais les sites

les plus importants sont le résidu de sérine (Ser1177) dans le domaine réductase et le

résidu thréonine (Thr495) dans le domaine de CaM-binding. Dans les cultures de cellules

endothéliales, la Ser1177 est non phosphorylée au niveau basal. Les forces de cisaillement, les

œstrogènes, le VEGF, l'insuline et la bradykinine phosphorylent la Ser1177 via des kinases

tel quel l’Akt (Dimmeler et al. 1999), la protéine kinase A (PKA, Boo et al. 2002),

l’adénosine monophosphate protéine-kinase (AMPK), la proteine kinase dépendante de

calcium/calmodulin (CaMKII, Fleming et al. 2001) facilitant le passage des électrons à

travers le domaine réductase de la eNOS augmentant ainsi l’activité de base de deux à trois

fois (McCabe et al. 2000).

La Thr495 est constitutivement phosphorylée dans les cellules endothéliales et constitue un

des sites de régulation négative. En effet la phosphorylation de la Thr495 est associée

à une diminution de l'activité enzymatique de la eNOS. Le lien entre la phosphorylation de la

Thr495 et la production de NO peut être expliqué par une interférence avec la liaison de la

CaM dans le domaine de CaM-binding, du fait qu’il y a moins d’affinité de la CaM à son site

7
de fixation quand la Thr495 est phosphorylée (Fleming et al. 2001). L’augmentation du [Ca]i

par divers stimuli (par exemple, la bradykinine, l'histamine et l’ionophore calcique A23187)

diminue la phosphorylation de la Thr495, augmentant l'activité de la eNOS de 10 à 20 fois le

niveau basal. L’activité de la eNOS n’est pas simplement déterminée par la formation du

complexe Ca+2/CaM, mais plutôt par le niveau de phosphorylation de la Ser1177 et de la

Thr495.

Les voies de signalisation impliquées dans la régulation de l’expression eNOS sont

très complexes. Des régulations de l’expression de la eNOS ont été rapportées par les facteurs

de transcription tel que NFκB, KLF-2 et FoxO1 ou par certaines microRNAs spécifiques

(Balligand et al. 2009). Les forces de cisaillement augmentent l’expression de la eNOS ainsi

que le niveau de son ARNm (Nishida et al. 1992). De plus, l’exercice physique, via

l’augmentation des forces de cisaillement, est capable d’augmenter la quantité d’ARNm de la

eNOS et donc de stimuler l’activité transcriptionnelle du gène de la eNOS aboutissant à une

augmentation de l’expression de la eNOS et de formation de NO (Sessa et al. 1994; Shen et

al. 1995; Woodman et al. 1997).

Le TGF-β1 (Transforming growth factor) joue un rôle important dans l’athérosclérose,

le remodelage vasculaire et l’angiogénèse (Pepper 1997). Le TGF-β1 augmente l’expression

et la stabilité de ARNm de la eNOS via le recrutement de facteurs de transcription smad2 et

NF-1(Inoue et al. 1995; Searles 2006). Le peroxyde d’hydrogène contrairement aux anions

superoxydes et aux radicaux hydroxyles augmente le niveau d’expression de l’ARNm de la

eNOS ainsi que l’expression de la protéine via l’activation de la CaMKII/JAK2 (Drummond

et al. 2000; Cai et al. 2001). Le TNF-α, les lipopolysaccharide et lipoprotéines de faible poids

moléculaire oxydées (exLDL) diminuent le niveau d’expression de l’ARNm de la eNOS

(Yoshizumi et al. 1993; Searles 2006).

8
1.2.1.2. Le facteur hyperpolarisant dérivé de l’endothélium (EDHF)

Concernant l’EDHF, sa nature exacte est toujours controversée et plus probablement

diffère en fonction de l’espèce de l’animal et du type de vaisseau sanguin; son rôle est plus

important dans les vaisseaux résistifs que dans les vaisseaux capacitifs (Shimokawa et al.

1996). L’EDHF est défini comme une hyperpolarisation du muscle lisse vasculaire

dépendante de l’endothélium, responsable de sa relaxation et qui est résistante à la

combinaison d’inhibiteurs de la eNOS et des COXs. La voie EDHF implique généralement

une hyperpolarisation des cellules endothéliales induite par activation des canaux potassiques

dépendant du calcium de moyenne (IKCa) et de faible conductance (SKCa) entraînant par la

suite une hyperpolarisation et une relaxation des cellules musculaires lisses (Edwards et al.

1998). Concernant les principales hypothèses portant sur la nature de l’EDHF, les études ont

montré l’implication soit du couplage électrique à travers les jonctions gap myoendothéliales

(Taylor et al. 1998), soit des ions potassium (K+) (Edwards et al. 1998). En effet, un transfert

direct de l’hyperpolarisation endothéliale au muscle lisse via les jonctions gap a été rapporté.

Les ions K+ à faible concentration provenant du courant potassique à travers les IKCa et SKCa

active la pompe Na+/K+ ATPase et les canaux KIR (canaux potassiques à courant rectifiant) au

niveau des cellules musculaires lisses favorisant l’hyperpolarisation (Figure 1). D’autres

facteurs EDHF n’impliquent pas une hyperpolarisation préalable de l’endothélium, sont

représentés par les acides époxyeicosatriénoïques (EETs) (Fisslthaler et al. 1999) ou encore

le peroxyde d’hydrogène (H2O2) (Matoba et al. 2000; Matoba et al. 2002). Il est important de

noter que bien que le rôle d’EDHF soit négligeable dans l’aorte et les artères de conductance,

c’est un facteur relaxant majeur dans la circulation coronarienne ainsi que dans les petites

artères et les artérioles telles que les artères mésentériques de second et troisième ordre

(Shimokawa et al. 1996).

9
1.2.1.3. La prostacycline (PGI2)

La prostacycline (PGI2) est un métabolite de l’acide arachidonique formée dans les

cellules endothéliales par la voie des cyclooxygénases (Moncada et al. 1976). Elle est libérée

en réponse aux forces de cisaillement (Rubanyi et al. 1986). La PGI2 active l’adénylyl cyclase

des cellules musculaires lisses entrainant une augmentation des concentrations de l’AMPc

responsable de la relaxation (Figure 1).

1.2.2. Les facteurs contracturants dérivés de l’endothélium

Dans certaines circonstances, l'endothélium peut aussi produire des substances

diffusibles qui activent le processus contractile dans les cellules musculaires lisses

vasculaires appelées sous l’acronyme EDCFs (endothelium-derived contracting factors) (De

Mey & Vanhoutte 1982). Généralement, les réponses contractiles dépendantes de

l’endothélium sont bloquées par des inhibiteurs de la cyclooxygénase ou des antioxydants

suggérant que les ECDFs correspondent essentiellement à des prostanoïdes (Miller &

Vanhoutte 1985; Luscher & Vanhoutte 1986; Yang et al. 2003) et/ou à des anions

superoxydes (Auch-Schwelk et al. 1989; Yang et al. 2002). Les cellules endothéliales peuvent

aussi produire d’autres vasoconstricteurs, tels que l’endothéline-1 (Ishikawa et al. 1988;

Yanagisawa & Masaki 1989) et l’angiotensine II (Saye et al. 1984). Mais, certains auteurs

considèrent que ces facteurs vasoconstricteurs ne participent pas aux changements

instantanés du tonus vasculaire qui est une caractéristique essentielle des EDCFs et que seuls

les produits endothéliaux vasoconstricteurs dérivés de la cyclooxygénase et les anions

superoxydes sont considérés comme de vrai EDCFs (Wong & Vanhoutte 2010).

10
1.2.2.1. Les dérivés de l’acide arachidonique vasoconstricteurs

Il existe deux isoformes de la cycloxygénase, COX-1 et COX-2. Elles ont une capacité

comparable à catalyser la transformation de l'acide arachidonique en prostaglandines. La

COX-1 est exprimée de façon constitutive dans la plupart des tissus, alors que la COX-2 est

inductible. Les deux isoformes peuvent jouer un rôle clé dans la génération de l'EDCF qui

diffère selon les espèces, le type de vaisseau sanguin et la pathologie associée (Tang &

Vanhoutte 2009). Les premières études ont démontré que les inhibiteurs non-sélectifs de la

COX entrainent une abolition des réponses contractiles dépendantes de l'endothélium (Miller

& Vanhoutte 1985). Les inhibiteurs sélectifs de la COX-1, contrairement à ceux de la COX-2

inhibent les réponses contractiles dépendantes de l'endothélium dans l'aorte du rat SHR (Ge et

al. 1995; Yang et al. 2003). De plus, les réponses contractiles dépendantes de l'endothélium

sont présentes dans l'aorte de souris transgéniques COX-2 knock-out, mais pas dans celles de

COX-1 knock-out (Tang et al. 2005). Pris conjointement, ces résultats suggèrent que les

prostanoïdes dérivées de la COX-1 sont responsables des contractions dépendantes de

l’endothélium dans les artères de rat et de souris (Wong & Vanhoutte 2010). Cependant, avec

le vieillissement la COX-2 peut être induite et contribue à la formation d'EDCF dans l’aorte

de souris (Tang et al. 2005) et d’hamster (Wong et al. 2009).

1.2.2.2. Les espèces réactives d’oxygène

Les espèces réactives d’oxygène (ROS) sont produites lors du métabolisme

physiologique de la cellule, mais leur surproduction entraîne un stress oxydant vasculaire qui

est couramment observé dans l'hypertension artérielle (Wang et al. 2004) et le diabète (Shi et

al. 2007b).

11
Les ROS impliqués dans le stress oxydant vasculaire sont : les anions superoxydes (O2●-), Les

radicaux hydroxyles (OH●-) et le peroxyde d'hydrogène (H2O2). Les ROS agissent soit

directement, en tant que vasoconstricteurs (Katusic et al. 1993), soit indirectement en

potentialisant les réponses contractiles en réduisant la biodisponibilité du NO (Rubanyi &

Vanhoutte 1986) ou par activation de COX dans les cellules musculaires lisses vasculaires

(Hibino et al. 1999). Divers systèmes enzymatiques produisent des ROS dans les vaisseaux.

Les principaux sont le cytochrome P450, les cycloxygénases, les lipoxygénases, la eNOS

découplée, ainsi que la xanthine oxydase et la NADPH oxydase. Les mécanismes

enzymatiques antioxydants sont : la superoxyde dismutase (SOD) qui catalyse la dismutation

de l’anion superoxydes (O2●-) en peroxyde d’hydrogène (H2O2), la catalase qui dégrade le

peroxyde d’hydrogène (H2O2) en eau (H2O) et oxygène moléculaire (O2) et la glutathion

peroxydase (GPx) qui dégrade le peroxyde d’hydrogène (H2O2) en eau. La GPx est la

principale défense anti-oxydante dans les cellules endothéliales (Thorin-Trescases et al.

2010). Les antioxydants endogènes non enzymatiques incluent les vitamines C et E, le

glutathion, les caroténoïdes et les flavonoïdes.

1.3. La dysfonction endothéliale liée au vieillissement

La dysfonction endothéliale est caractérisée par une atténuation des relaxations

dépendantes de l’endothélium ; c’est une altération que l’on retrouve dans la plupart des

maladies cardiovasculaires telles que l’hypertension artérielle, l’hypercholestérolémie et le

diabète, aussi bien chez l’homme que chez les animaux. De plus, comme la dysfonction

endothéliale précède souvent les changements structuraux de la paroi artérielle, il semblerait

qu’elle joue un rôle important dans l’initiation et le développement des pathologies

cardiovasculaires.

12
 Le vieillissement est caractérisé par une détérioration progressive du système

cardiovasculaire avec notamment une altération de la fonction endothéliale (Labinskyy et al.

2006). Une diminution, au cours du vieillissement, de la relaxation endothélium-dépendante

induite par l’acétylcholine a été observée dans plusieurs lits vasculaires tels que l’artère

brachiale humaine (Taddei et al. 1995), l’aorte (Kung & Luscher 1995), l’artère carotide

(Hongo et al. 1988) et l’artère mésentérique de rat (Atkinson et al. 1994a).

De nombreux mécanismes moléculaires sont impliqués dans la dysfonction

endothéliale liée à l’âge : La diminution des facteurs vasodilatateurs endothéliaux (NO, EDHF

et prostacycline), stress oxydant et souvent aussi une augmentation de l’EDCF.

1.3.1. Diminution des facteurs vasodilatateurs dérivés de l’endothélium

1.3.1.1. Diminution de la composante NO de relaxation

Des études ont montré une diminution des relaxations dépendantes du NO chez les

sujets âgés de plus de 60 ans (Taddei et al. 1995; Al-Shaer et al. 2006) et chez les rats âgés

(Kung & Luscher 1995). Cette diminution peut être expliquée soit par une diminution de

formation et/ou une augmentation de sa dégradation.

Une diminution de la formation du NO peut s’expliquer soit par une diminution de la

disponibilité de son précurseur (L-arginine) ou des cofacteurs de sa synthèse (BH4, NADPH)

soit par une diminution de l’expression ou de l’activité de la eNOS. L’augmentation de la

dégradation du NO est due à son interaction avec des espèces réactives de l’oxygène.

13
 1.3.1.1.1. Diminution de la disponibilité du précurseur L-arginine

L’arginine, est un précurseur limitant de la synthèse du NO par la eNOS. Plusieurs

études ont montré une diminution de la formation du NO associée à une augmentation de

l’expression ou de l’activité de l’arginase, une enzyme du cycle de l’urée qui dégrade L-

arginine en L-ornithine et urée. Dans l’aorte de rat âgé, la diminution de l’activité de la eNOS

est associée à une augmentation de l’expression et de l’activité de l’arginase et l’inhibition de

cette dernière diminue le découplage de la eNOS (Kim et al. 2009) et restore son activité

(Berkowitz et al. 2003). La diminution de l’expression de l’arginase-I dans des anneaux

d’aorte par des oligonucléotides antisens augmente également la biodisponiblité du NO chez

le rat âgé (White et al. 2006). De plus, l’administration de L-arginine améliore la dysfonction

endothéliale liée au vieillissement chez l’homme (Chauhan et al. 1996) et le rat (Moody et al.

1997).

1.3.1.1.2. Diminution de la disponibilité du cofacteur BH4

La tétrahydrobioptérine (BH4) est un cofacteur important dans la synthèse du NO par

la eNOS. Son absence est associée à un découplage de la eNOS entrainant la formation

d’anions superoxydes par la eNOS (Figure 3-B) (Vasquez-Vivar et al. 1998). Chez la souris,

le vieillissement est associé à une diminution de BH4 suite à une diminution des GTP

cyclohydrolase I et sepiaptérine reductase, enzymes inpliquées dans la synthèse de BH4 (Yang

et al. 2009b). L’administration de BH4 chez des patients âgés améliore les relaxations

dépendantes de l’endothélium (Higashi et al. 2006).

1.3.1.1.3 Augmentation des inhibiteurs endogènes de la eNOS

Des études cliniques et expérimentales suggèrent qu’une accumulation des inhibiteurs

endogènes de la eNOS, comme la diméthylarginine asymétrique (ADMA) et la NG

14
monométhyl-L-arginine (L-NMMA) contribue à la dysfonction endothéliale observée dans

diverses pathologies telles que l’hypertension artérielle et l’obésité (Boger & Bode-Boger

2000). ADMA et L-NMMA sont des produits de dégradation de résidus arginine méthylée

sur diverses protéines. La méthylation est effectuée par un groupe d’enzymes dénommées

méthyl transférase jouant un rôle important dans les régulations post-traductionnelles. Des

études ont montré une augmentation de l’ADMA avec l’âge chez l’homme (Kielstein et al.

2003) et le rat (Xiong et al. 2001).

II.1.1.3.4. Diminution de l’activité de la eNOS

Des études ont montré qu’une diminution de l’activité de la eNOS associée à

diminution de l’expression de la eNOS chez les animaux âgés (Csiszar et al. 2002; Wu et al.

2007) ou, par contre, à une augmentation de l’expression de la eNOS (Matz et al. 2000a; van

der Loo et al. 2000; Briones et al. 2005). Cette augmentation d’expression peut être expliquée

par un mécanisme de compensation, suite à diminution de la formation du NO (Herrera et al.

2010). Ces discordances entre les différents travaux, montrant parfois une diminution de

l’expression la eNOS au cours du vieillissement et parfois une augmentation de cette dernière,

pourraient être attribuées à l'utilisation de différentes souches de rats, de différents lits

artériels et, surtout à la différence d'âge entre les rats âgés et de leurs contrôles plus jeunes

(Herrera et al. 2010).

Le vieillissement est associé également à une formation accrue d’anions superoxydes

qui réagissent avec le NO et diminuant ainsi sa biodisponibilité. Ceci aboutit à la formation

consécutive de peroxynitrite responsable d’un découplage de la eNOS, probablement suite à

une oxydation de la BH4 et résulte en une diminution de l’activité de la eNOS et de la

formation du NO et une formation accrue d’espèce réactives de l’oxygène (Katusic 2007).

15
1.3.2. Diminution de l’EDHF

Une diminution de la composante EDHF de relaxation a été décrite dans l’artère

mésentérique supérieure et dans l’artère rénale de rat (Goto et al. 2000; Long et al. 2005; Dal-

Ros et al. 2011), et dans l’artère gastro-épiploique distale chez l’homme (Urakami-Harasawa

et al. 1997). Le traitement préventif avec l’apocynine, inhibiteur de la NADPH oxydase,

enpêche la diminution de la composante EDHF de relaxation indiquant que les ROS joue un

rôle important dans cette dysfonction (Dal-Ros et al. 2011). D’autres auteurs ont montré que

la composante EDHF de relaxation n’est pas modifiée au cours du vieillissement au niveau de

l’artère rénale de rat (Bussemaker et al. 2003) ou même augmentée dans la microcirculation

cutanée en réponse à une diminution de la composante NO (Gaubert et al. 2007).

1.3.3. Augmentation de l’EDCF

Le vieillissement est associé à une augmentation des réponses contractiles dépendantes

de l’endothélium dans l’aorte de rat (Koga et al. 1989; Abeywardena et al. 2002). Dans

l’artère fémorale de rat âgé, les réponses contractiles dépendantes de l’endothélium sont

inhibées par les inhibiteurs de COX-1 et de COX-2 (Shi et al. 2008). Il a été montré que la

prostaglandine F2alpha dérivé de la cyclooxygénase 2 était seule responsable de réponses

contractiles dépendantes de l’endothélium dans l’aorte de hamster âgé (Wong et al. 2009).

L’analyse génomique par RT-PCR a révélé une augmentation de l’expression de COX-1, de

COX-2, de thromboxane synthase, de prostacycline synthase et de prostanglandine synthase

dans l’endothélium de rat âgé (Tang & Vanhoutte 2008). Le vieillissement est associé à une

16
augmentation de la formation de prostacycline, dont l’activité balance d’un effet

vasodilatateur par activation des récepteurs IP vers un effet plutôt vasoconstricteur par

activation des récepteurs TP (Rapoport & Williams 1996). L’endothéline peut jouer un rôle

important dans la dysfonction endothéliale liée au vieillissement (Bohm & Pernow 2007). Son

expression augmente chez le rat et la souris âgés (Donato et al. 2009; Goel et al. 2010) ainsi

que sa synthèse et sa libération dans les artères de rats âgés (Goel et al. 2010). Chez l’homme,

l’exacerbation des réponses contractiles à l’endothéline chez les sujets âgés (Van Guilder et

al. 2007) a été attribuée à l’augmentation de la sensibilité des récepteurs ETA (Donato et al.

2007).

1.3.4. Augmentation du stress oxydant

Il est largement connu que le vieillissement vasculaire est associé à un stress oxydant

(Hamilton et al. 2001). L’augmentation de la formation des ROS au cours du vieillissement

vasculaire est considérée comme la cause majeure de la dysfonction endothéliale liée au

vieillissement chez l’homme (Donato et al. 2007) et l’animal (Hamilton et al. 2001; Rippe et

al. 2010). L’augmentation du stress oxydant vasculaire entraine une inactivation du NO par

interaction avec les anions superoxydes et une diminution de la composante NO de relaxation

dépendante de l’endothélium. Le stress oxydant vasculaire peut aussi être dû à une diminution

de l’expression d’enzymes antioxydantes. En effet, une diminution de la superoxyde

dismutase extracellulaire (EC-SOD), associée à une dysfonction endothéliale, a été observée

chez les rats âgés (Alvarez de Sotomayor et al. 2007). L’augmentation de l’expression de la

EC-SOD améliore la dysfonction endothéliale liée au vieillissement (Brown et al. 2006). Les

sources enzymatiques du stress oxydant observé lors du vieillissement vasculaire sont la

17
NADPH oxydase (Oudot et al. 2006), la eNOS découplée (Yang et al. 2009b), la xanthine

oxydase (Chung et al. 1999) et la chaine respiratoire mitochondriale (Ungvari et al. 2007).

Plusieurs études on montré l’implication de l’activation du système rénine-

angiotensine locale dans l’induction du stress oxydant vasculaire responsable de la

dysfonction endothéliale dans diverses pathologies telles que le diabète (Wong et al. 2010),

l’hypertension artérielle (Schulz et al. 2011) et l’hypertension pulmonaire (Dal-Ros et al.

2010), ou lors du processus physiologique de la ménopause (Yung et al. 2011) et du

vieillissement (Modrick et al. 2009). L’angiotensine II induit une augmentation de la

formation de ROS par la chaine respiratoire mitochondriale (Doughan et al. 2008) ou par

activation de la NADPH oxydase (Chrissobolis et al. 2012) . Le stress oxydant vasculaire peut

activer le facteur de transcription NFκB responsable de la synthèse de facteurs pro-

inflammatoires tels que la iNOS et les facteurs d’adhésion (Ungvari et al. 2007).

1.4. La sénescence endothéliale

La sénescence cellulaire a été décrite pour la première dans des cultures de fibroblastes

cutanés (Hayflick & Moorhead 1961). Contrairement à l’idée que les cellules en culture se

divisent indéfiniment, ces chercheurs ont observé que, lors du repiquage en série que les

cellules s’arrêtent de se diviser après un nombre fini de doublements de populations, appelé

par la suite la limite de Hayflick. Ils ont également montré que les cellules de donneurs âgés

présentent moins de divisions que celles provenant de sujet jeunes (Hayflick 1965). Sur la

base de ces conclusions, il a été proposé que les cellules somatiques ont une capacité

réplicative intrinsèque limitée et que cette propriété correspond au vieillissement au niveau

cellulaire. Le mécanisme moléculaire de la sénescence réplicative a été partiellement

18
élucidé lorsqu’un certain nombre de laboratoires ont montré un raccourcissement de

télomères lors de la sénescence. Les télomères sont les extrémités physiques de chromosomes.

Dans les cellules de mammifères, ils sont constitués d'une séquence d’ADN

répétée (TTAGGG) qui s'étend sur une longueur de plusieurs milliers de paires de bases (pb).

Au cours de la division cellulaire, des motifs TTAGGG sont perdus en raison de l’incapacité

de la machinerie conventionnelle à répliquer les extrémités linéaires des chromosomes. Cette

perte d’ADN télomérique est compensée par une transcriptase inverse spécialisée, la

télomérase, qui assure la synthèse de novo des répétitions télomériques sur les télomères

préexistants, maintenant ainsi leur longueur. L’activité télomérase est présente dans les

cellules germinales (qui ne subissent pas de sénescence) et les lignées cellulaires immortelles

(qui ont échappé à la sénescence). À l’inverse, la majorité des cellules somatiques n’ont pas

suffisamment d’activité télomérase et leurs télomères raccourcissent donc à chaque

réplication chromosomique. Quand la longueur du télomère atteint une taille critique, la

prolifération de la cellule en réponse à des stimuli mitotiques s’arrête. Les cellules entrent

dans un état irréversible d’arrêt de cycle cellulaire avec des modifications morphologiques et

fonctionnelles, mais demeurent métaboliquement actives (Thorin-Trescases et al. 2010). C’est

ce que l’on appelle la sénescence réplicative.

La sénescence peut être induite également suite à altération de l’ADN par un stress

oxydant. Une telle sénescence se produit sans une division excessive de la cellule et

généralement sans raccourcissement de télomères. La sénescence est dite sénescence

prématurée induite par stress (stress-induced premature senescence, SIPS). Divers stimuli sont

impliqués : les oncogènes, les rayons UV, les irradiations et le stress oxydant (Toussaint et al.

2000).

Il a été montré que la sénescence des cellules endothéliales se produit in vivo dans les

plaques d’athérosclérose et que la sénescence endothéliale joue un rôle important dans le

19
développement de l’athérosclérose (Fenton et al. 2001; Chen et al. 2002; Minamino et al.

2002). De plus, la sénescence des cellules endothéliales est associée à une diminution de la

formation du NO et l’expression de la eNOS (Matsushita et al. 2001) et à une augmentation

de formation de ROS (Haendeler et al. 2004). La sénescence endothéliale peut être accélérée

par l’angiotensine II (Imanishi et al. 2005). L’ensemble de ces données suggère que la

sénescence endothéliale au niveau cellulaire est à la base de la dysfonction endothéliale liée

au vieillissement au niveau vasculaire. De plus, les cellules endothéliales sénescentes sont

pro-inflammatoires, pro-thrombotiques et pro-athérosclérotiques soulevant la question de

l’implication de la sénescence endothéliale dans l’athérosclérose.

Le rôle du NO dans la modulation de la sénescence endothéliale est controversé. Le

groupe de Dimmeler et d’autres ont montré que le NO augmente l’activité de la télomérase

retardant ainsi la sénescence des cellules endothéliales (Vasa et al. 2000; Hayashi et al. 2006).

Par contre, celui d’Erusalimsky a montré que le NO à des concentrations physiologiques ne

module ni l’activité de la télomérase, ni la capacité réplicative des cellules endothéliales et

que interférence de la eNOS par siRNA n’accélère pas la sénescence (Hong et al. 2007). Ce

même groupe a montré plus tard que le NO à fortes concentrations accélère le développement

de la sénescence (Erusalimsky & Skene 2009).

1.5. Propriétés protectrices des composés polyphénoliques vis-à-vis le système

cardiovasculaire.

Des études épidémiologiques suggèrent que la consommation régulière de polyphénols

contenus dans certains aliments (le vin rouge, le thé, les grains et certains fruits) est associée à

20
une réduction de la morbidité et la mortalité cardiovasculaires (Dauchet et al. 2006; He et al.

2006; Sofi et al. 2008).

Les effets protecteurs des composés polyphénoliques pourraient être attribués, du

moins en partie, à leurs propriétés antioxydantes permettant de neutraliser les espèces

réactives de l’oxygène (ROS), de stimuler des enzymes antioxydantes et d’inhiber la xanthine

oxydase et la NADPH oxydase. Ainsi, il a été proposé que ces propriétés antioxydantes

améliorent la biodisponibilité du NO en le protégeant de sa dégradation par les anions

superoxydes (Stoclet et al. 2004).

De plus, la protection vasculaire pourrait résulter de l’effet direct des polyphénols sur

les vaisseaux sanguins. En effet, les polyphénols du vin sont de puissants stimulateurs des

relaxations endothélium-dépendantes par l’intermédiaire d’une formation accrue de NO et

d’induction de relaxations médiées par l’EDHF (Ndiaye et al. 2003; Ndiaye et al. 2004;

Ndiaye et al. 2005). Les polyphénols augmentent la production de NO par l’intermédiaire

d’une activation rédox-sensible de la voie PI3-kinase/Akt conduisant à la phosphorylation de

la eNOS à son site d’activation la Ser 1177 mais également par un mécanisme dépendant de la

concentration cytosolique en calcium libre (Andriambeloson et al. 1997; Ndiaye et al. 2003;

Ndiaye et al. 2004; Ndiaye et al. 2005) ou par déphosphorylation du site d’inhibition de la

eNOS Thr495 (Anter et al. 2004). La réponse médiée par l’EDHF est essentiellement

dépendante de la voie PI3/kinase/Akt. Les composés polyphénoliques inhibent la synthèse de

l’endothéline 1 (Corder et al. 2001; Reiter et al. 2010). En réponse à une administration

chronique, les composés polyphénoliques sont également capables d’augmenter l’expression

de la eNOS et la formation du NO (Rathel et al. 2007).

21
 Cet ensemble de mécanismes pourrait expliquer les propriétés hypotensives, anti-

hypertensives et anti-athérosclérotiques des composés polyphénoliques. Mais, peu d’études se

rapportent aux propriétés protectrices des polyphénols contre la dysfonction endothéliale liée

au vieillissement.

Les polyphénols sont des molécules naturelles issues du métabolisme secondaire des

plantes. Plus de 8000 structures phénoliques sont actuellement connues, allant de molécules

phénoliques simples de bas poids moléculaire tels les acides phénoliques à des composés

hautement polymérisés comme les tannins. Ils peuvent être conjugués avec un ou plusieurs

résidu(s) sucré(s) lié(s), formes sous lesquelles seront souvent retrouvées. Les polyphénols

sont caractérisés par la présence d’au moins un noyau aromatique avec un ou plusieurs

groupements hydroxyles attachés (Crozier et al. 2009). Les polyphénols naturels peuvent être

répartis en deux groupes, les flavonoïdes et les non- flavonoïdes (Crozier et al. 2009).

1.5.1. Les flavonoïdes :

Les flavonoïdes sont caractérisés par un squelette de base à 15 atomes de carbone

formé par deux cycles aromatiques reliés par une chaine de trois carbones : C6-C3-C6, chaine

souvent fermée sous forme d’un hétérocycle oxygéné hexa- ou pentagonal. Ils sont largement

distribués dans le règne végétal et sont particulièrement présents dans l’épiderme des feuilles

et dans la peau des fruits.

22
 O O
O

Flavone
OH

O O
3’ O
2’ 4’
Flavonol Isoflavone
8
B
1’
9 O 2 5’
7
6’
A C
6 3
10
5 4

O
O
OH

Flavanone Flavan-3-ol
O
Anthocyane

Figure 5 : Représentation de la famille de flavonoïdes.

Les principales classes de flavonoïdes retrouvées dans notre alimentation sont : les flavonols,

les flavones, les flavan-3-ols, les anthocyanes, les flavanones, et les isoflavones (Figure 5).

D’autres classes minoritaires peuvent être aussi retrouvées telles que les dihydroflavonols, les

flavan-3,4-diols, les coumarines, les chalcones, les dihydrochalcones et les aurones.

1- Les flavonols sont les flavonoïdes les plus répandus dans le règne végétal, à

l'exception des algues et des champignons. Les principaux flavonols alimentaires sont

retrouvés sous forme glycosylée et sont représentés par le kaempférol, la

quercétine, l’isorhamnétine et la myricétine.

2- Les flavones, telles que l'apigénine et de la lutéoline, n'ont pas de groupement

hydroxyle en position C3. Les flavones sont présentes dans le céleri, le persil et

l’aubépine.

3- Les flavane-3-ols constituent la classe la plus complexe des flavonoïdes allant de

monomères simples (tels que les catéchines, les épicatéchines et les catéchines

23
 gallates) à des structures plus complexes d’oligomères et de polymères de pro-

anthocyanes qui sont également connu sous le nom de tanins condensés.

4- Les anthocyanes sont présentes dans de nombreuses plantes, particulièrement dans les

fruits rouges et les fleurs où elles sont responsables de la couleur rouge, bleu ou

violette. elles ont été également isolées dans les feuilles, les tiges, les graines et les

tissus des racines. Les anthocyanes les plus communes sont : pélargonidine, cyanidine,

delphinidine, péonidine, pétunidine et malvidine.

5- Les flavanones sont présentes à des concentrations particulièrement élevées dans les

agrumes. La flavanone la plus courante est l’hespéridine, qui, avec la naringénine se

trouvent dans la peau du citron.

6- Les isoflavones présentent la caractéristique de posséder le cycle B fixé sur le carbone

C3 au lieu du carbone C2 comme pour les autres flavonoïdes. Elles se trouvent

presque exclusivement dans les plantes légumineuses, avec des concentrations

particulièrement élevées dans le soja. La génistéine et daidzéine sont les principaux

flavonoïdes du soja.

1.5.2. Les non-flavonoïdes

Les principaux polyphénols non-flavonoïdes d’origine alimentaire sont l’acide

gallique (acide phénolique C6-C1) (Figure 6), les acides hydroxycinnamiques (C6-C3)

et leurs dérivés conjugués, et les stilbènes (structure C6-C2-C6). Les acides

hydroxycinnamiques sont représentés par acide p-coumarique, l'acide caféique

(Figure 6), l'acide férulique et l'acide sinapique. Les stilbènes sont produits par des

plantes en réponse à des attaques de pathogènes extérieurs (insectes, micro-

organismes). La principale source alimentaire de stilbènes est le resvératrol (Figure 6)

à partir de vin rouge et les arachides.

24
 COOH
OH
HO

OH
HO OH OH
HO
HOOC
OH

Acide gallique Acide caféique Trans Resveratrol

Figure 6 : Représentation de polyphénols non flavonoïdes majeurs dans l’alimentation

V. La thymoquinone : rôle dans la protection cardiovasculaire

La thymoquinone (TQ) est le constituant le plus actif d’huile des graines de nigelle

(Nigella sativa) appelé communément cumin noir (Ragheb et al. 2009). L’huile de graines de

nigelle a été longtemps utilisée dans la médecine traditionnelle pour le traitement les maladies

inflammatoires et hépatiques ainsi que l’arthrite (Khader et al. 2009). Elle contient des acides

aminés, des protéines, des glucides, des alcaloïdes, des saponines et de nombreux autres

composés. Les composants actifs de l'huile volatile de cumin noir ont été identifiés par El-

Dakhakhany en 1963 montrant que la thymoquinone est le constituant actif principal malgré

la présence d’autres composés analogues tels que le thymol et un dimère de thymoquinone

nommé dithymoquinone (Figure 7) (el-Dakhakhny 1965).

La TQ est connue pour ses effets antioxydants (Butt & Sultan 2010; Tesarova et al.

2011). En effet, la TQ est un piégeur d’anions superoxyde et de radicaux hydroxyles (Burits

& Bucar 2000; Badary et al. 2003). Elle diminue le stress oxydant dans le modèle de rat

diabétique induit par la streptozotocine (Sankaranarayanan & Pari 2011) et dans

l’hypercholestérolémie chez le lapin (Attia et al. 2010). La TQ diminue la cardiotoxicité de la

cyclophosphamide par diminution du stress oxydant et amélioration de la fonction

mitochondriale (Nagi et al. 2011). De plus, le traitement de rats avec la thymoquinone

prévient l’hypertension artérielle induite par un traitement avec L-NAME (inhibiteur de la

25
eNOS) (Khattab & Nagi 2007). La TQ peut agir comme un vasodilatateur en induisant des

relaxations d’anneaux d’artère pulmonaire de rats précontractés à la phénylephrine (Suddek

2010). La TQ est également connu pour ses propriétés anticancéreuses et anti-inflammatoires

(voir revue; Woo et al. 2012).

Thymoquinone Thymol Dithymoquinone

Figure 7. Structure chimique de la thymoquinone et ses analogues.

26
 2. OBJECTIFS DES ETUDES EXPERIMENTALES

Nous avons vu à travers ce travail bibliographique que la dysfonction endothéliale est un

facteur prédicteur et marqueur précoce des maladies vasculaires. La dysfonction endothéliale

caractéristique du vieillissement se traduit par une baisse de la capacité vasodilatatrice de

l’endothélium suite un déséquilibre entre les facteurs endothéliaux dilatateurs (NO, EDHF,

PGI2) et constricteurs (endothéline-1, thromboxane A2, angiotensine II, anion superoxyde)

qui contrôlent le tonus vasculaire et un stress oxydant. Nous avons vu que les polyphénols

végétaux possèdent un potentiel thérapeutique considérable dans le domaine cardiovasculaire

au moins par leurs effets vasodilatateurs et antioxydants.

Ces travaux de thèse ont pour objectifs

1) de caractériser l’effet du vieillissement sur la fonction endothéliale en évaluant, ex vivo,

les réponses vasodilatatrices et contractiles dépendantes de l’endothélium au niveau de

l’artère mésentérique chez le rat.

2) d’évaluer l’effet protecteur potentiel d’un traitement préventif ou curatif avec les

extraits polyphénoliques (extrait du vin rouge ou extrait d’aubépine).

3) d’élucider, à l’aide de marquages tissulaires, les mécanismes moléculaires de la

dysfonction endothéliale induite par le vieillissement ainsi que les mécanismes moléculaires

associés aux effets protecteurs des polyphénols.

4) d’étudier les effets d’un extrait polyphénolique sur la sénescence réplicative des

cellules endothéliale.

27
 3. PUBLICATION SCIENTIFIQUES

Les études expérimentales menées dans ce travail de thèse sont présenté ici sous la forme
de quatre articles scientifique originaux.

Article I :
Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger and Valérie B. Schini-Kerth. Intake of
grape-derived polyphenols causes a persistent improvement of aging-related endothelial dysfunction in
the rat mesenteric artery. PLoS One. 2012;7(2):e32039. Epub 2012 Feb 27.

Article II :
Noureddine Idris Khodja and Valérie B. Schini-Kerth. Thymoquinone improves aging-related
endothelial dysfunction in the rat mesenteric artery. (Naunyn Schmiedebergs Arch Pharmacol, sous
presse, 2012)

Article III :
Noureddine Idris Khodja, Cyril Auger, Egon Koch and Valérie B Schini-Kerth. Crataegus special
extract WS®1442 prevents aging-related COX-mediated endothelium-dependent contractions.
(Phytomedicine 2012, sous presse)

Article VI :
Noureddine Idris Khodja, Marouane Kheloufi and Valérie B. Schini-Kerth. Crataegus special extract
WS®1442 delays replicative senescence in coronary artery endothelial cells: role of eNOS-
derived NO (en préparation, 2012)

28
Article 1

Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger and Valérie B. Schini-Kerth. Intake of
grape-derived polyphenols causes a persistent improvement of aging-related endothelial dysfunction in
the rat mesenteric artery. PLoS One. 2012;7(2):e32039. Epub 2012 Feb 27.

Le vieillissement vasculaire est associé à une dysfonction endothéliale caractérisée par

une diminution des relaxations dépendantes de l’endothélium dans plusieurs lits vasculaires

comme l’artère brachiale chez l’homme (Celermajer et al. 1994) et l’aorte (Kung & Luscher

1995), la carotide (Hongo et al. 1988) et l’artère mésentérique de rats (Atkinson et al. 1994b).

La dysfonction endothéliale liée au vieillissement est également associée à un stress oxydant

(Hamilton et al. 2001; Donato et al. 2007) vraisemblablement dû à une activation du système

angiotensine. En effet, les antagonistes des récepteurs AT1 et les inhibiteurs de l’enzyme de

conversion de l’angiotensine préviennent la dysfonction endothéliale liée au vieillissement

(Kansui et al. 2002; Goto et al. 2004). De plus, les souris transgéniques qui n’expriment pas

le récepteur AT1 ne présentent pas une dysfonction endothéliale liée au vieillissement

(Modrick et al. 2009). Il est bien connu que la consommation régulière d’aliments riches en

polyphénols est associée à une diminution du risque cardiovasculaire (Schini-Kerth et al.

2011). Les effets bénéfiques des polyphénols comme ceux du vin rouge s’explique en partie

par leur action sur les vaisseaux sanguins. En effet, les polyphénols entrainent des relaxations

vasculaires dépendantes de l’endothélium en stimulant la formation du NO et des

hyperpolarisations dépendantes de l’endothélium. Les polyphénols sont aussi des puissants

antioxydants qui peuvent protéger contre le stress oxydant vasculaire impliqué dans la

dysfonction endothéliale liée au vieillissement.

29
L’objectif de notre étude est :

 Evaluer la cinétique d’induction et de disparition des effets protecteurs d’un

extrait de polyphénols du vin rouge sur la dysfonction endothéliale liée au

vieillissement chez des rats Wistar âgés de 46 semaines en comparaison à des rats

jeunes âgés de 16 semaines. La fonction endothéliale est évaluée par la mesures

des composantes NO et EDHF de relaxation dans les anneaux d’artère

mésentérique suspendus dans des chambres à organes isolés.

 Déterminer le rôle du stress oxydant dans la dysfonction endothéliale liée au

vieillissement ainsi que la nature et les sources enzymatiques des espèces réactive

d’oxygène.

 Identifier les mécanismes moléculaires contribuant à l’altération des composantes

NO et EDHF de relaxation au cours du vieillissement ainsi que le rôle du système

angiotensine locale, à l’aide de marquages tissulaires.

 Etudier les effets curatifs des polyphénols du vin sur le stress oxydant vasculaire

et le système angiotensine locale.

30
Grape-Derived Polyphenols Improve Aging-Related
Endothelial Dysfunction in Rat Mesenteric Artery: Role of
Oxidative Stress and the Angiotensin System
Noureddine Idris Khodja, Thierry Chataigneau, Cyril Auger, Valérie B. Schini-Kerth*
UMR CNRS 7213 - Laboratoire de Biophotonique et Pharmacologie, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France

Abstract
Aging is characterized by the development of an endothelial dysfunction, which affects both the nitric oxide (NO)- and the
endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations, associated with vascular oxidative stress and the
activation of the angiotensin system. This study investigated whether red wine polyphenols (RWPs), antioxidants and potent
stimulators of NO- and EDHF-mediated relaxations improve aging-related endothelial dysfunction, and, if so, examined the
underlying mechanism. Mesenteric artery reactivity was determined in organ chambers, vascular oxidative stress by
dihydroethidine and MitoSOX staining, and expression of target proteins by immunohistochemical staining. Control young
rats (16 weeks) received solvent (ethanol, 3% v/v), and middle-aged rats (46 weeks) either solvent or RWPs (100 mg/kg/day)
in the drinking water. The acetylcholine-induced endothelium-dependent NO component was slightly reduced whereas the
EDHF component was markedly blunted in rings of middle-aged rats compared to young rats. The endothelial dysfunction
was associated with oxidative stress, an upregulation of angiotensin II and AT1 receptors and a down-regulation of SKCa,
IKCa, and angiotensin converting enzyme. Intake of RWPs for either one or two weeks improved the NO and the EDHF
components of the relaxation, and normalized oxidative stress, the expression of SKCa, IKCa and the components of the
angiotensin system. The protective effect of the 2-week RWPs treatment persisted for one and two weeks following
stopping intake of RWPs. Thus, intake of RWPs caused a persistent improvement of the endothelial function, particularly the
EDHF component, in middle-aged rats and this effect seems to involve the normalization of the expression of SKCa, IKCa and
the angiotensin system.

Citation: Idris Khodja N, Chataigneau T, Auger C, Schini-Kerth VB (2012) Grape-Derived Polyphenols Improve Aging-Related Endothelial Dysfunction in Rat
Mesenteric Artery: Role of Oxidative Stress and the Angiotensin System. PLoS ONE 7(2): e32039. doi:10.1371/journal.pone.0032039
Editor: Christian Schulz, Heart Center Munich, Germany
Received December 1, 2011; Accepted January 18, 2012; Published February 27, 2012
Copyright: ß 2012 Idris Khodja et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in part, by the Office National Interprofessionnel des Fruits, des Légumes, des Vins et de l’Horticulture (Action Vin & Santé,
France). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external
funding was received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: valerie.schini-kerth@unistra.fr

Introduction atherosclerosis and hypertension. Aging-related endothelial dys-

The endothelium is a key regulator of vascular homeostasis
mostly through the release of several potent vasoactive factors that
control vascular tone, blood fluidity, inflammation and smooth
muscle cell proliferation. The endothelium-derived relaxing
factors, which promote vascular protection, include nitric oxide
(NO), prostacyclin, and endothelium-derived hyperpolarizing
factor (EDHF) [1]. The importance of the EDHF phenomenon
increases as the arterial diameter decreases and, hence, it has been
suggested to play a significant role in the regulation of peripheral
vascular resistance [2]. In the mesenteric artery as well as in
several other types of arteries, EDHF-mediated responses involve
the activation of endothelial SKCa and IKCa channels (small and
intermediate conductance Ca2+-activated K+ channels, respective-
ly) inducing hyperpolarization of the endothelium which is
thereafter transmitted, in part, to the underlying vascular smooth
cells via myo-endothelial gap junctions with subsequent relaxation
[3].
Vascular aging is associated with the development of an
endothelial dysfunction, which may contribute to the initiation
and development of major cardiovascular diseases such as
function has been described in different vascular beds including
the human brachial artery [4], the rat aorta [5], the rat carotid
artery [6] and the rat perfused mesenteric bed [7]. The aging-
related endothelial dysfunction often involves a decreased NO- [8]
and EDHF-mediated relaxations [9,10], and also, in some blood
vessels, the development of endothelium-dependent contractile
responses [11]. It is associated also with an excessive vascular
formation of reactive oxygen species (ROS), in particular
superoxide anions, which, in turn, can inactivate NO [12,13].
Potential sources of ROS in old arteries include NADPH oxidase
[12], mitochondrial respiration chain [14], xanthine oxidase [15],
and uncoupled endothelial NO synthase [16]. Although the
mechanism underlying the aging-related oxidative stress is unclear,
recent evidence suggests a role for the angiotensin system. Indeed,
both an angiotensin-converting enzyme inhibitor and an AT1
receptor antagonist have been shown to prevent the aging-related
endothelial dysfunction [10,17]. In addition, angiotensin II, which
is a potent inducer of vascular oxidative stress via the AT1
receptor-dependent upregulation of NADPH oxidase [18], has
also been shown to induce a severe inhibition of EDHF-mediated
relaxations in the mesenteric artery [19].

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 Polyphenols and Endothelial Dysfunction in Aging

Numerous vascular reactivity studies indicate that several
polyphenol-rich sources such as red wine polyphenols and green
tea catechins are potent inducers of both NO- and EDHF-
mediated endothelium-dependent relaxations [20,21,22]. In
addition, chronic intake of red wine polyphenols improved the
angiotensin II-induced hypertension and endothelial dysfunction
in rats [18]. The beneficial effect of red wine polyphenols involves
their ability to prevent vascular oxidative stress, in part, by
reducing the expression of NADPH oxidase [18]. Moreover,
chronic intake of red wine polyphenols by young rats prevented
aging-related endothelial dysfunction in the mesenteric artery and
decline in physical performance [23]. Therefore, the aim of the
present study was to determine whether established aging-related
endothelial dysfunction can also be improved by the intake of red
wine polyphenols, and, if so, to determine the time-course of the
induction of the vascular protective effect and its removal after
stopping intake of red wine polyphenols, as well as the role of
oxidative stress and the angiotensin system.
Results
Aging is associated with blunted NO- and EDHF-
mediated relaxations in the mesenteric artery
In mesenteric artery rings with endothelium, acetylcholine-
induced NO-mediated relaxations (determined in the presence of
indomethacin, and charybdotoxin plus apamin to prevent the
formation of vasoactive prostanoids and EDHF, respectively)
were slightly but significantly reduced in mature-adult (25-week
old) and middle-aged (46-week old) rats compared to young (16-
week old) rats (Figure 1a). In addition, acetylcholine-induced
EDHF-mediated relaxations (determined in the presence of
indomethacin and L-NA to prevent the formation of vasoactive
prostanoids and NO, respectively) were reduced significantly to
some extent in mature-adult rats and markedly blunted in
middle-aged rats in comparison to young rats (Figure 1b). In
contrast, relaxations in mesenteric artery rings without endothe-
lium to the exogenous donor of NO, sodium nitroprusside (EC50
were 3.060.5 nM, 1.860.9 nM, 2.560.9 nM for 16, 25 and 46-
week old rats, respectively; n = 5–6) and the ATP-sensitive K+
channel opener, levcromakalim (EC50 were 118.3635.4 nM,
274.2695.2 nM, 154.9652.7 nM for 16-, 25- and 46 week-old
rats, respectively; n = 5–6) were similar in the three groups of
age.
Intake of RWPs improves NO- and EDHF-mediated
relaxations in middle-aged rats
Intake of RWPs (100 mg/kg/day) in the drinking water for
either 7 or 14 days improved slightly but significantly NO-
mediated relaxations and markedly EDHF-mediated relaxations
in the mesenteric artery of middle-aged rats (Figure 2). The 7- and
14-day RWPs treatments did affect neither relaxations to sodium
nitroprusside nor those to levcromakalim in mesenteric artery
rings without endothelium (data not shown).
Persistent protective effect of the RWPs treatment on
NO- and EDHF-mediated relaxations in middle-aged rats
In order to determine whether the protective effect of a 14-day
treatment of middle-aged rats with RWPs (100 mg/kg/day) on the
endothelial function persists after stopping their intake, rats were
treated with RWPs for 14 days followed by a 7- or 14-day intake of
solvent and then the reactivity of the mesenteric artery was
determined. Intake of RWPs for either 21 or 28 days was
associated with a slight but significant improvement of the
acetylcholine-induced NO-mediated relaxation and a marked
improvement of the EDHF-mediated relaxation (Figure 3). The
protective effect of the 14-day RWPs treatment on the NO
component was also observed one but not two weeks after stopping
the intake of RWPs whereas that on the EDHF component
persisted thereafter for two weeks (Figure 3). Thus, these data
indicate that the protective effect of the RWPs treatment on the
endothelial function is a sustained event, which persists, at least,
two weeks after stopping their intake.

Figure 1. Acetylcholine-induced NO- and EDHF-mediated relaxations decrease with increasing age. Mesenteric artery rings from young
(16-week old), mature-adult (25-week old) and middle-aged (46-week old) rats were contracted with phenylephrine (1 mM) in the presence of
indomethacin (10 mM, to inhibit the formation of prostanoids) and (a) charybdotoxin (CTX, 100 nM) plus apamin (APA, 100 nM) to inhibit the
participation of EDHF, or (b) Nv-nitro-L-arginine (L-NA, 300 mM) to rule out the formation of NO before a concentration-relaxation curve to
acetylcholine was constructed. Results are shown as mean 6 SEM of 5 to 6 different rats. *P,0.05 indicates a significant difference versus young rats
and #P,0.05 indicates a significant difference versus mature-adult rats.
doi:10.1371/journal.pone.0032039.g001

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 Polyphenols and Endothelial Dysfunction in Aging

Figure 2. Intake of RWPs improves blunted NO- and EDHF-mediated relaxations in the mesenteric artery of middle-aged rats. Young
(16-week old) and middle-aged (46-week old) control rats received solvent (ethanol, 3% v/v) in the drinking water and middle-aged treated rats
received RWPs (100 mg/kg/day) for either 7 (a, b) or 14 days before sacrifice (c, d). NO-mediated relaxations were determined in rings contracted with
phenylephrine (1 mM) in the presence of indomethacin (10 mM) and charybdotoxin (CTX, 100 nM) plus apamin (APA, 100 nM) to inhibit the
participation of prostanoids and EDHF, respectively (a, c). EDHF-mediated relaxations were determined in the presence of indomethacin (10 mM) and
Nv-nitro-L-arginine (L-NA, 300 mM) to rule out the participation of prostanoids and NO, respectively (b, d). Results are shown as mean 6 SEM of 5 to 6
different rats. *P,0.05 indicates a significant difference versus the young rat control group and #P,0.05 a significant difference versus the middle-
aged rat control group.
doi:10.1371/journal.pone.0032039.g002

RWPs improve aging-related down-regulation of the
expression of eNOS, SKCa and IKCa, in the mesenteric
artery of middle-aged rats
Immunohistochemical staining of eNOS in mesenteric artery
sections indicated a slightly but significantly reduced staining in
middle-aged rats compared to young rats, an effect which was
improved by intake of RWPs for either 2 or 4 weeks but not after a
2-week RWPs treatment period followed by a 2-week washout
period (Figure 4).
Since blunted EDHF-mediated relaxations have been associated
with a reduced expression of SKCa and IKCa [24], the expression
level of both SKCa and IKCa was assessed in mesenteric artery
sections by immunohistochemical staining. Strong SKCa and IKCa
fluorescence signals were observed throughout the arterial wall in
young rats (Figure 4). Both the SKCa and IKCa fluorescence signals
were significantly reduced in the mesenteric artery from middle-
aged compared to young rats (Figure 4). Chronic intake of RWPs
for either 2 or 4 weeks improved the expression level of SKCa and
IKCa in the mesenteric artery of middle-aged rats whereas no such
effect was observed following a 2-week RWPs treatment period
followed by a 2-week washout period (Figure 4).
RWPs reduce oxidative stress and the expression of
NADPH oxidase subunits nox1 and p22phox in the
mesenteric artery of middle-aged rats
Since aging is associated with increased vascular oxidative stress,
and oxidative stress with a reduced expression of SKCa and IKCa
in the rat mesenteric artery [14,24], the possibility that the RWPs
treatment improves the level of oxidative stress in the mesenteric
artery of middle-aged rats was assessed using the redox-sensitive
fluorescent probe dihydroethidine (DHE). The DHE fluorescence
signal was markedly increased throughout the arterial wall in
middle-aged compared to young rats (Figure 5a). Intake of RWPs
for 2 weeks, 4 weeks or 2 weeks followed by a 2-week washout

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 Polyphenols and Endothelial Dysfunction in Aging

Figure 3. The RWPs-induced Improvement of endothelial dysfunction in the mesenteric artery persists after 7 and 14-day washout
periods. Middle-aged rats (46-week old) received RWPs (100 mg/kg/day) in the drinking water for either 21 days or 14 days followed by a 7-day
washout period (a, b), and for 28 days or 14 days followed by a 14-day washout period (c, d). NO-mediated relaxations were determined in rings
contracted with phenylephrine (1 mM) in the presence of indomethacin (10 mM) and charybdotoxin (CTX, 100 nM) plus apamin (APA, 100 nM) to
inhibit the participation of prostanoids and EDHF, respectively (a, c). EDHF-mediated relaxations were recorded in the presence of indomethacin
(10 mM) and Nv-nitro-L-arginine (L-NA, 300 mM) to rule out the participation of prostanoids and NO, respectively (b, d). Results are shown as means 6
SEM of 5 to 6 rats. *P,0.05 indicates a significant difference versus the middle-aged rat control group.
doi:10.1371/journal.pone.0032039.g003

period was associated with a significant reduction in the DHE
fluorescence signal in the mesenteric artery of middle-aged rats
(Figure 5a).
In order to determine the nature and source of ROS,
mesenteric artery sections from middle-aged rats were treated
with different inhibitors before DHE staining. The DHE
fluorescence signal in mesenteric artery sections of 46-week old
rats was markedly reduced by membrane-permeant analogues of
superoxide dismutase (MnTMPyP) and catalase (PEG-catalase)
indicating the involvement of both superoxide anions and
hydrogen peroxide (Figure 5b). In addition, the increased DHE
fluorescence signal was also markedly reduced by apocynin (a
NADPH oxidase inhibitor), L-NA (an endothelial NO synthase
inhibitor), sulfaphenazol (a cytochrome P450 inhibitor), indo-
methacin (a cyclooxygenase inhibitor) and by a combination of
inhibitors of the mitochondrial respiration chain (KCN, myx-
othiazol, and rotenone) indicating the involvement of NADPH
oxidase, uncoupled eNOS, cytochrome P450, cyclooxygenases
and the mitochondrial respiration chain (Figure 5b). In addition,
the level of oxidative stress in the mesenteric artery was also
assessed by nitrotyrosine staining, an indicator of the formation of
peroxynitrites. As indicated in Figure 5a, an increased signal was
observed in the mesenteric artery of middle-aged rats compared to
young rats, an effect which was prevented by the RWPs treatment
for either 2 or 4 weeks (Figure 5a). Moreover, the level of
mitochondrial oxidative stress was assessed using the redox-
sensitive dye MitoSOX. As indicated in Figure 5a, an increased
staining was observed in the mesenteric artery of middle-aged rats
compared to young rats, and this effect was prevented by the
intake of RWPs for either 2, 4 or 2 weeks followed by a 2-week
washout period (Figure 5a).
Since the characterization of the enzymatic sources of ROS in
the mesenteric artery of middle-aged rats has indicated the
involvement of NADPH oxidase and cyclooxygenases, their
expression level was determined by immunohistochemical stain-
ing. Both the signal for NAPDH oxidase subunits nox1 and
p22phox were significantly increased in middle-aged rats com-
pared to young rats, and this effect was prevented by the RWPs
treatments (2 weeks, 4 weeks and 2 weeks followed a 2-week
washout period; Figure 5a). In contrast, the COX-1 signal was

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 Polyphenols and Endothelial Dysfunction in Aging

Figure 4. RWPs improve aging-related down-regulation of eNOS, SKCa and IKCa expression in the mesenteric artery. The expression
level of eNOS, SKCa and IKCa (small- and intermediate-conductance Ca2+-activated K+ channel, respectively) was determined in mesenteric artery
sections using purified polyclonal antibodies and a fluorescence-tagged secondary antibody by confocal microscopy. Left panels show representative
immunofluorescence staining and right panels corresponding cumulative data. Results are shown as mean 6 SEM of 4 different rats. *P,0.05
indicates a significant difference versus the young rat control group and #P,0.05 versus the middle-aged rat control group. All micrographs were
taken at the same magnification.
doi:10.1371/journal.pone.0032039.g004

similar in all groups (Figure 5a), and that of COX-2 was barely
detectable (data not shown).
RWPs normalize the aging-related alterations of the
angiotenin system
Since previous studies have suggested the involvement of the
angiotensin system in the aging-related endothelial dysfunction
[10,17], the expression level of angiotensin II, AT1 receptors, AT2
receptors and angiotensin-converting enzyme (ACE) was assessed
by immunohistochemical staining in the mesenteric artery of
young and middle-aged rats. The expression level of angiotensin II
and AT1 receptors was predominantly associated with the luminal
surface and also, to some extent, the adventitia in the mesenteric
artery of young rats (Figure 6). The expression level of both Ang II
and AT1 receptors was significantly increased in the mesenteric
artery of middle-aged rats (Figure 6). The Ang II fluorescence was
associated predominantly with the luminal surface whereas that of
AT1 receptors was observed throughout the arterial wall (Figure 6).
Strong AT2 receptor and ACE fluorescence signals were observed
predominantly at the luminal surface of the mesenteric artery of
young rats whereas they were markedly reduced in the mesenteric
artery of middle-aged rats (Figure 6).
Intake of RWPs for either 2 or 4 weeks was associated with
expression levels of Ang II and AT1 receptors in the mesenteric
artery of middle-aged rats similar to those observed in young rats
(Figure 6). The RWPs treatment improved also the AT2 receptor
and ACE signals but only after a 4-week treatment period
(Figure 6). The beneficial effect of the RWPs treatment on Ang II,
AT1 receptors, AT2 receptors and ACE levels were not observed
after 2 weeks of RWPs intake followed by a 2-week washout period
(Figure 6).
Discussion
The major findings of the present study indicate that established
aging-related blunted endothelium-dependent NO- and EDHF-
mediated relaxations are improved by oral intake of RWPs; such
an effect is observed already 7 days after intake of RWPs and is
maintained when the RWPs intake period is extended up to 28
days. Moreover, the beneficial effect of a 14-day intake of RWPs
on EDHF and NO components is still observed 7 and/or 14 days
after stopping intake of RWPs indicating that the protective effect
of RWPs on aging-related endothelial dysfunction is a sustained
event. The present findings further indicate that aging is associated
with an increased vascular formation of ROS, in particular
superoxide anions and hydrogen peroxide, which appears to
involve several sources including NADPH oxidase, uncoupled
eNOS, cytochrome P450, cyclooxygenases and the mitochondrial
respiration chain. Vascular aging is also associated with an
increased activation of the angiotensin system as indicated by
enhanced immunohistochemical staining of angiotensin II and
AT1 receptors in the arterial wall. Since angiotensin II is a strong
activator of vascular oxidative stress via AT1 receptor and an
inducer of endothelial dysfunction, it is likely that the protective
effect of the RWPs treatment on aging-related endothelial
dysfunction is due, at least in part, to its ability to normalize the
expression of angiotensin II and AT1 receptors in the mesenteric
arterial wall of middle-aged rats.
The study of the endothelial function in the mesenteric artery as
a function of aging has indicated a major impairment of the
EDHF component and also, to some extent, the NO component.
Aging-related blunted NO-mediated relaxations have also been
observed in the second and the third branch of the superior
mesenteric artery [8], the aorta [14], the carotid artery [6], the

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Figure 5. RWPs treatment reduces oxidative stress in the mesenteric artery of middle-aged rats: several sources are involved.
Oxidative stress in mesenteric artery sections was determined using the redox-sensitive fluorescent dye dihydroethidine (DHE), nitrotyrosine
immunohistochemical staining and the mitochondrial redox-sensitive dye, MitoSOX. In addition, immunohistochemical staining of the NADPH
oxidase subunits nox1 and p22phox, and COX-1 is also shown. a) Left panels show representative photographs; right panels corresponding
cumulative data. b) Mesenteric artery sections from middle-aged rats were exposed either to MnTMPyP (membrane-permeant superoxide dismutase
mimetic), PEG-catalase (membrane-permeant catalase), apocynin (NADPH oxidase inhibitor), L-NA (NO synthase inhibitor), sulfaphenazol (cytochrome
P450 inhibitor), indomethacin (cyclooxygenase inhibitor) or inhibitors of the mitochondrial respiration chain (KCN, myxothiazol, and rotenone) for
30 min before DHE staining. Upper panel represents ethidium staining; lower panel corresponding cumulative data. Results are shown as mean 6
SEM of 4 different rats. *P,0.05 indicates a significant difference versus the young rat control group (a) and the middle-aged rat control group (b),
and #P,0.05 versus the middle-aged rat control group. All micrographs were taken at the same magnification.
doi:10.1371/journal.pone.0032039.g005

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Figure 6. RWPs improve the aging-related over-expression of several components of angiotensin system in the mesenteric artery.
The expression level of angiotensin II (Ang II), AT1 receptor (AT1R), AT2 receptors (AT2R) and angiotensin-converting enzyme (ACE) was determined
by immunohistochemical staining. Left panels show representative immunofluorescence staining; right panels corresponding cumulative data.
Results are shown as mean 6 SEM of 4 different rats. *P,0.05 indicates a significant difference versus the young rat control group and #P,0.05
versus the middle-aged rat control group. All micrographs were taken at the same magnification.
doi:10.1371/journal.pone.0032039.g006

coronary arterioles [25] and the perfused kidney [26] in the rat. In
aging, an impairment of EDHF-mediated relaxations has been
observed in the superior mesenteric artery and the perfused kidney
of rats [9,23,26], and the human gastroepiploic distal artery [27].
Although the aging-related endothelial dysfunction has been
consistently observed, great variations in the extent of the
impairment of the NO and/or the EDHF components have been
reported and have been attributed to the use of different rat
strains, arterial types and the difference of age of the old rats
[5,6,8,9,10,17,26]. In addition, we have observed a pronounced
impairment of the NO component in the rat mesenteric artery of
40-week old Wistar rats in a previous study whereas only a small
impairment was observed in the mesenteric artery of 46-week old
Wistar rats in the present study [23]. Although surprising such a
difference may be due to the fact that the former rats were from
our inbred strain whereas the latter ones were bought from a
commercial supplier.
The present findings indicate that intake of RWPs (100 mg/kg/
day) improved both NO- and EDHF-mediated relaxations in the
mesenteric artery of middle-aged rats and that this effect is
observed already after a 7-day treatment period and persisted up
to a 28-day treatment period. The dose of 100 mg/kg/day RWPs
corresponds to a human equivalent dose of ,1000 mg/day [28].
Our previous study has also indicated that chronic intake of low
doses of RWPs (25 or 75 mg/kg/day) by 16 week-old young rats
for 24 weeks is associated with improved NO and EDHF-
mediated relaxations in the rat mesenteric artery [23]. Thus,
RWPs appear to be able to retard the development of an
endothelial dysfunction and to improve an established endothelial
dysfunction affecting both NO and EDHF related to aging. Since
the improvement of the NO component was still observed one
week after stopping intake of RWPs and that of the EDHF
component persisted up to 2 weeks in middle-aged rats, the
beneficial effect of RWPs on the endothelial function appears to be
a long lasting event, most likely, due to their ability to accumulate
into tissues [29].
The aging-related impairment of the EDHF component seems
to be mostly due to a reduced expression of SKCa and IKCa
channels, which mediate EDHF-dependent hyperpolarization and
relaxation in the rat mesenteric artery [3]. A reduced expression
level of SKCa and IKCa associated to blunted EDHF-mediated
relaxations has also been observed in the rat mesenteric artery
following angiotensin II-induced hypertension [30] or common
bile duct ligation [24], and in the rat cavernous tissue following
streptozotocin-induced diabetes [31]. The present findings suggest
that the beneficial effect of the RWPs treatment on the EDHF
component might be due, at least in part, to their ability to restore
the expression of SKCa and IKCa to a level similar to that observed
in young adults. The RWPs treatment also increased the reduced
eNOS fluorescence signal in the mesenteric artery of middle-aged
rats; such an effect might contribute to explain the improved NO-
mediated relaxation. Lower levels of eNOS expression in old
blood vessels have also been observed previously in some studies
[25,32] whereas others reported an increased eNOS expression
level [8,14,23]. Such a heterogeneous effect might be related to the
use of different types of arteries, the difference of age of the old
rats, and possibly also to the fact that aged rats were from different
suppliers.

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37

Several studies have indicated that vascular oxidative stress
plays a major role in aging-related endothelial dysfunction
[14,25,33]. Indeed, an increased level of superoxide anions
[14,25,33], and also hydrogen peroxide as shown in the present
study, is observed in the aged arterial wall. Moreover, several
sources of reactive oxygen species have been involved and, in
particular, NADPH oxidase in the rat aorta and mesenteric artery
[33,34], xanthine oxidase in the rat aorta [35], cyclooxygenase-2
in pig pial arteries [36], and uncoupled endothelial NO synthase in
the mouse mesenteric artery [37]. The present findings suggest
that, besides NADPH oxidase, cyclooxygenases, and uncoupled
endothelial NO synthase, cytochrome P450 monoxygenases and
the mitochondrial respiration chain contribute also to the
increased oxidative stress in the mesenteric artery of middle-aged
rats. Superoxide anions are known to reduce the bioavailability of
NO both by reacting with NO to form peroxynitrite and by
oxidizing tetrahydrobiopterin, an essential co-factor of NO
synthase [38,39]. Moreover, the beneficial effect of the RWPs
treatment on aging-related endothelial dysfunction appears to
involve their ability to prevent the excessive oxidative stress in the
aged arterial wall. The antioxidant effect may due to their ability
to directly interact with superoxide anions and other reactive
oxygen species such as hydroxy and peroxy radicals [40]. It may
also be due to their ability to prevent the aging-related
upregulation of the NADPH oxidase subunits nox1 and p22phox,
and to improve the mitochondrial formation of ROS as shown in
the present study. Previous studies have also shown that RWPs
prevent the expression of NADPH oxidase in the aorta
subsequently to the infusion of a hypertensive dose of angiotensin
II to rats [18]. Moreover, tea polyphenols prevented the
expression of NADPH oxidase and up-regulated that of catalase
in vascular cells [41,42].
Although the mechanism underlying the aging-related oxidative
stress-mediated endothelial dysfunction remains to be determined,
several lines of evidence support a role for the angiotensin system.
Indeed, angiotensin II is a potent inducer of endothelial
dysfunction and vascular oxidative stress [19,43]. Moreover,
treatment of rats with either an angiotensin-converting enzyme
inhibitor or an AT1 receptor antagonist effectively ameliorated
endothelial dysfunction, both the NO component and the EDHF
component, in aged blood vessels, in part, by decreasing vascular
oxidative stress [7,9,10,44]. Moreover, in old AT1-receptor
deficient mice, endothelium-dependent relaxations in the basilar
artery were normal whereas those in wild-type mice were reduced
by about 50% [45]. The present findings also support a role for the
angiotensin system in the aging-related endothelial dysfunction
since aging was associated with an increased expression level of
angiotensin II and AT1 receptors. In addition, aging was also
associated with reduced expression of AT2 receptors, which have
been shown to stimulate the endothelial formation of NO [46].
Although the source of angiotensin II remains to be clarified, it
does not seem to involve the angiotensin-converting enzyme since
its expression level appears to be down-regulated in the mesenteric
artery of middle-aged rats. The possibility that the local
angiotensin system contributes to the increased expression level
of angiotensin II in aged blood vessels still remains to be studied.
Such a possibility might involve chymases, which have been shown
to mediate the high glucose-induced formation of angiotensin II in
rat vascular smooth muscle cells [47]. The present findings
indicate that the intake of RWPs is able to normalize the
expression level of angiotensin II, AT1 receptors, AT2 receptors
and angiotensin-converting enzyme in the mesenteric artery of
aged rats to a level similar to that observed in young rats. In
addition, intake of RWPs by rats starting at week 16 until week 40
PLoS ONE | www.plosone.org
Polyphenols and Endothelial Dysfunction in Aging
prevented also the expression of AT1 receptors in the mesenteric
artery [23]. The increased formation of angiotensin II in aged
blood vessels most likely contributes, besides oxidative stress, to the
down-regulation of SKCa and IKCa since a reduced expression
level of both channels is observed in the rat mesenteric artery
during angiotensin II-induced hypertension [30].
In conclusion, aging is associated with the development of an
endothelial dysfunction in the rat mesenteric artery, which affects
markedly the EDHF component and also, to some extent, the NO
component. Intake of RWPs in the drinking water improves the
established aging-related endothelial dysfunction most likely by
normalizing the local activation of the angiotensin system and
preventing the oxidative stress-mediated impairment of both the
NO and the EDHF components.
Materials and Methods
Ethics statement
This study conforms to the Guide of Care and the Use of
laboratory Animals published by the US National Institutes of
Health (NIH publication No. 85-23, revised 1996) and the present
protocol was approved by the local ethics committee (Comité
Régional d’Éthique en Matière d’Expérimentation Animale,
approval AL/01/09/09/05).
Preparation of red wine polyphenols (RWPs)
RWPs dry powder, obtained from French red wine (Corbières
A.O.C., France), was provided by Dr. M. Moutounet (Institut
National de la Recherche Agronomique, Montpellier, France) and
analyzed by Pr. P.-L. Teissedre (Université de Bordeaux, France).
One liter of red wine produced 2.9 g of RWPs, which contained
471 mg/g total phenolic compounds expressed as gallic acid
equivalents. The extract contained 8.6 mg/g catechin, 8.7 mg/g
epicatechin, dimers (B1, 6.9 mg/g; B2, 8.0 mg/g; B3, 20.7 mg/g;
B4, 0.7 mg/g), anthocyanins (malvidin-3-glucoside, 11.7 mg/g;
peonidin-3-glucoside, 0.66 mg/g; cyanidin-3-glucoside, 0.06 mg/
g), and phenolic acids (gallic acid, 5.0 mg/g; caffeic acid, 2.5 mg/
g; caftaric acid, 12.5 mg/g).
In vivo treatment of rats
Food and water were given ad libitum. The study was conducted
in 12 young (16 weeks), 6 mature-adult (25 weeks) and 48 middle-
aged (46 weeks) male Wistar rats. Rats were divided into groups of
6 rats receiving either solvent (3% ethanol v/v) or red wine
polyphenols (100 mg/kg/day) in the drinking water for different
periods as indicated. Before sacrifice, rats were anaesthetized with
pentobarbital (50 mg/kg, intraperitoneally). After excision, the
mesenteric artery was placed in Krebs bicarbonate solution for the
subsequent determination of vascular reactivity using organ
chambers, immunohistochemical staining and measurement of
vascular oxidative stress.
Vascular reactivity studies
The main superior mesenteric artery was cleaned of connective
tissue and cut into rings (2–3 mm in length). This type of blood
vessel has been selected due to the fact that endothelium-
dependent relaxations involve both NO and EDHF as in human
coronary arteries, which are the major site for the development of
cardiovascular diseases such as atherosclerosis [48]. In some
preparations, the endothelium was removed by rubbing the
intimal surface of rings with a pair of forceps. Rings were
suspended in organ baths containing oxygenated (95% O2, 5%
CO2) Krebs bicarbonate solution (mM: NaCl 119, KCl 4.7,
KH2PO4 1.18, MgSO4 1.18, CaCl2 1.25, NaHCO3 25 and d-
8 February 2012 | Volume 7 | Issue 2 | e32039
38

glucose 11, pH 7.4, 37uC) for the determination of changes in
isometric tension. The rings were stretched step by step until an
optimal resting tension of 1 g was reached and then allowed to
equilibrate for at least 60 min. After the equilibration period, the
rings were exposed to Krebs bicarbonate solution containing a
high concentration of potassium (80 mM) until reproducible
contractile responses were obtained. After washing with Krebs
bicarbonate solution, the rings were precontracted with phenyl-
ephrine (PE, 1 mM) to about 80% of the maximal contraction by
high K+ solution before addition of acetylcholine (ACh, 1 mM) to
check the presence of a functional endothelium. After washout and
a further 30-min equilibration period, rings were again contracted
with PE before the application of increasing concentrations of
either ACh, sodium nitroprusside (an exogenous NO donor) or
levcromakalim (an ATP-sensitive K+ channel opener) to construct
concentration-response curves. Sodium nitroprusside- and levcro-
makalim-induced relaxations were examined in endothelium-
denuded rings of mesenteric artery. In some experiments, rings
were exposed to an inhibitor for 30 min before contraction with
PE. The NO-mediated component of relaxation was determined
in the presence of indomethacin (10 mM) and charybdotoxin
(CTX, 100 nM) plus apamin (APA, 100 nM) to inhibit the
participation of prostanoids and EDHF, respectively. The EDHF-
mediated component of relaxation was determined in the presence
of indomethacin (10 mM) and Nv-nitro-L-arginine (L-NA,
300 mM) to inhibit the formation of prostanoids and NO,
respectively. Relaxations were expressed as percentage of the
contraction induced by PE.
Immunohistochemical determination of target proteins
in the mesenteric arterial wall
Segments of the main mesenteric artery were removed,
embedded in OCT compound (Tissue-Tek, Sakura Finetek) and
snap-frozen in liquid nitrogen. Frozen arteries were cryosec-
tionned at 14 mm. Sections were air-dried for 15 min and stored at
280uC until use. Sections were first fixed with paraformaldehyde
at 4%, washed and treated with 10% milk or 5% goat serum in
PBS containing 0.1% Triton 6100 for 1 h at room temperature to
block non-specific binding. Sections were then incubated over-
night at 4uC with an antibody directed against either eNOS (1/
100), calcium-dependent potassium channels (SKCa, IKCa, 1/200),
angiotensin II (1/500), AT1 receptor (1/400), AT2 receptor (1/
400), angiotensin-converting enzyme (1/200), nitrotyrosine (1/
200), nox1 (1/200), p22phox (1/200), cyclooxygenase-1 or -2
(1/200), and nitrotyrosine (1/100). Sections were then washed
with PBS, incubated with the secondary antibody (Alexa 488-
conjugated goat anti-rabbit IgG or Alexa 637-conjugated goat
anti-rabbit) diluted (1/400) in the same buffer for 2 h at room
temperature in the dark, and washed before being mounted in
Vectashield (mounting medium for fluorescence, Vector Labora-
tories, Inc. Burlingame, CA 94010) and cover-slipped. For
negative controls, primary antibodies were omitted. The samples
were observed using a confocal laser-scanning microscope (Leica
SP2 UV DM IRBE) with 406 magnification lens. Quantification
of proteins levels was performed using Image J 1,4 2q software
(National Institutes of Health, USA).
Determination of vascular and mitochondrial ROS
formation
The redox-sensitive fluorescent dye dihydroethidine was used to
evaluate in situ formation of reactive oxygen species (ROS)
PLoS ONE | www.plosone.org
Polyphenols and Endothelial Dysfunction in Aging
following the method previously described by Sarr et al. [18].
Mesenteric arterial rings (3 to 4 mm length) were embedded in
OCT compound (Tissue-Tek), and frozen in a nitrogen bath for
cryostat sections. Dihydroethidine (2.5 mM, Sigma) was applied
onto 25 mm unfixed cryosections of mesenteric arteries for 30 min
at 37uC in a light-protected humidified chamber to determine the
in situ ROS formation. To determine the nature and the source of
ROS, sections were incubated with either MnTMPyP (membrane-
permeant superoxide dismutase mimetic, 100 mM), polyethylene
glycol-catalase (membrane-permeant catalase, 500 UI/ml), L-NA
(NO synthase inhibitor, 300 mM), apocynin (NADPH oxidase
inhibitor, 300 mM), sulfaphenazol (cytochrome P450 inhibitor,
100 mM), indomethacin (cyclooxygenase inhibitor, 10 mM) or
inhibitors of the mitochondrial respiration chain (myxothiazol,
0.5 mM+rotenone, 1 mM+KCN, 1 mM) for 30 min at 37uC before
addition of dihydroethidine. MitoSox (Invitrogen, Carlsbad, CA),
a mitochondrion-specific hydroethidine-derivative fluorescent dye,
was used to assess mitochondrial ROS in situ. Briefly, 14 mm
unfixed cryosections of mesenteric arteries were incubated with
MitoSox (5 mM at 37uC for 60 min) in a light-protected
humidified chamber. Sections were then washed three times,
mounted in Vectashield and cover-slipped. Images were obtained
with a Leica SP2 UV DM IRBE laser scanning confocal
microscope. Quantification of staining levels was performed using
Image J 1,42q software.
Materials
Antibodies were purchased as indicated: mouse anti-eNOS
(BD Biosciences Pharmingen, San Diego, California, USA),
anti-KCa3.1 (intermediate conductance Ca2+-activated K+
channel 4, IKCa) and anti-KCa2.3 (small conductance Ca2+-
activated K+ channel 3, SKCa) (Alomone Labs, Jerusalem,
Israel), ACE antibody (Abbiotec, San Diego, CA, USA), rabbit
anti-angiotensin II (Peninsula laboratories, San Carlos, CA,
USA), rabbit anti-AT1 receptor (Santa Cruz Biotechnology),
rabbit anti-AT2 receptor (Santa Cruz Biotechnology), mouse
anti-nitrotyrosine (United States Biological), mouse anti-nox1
(gp91phox) (BD Biosciences Pharmingen, San Diego, California,
USA), rabbit anti-p22phox (Santa Cruz Biotechnology), COX-1
Monoclonal Antibody, COX-2 polyclonal antibody (Cayman
chemical company, Michigan-USA). Alexa fluor-488 or 637
labelled goat anti-rabbit IgG (Invitrogen, Molecular Probes).
Apamin and charybdotoxin were obtained from Latoxan
(Valence, France) and Nv-nitro-L-arginine, indomethacin,
acetylcholine, sodium nitroprusside, levcromakalim from Sig-
ma-Aldrich.
Statistical analysis
Data are presented as mean 6 SEM. of n experiments. Mean
values were compared by ANOVA followed by the Bonferroni
post-hoc test to identify significant difference between treatments,
using GraphPad Prism (version 5 for Microsoft windows.
GraphPad software, Inc, San Diego, CA, USA). The difference
was considered to be significant when the P value was less than
0.05.
Author Contributions
Conceived and designed the experiments: TC VBSK. Performed the
experiments: NIK. Analyzed the data: NIK CA TC VBSK. Wrote the
paper: NIK TC CA VBSK.
9 February 2012 | Volume 7 | Issue 2 | e32039
39

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Synthèse des résultats de l’article 1

Les données montrent que le vieillissement vasculaire chez le rat Wistar agé de 46 semaines

est associé à une dysfonction endothéliale caractérisée par une diminution faible mais

significative de la composante NO de relaxation et une altération importante de la composante

EDHF de relaxation dans l’artère mésentérique et ce en comparaison avec des rats jeunes de

16 semaines. Le facteur NO est formé à partir de L-arginine par la eNOS alors que l’EDHF,

dont la nature diffère en fonction des espèces et de type d’artères, implique souvent une

activation des SKCa et IKCa induisant une hyperpolarisation des cellules endothéliales qui est

ensuite transmise aux cellules musculaires lisses via les jonctions gap entrainant une

relaxation. L’analyse de coupes d’artères mésentériques par immunomarquage indique une

diminution de l’expression de la eNOS, de SKCa, et de IKCa au niveau de l’endothélium de

l’artère mésentérique de rats âgés de 46 semaines en comparaison avec des rats âgés de 16

semaines. La diminution de l’expression de la eNOS et des canaux potassiques responsables

de l’hyperpolarisation dépendante de l’endothélium vraisemblablement contribue à la

diminution des composantes NO et EDHF de relaxation. La dysfonction endothéliale liée au

vieillissement est associée également à une augmentation de la formation de ROS. L’analyse

de la nature et des sources du stress oxydant vasculaire indique qu’il s’agit essentiellement

d’anions superoxydes et de peroxyde d’hydrogène produits par différentes sources

enzymatiques incluant la NADPH oxydase, la eNOS découplée, le cytochrome P450, la

cyclooxygénase et la chaine respiratoire mitochondriale. Ces résultats sont soutenus par une

augmentation de l’expression des sous-unités de la NADPH oxydase (nox1 et p22phox), par

un stress mitochondrial évalué à l’aide d’une sonde sélective de la formation de ROS au

niveau de la mitochondrie (MitoSOX) et par un marquage plus important de la nitrotyrosine

au niveau de l’endothélium, un indicateur indirect du stress oxydant. Nous avons également

41
montré que la dysfonction endothéliale liée au vieillissement est associée à un excès

d’expression de l’angiotensine II et de son récepteur AT1. Le traitement de rats âgés de 46

semaines avec un extrait polyphénolique du vin rouge à raison de 100 mg/kg/j améliore les

composantes NO et EDHF de relaxation. Cet effet curatif est observé après une semaine de

traitement pour atteindre son maximum après deux semaines de traitement. Le traitement des

rats pendant 3 ou 4 semaines n’entraine pas une amélioration supérieure à celle d’un

traitement de deux semaines. De plus, l’effet curatif d’un traitement de deux semaines avec

les polyphénols du vin est observé même après deux semaines d’arrêt de traitement. Cet effet

curatif de polyphénols est associé à une amélioration de l’expression de la eNOS, de SKCa et

de IKCa dans les coupes d’artères mésentériques de rats âgés, ce qui peut expliquer

l’amélioration des composantes NO et EDHF de relaxation. Le traitement des rats âgés avec

des polyphénols du vin diminue également la formation de ROS dans toute la paroi

vasculaire. Cet effet est associé à une diminution du stress mitochondrial, de l’expression des

sous-unités de la NADPH oxydase (nox1 et p22phox) et du marquage de la nitrotyrosine. Le

traitement avec les polyphénols diminue également l’activation du système angiotensine

locale comme le montre la diminution de l’expression de l’agiotensine II et de l’AT1. Pour la

plupart de ces paramètres, l’effet curatif de polyphénols est observé au moins une semaine

voir deux semaines après l’arrêt du traitement, indiquant que l’effet curatif des polyphénols

est un effet soutenu dans le temps et qui persiste même après deux semaine d’arrêt de

traitement.

En conclusion, le traitement de deux semaines avec les polyphénols du vin rouge

améliore la dysfonction endothéliale liée au vieillissement dans l’artère mésentérique de rat

avec un effet bénéfique qui est maintenu même après deux semaines d’arrêt de traitement. Le

traitement avec les polyphénols augmente les composante NO et EDHF de relaxation,

diminue le stress oxydant vasculaire et diminue l’activation du système angiotensine locale.

42
Article 2

Noureddine Idris Khodja and Valérie B. Schini-Kerth. Thymoquinone improves aging-related

endothelial dysfunction in the rat mesenteric artery. (Naunyn Schmiedebergs Arch Pharmacol, sous
presse, 2012)

Lors de notre première étude, nous avons montré que l’extrait de polyphénol du vin

améliore la dysfonction endothéliale liée au vieillissment chez le rat Wistar. Cet effet est dû,

au moins en partie, à l’effet antioxydant des polyphénols et à l’inhibition du système

angiotensine locale. L’extrait du vin contient des centaines voir des milliers de composés

chimiques différents et son effet bénéfique sur la fonction vasculaire est attribué à plusieurs

molécules impliquant des procyanidines and anthocyanines (Auger et al. 2010). L’objectif de

notre deuxième étude est d’évaluer l’effet d’une substance antioxydante définie sur le

vieillissement vasculaire. Notre choix de la thymoquinone est orienté par le fait que cette

substance, de structure bien connu et d’origine naturelle, est connue pour ses effets bénéfique

anticancéreux et anti-inflammatoire (Woo et al. 2012) mais surtout de sa bonne tolérance en

clinique (Akhondian et al. 2011). Elle était utilisée pour le traitement d’appoint des crises

épileptique réfractaire chez l’enfant à raison de 1 mg/kg/j (Akhondian et al. 2011). Elle est

extraite d’huile de graine de nigelle (Nigella Sativa) qui était longtemps utilisé en médecine

traditionnelle dans le traitement de l’arthrite et des maladies inflammatoires et hépatiques

(Khader et al. 2009).

La dysfonction endothéliale liée au vieillissement est un signe précoce de pathologies

cardiovasculaire (Schachinger et al. 2000; Halcox et al. 2002; Widlansky et al. 2003) et

l’amélioration de cette dysfonction endothéliale pourraient retarder le développement de

pathologies cardiovasculaires (Widlansky et al. 2003).

43
 L’objectif de notre étude est de déterminer si le traitement de rats males Wistar, avec

10 mg/kg/j de thymoquinone dans l’eau de boisson pendant deux semaines, est capable de

corriger la dysfonction endothéliale impliquant une diminution des composantes NO et EDHF

de relaxation, ainsi que le niveau d’expression de la eNOS, de SKCa et de IKCa, protéines

impliquées dans la relaxation dépendante de l’endothélium.

Nous avons également caractérisé le potentiel antioxydant de la thymoquinone contre

le stress oxydant vasculaire, le stress mitochondrial et le découplage de la eNOS induits par le

vieillissement.

Enfin, vu que le système angiotensine locale est fortement impliqué dans la

dysfonction endothéliale liée au vieillissement, nous nous sommes intéressés à l’effet du

traitement avec la thymoquinone sur l’expression de l’angiotensine II et de son récepteur AT1

dans l’artère mésentérique de rat.

44
Naunyn-Schmiedeberg's Arch Pharmacol
DOI 10.1007/s00210-012-0749-8
ORIGINAL ARTICLE
Thymoquinone improves aging-related endothelial
dysfunction in the rat mesenteric artery
Noureddine Idris-Khodja & Valérie Schini-Kerth
Received: 14 January 2012 / Accepted: 22 March 2012
# Springer-Verlag 2012
Abstract Aging-related endothelial dysfunction is character-
ized by blunted nitric oxide (NO)- and endothelium-derived
hyperpolarizing factor (EDHF)-mediated relaxations in arter-
ies, which may be due, at least in part, to increased oxidative
stress. Endothelial dysfunction will promote the initiation and
development of major cardiovascular diseases such as athero-
sclerosis and hypertension. Thymoquinone (TQ) is the most
active constituent of the volatile oil of Nigella sativa seeds
with well-documented antioxidative properties and vasodila-
tor effects. This study determined whether TQ improves the
endothelial function in middle-aged rats. Control young rats
(16 weeks) received solvent (ethanol, 3 % v/v), and middle-
aged rats (46 weeks) either solvent or TQ (10 mg/kg/day) in
the drinking water. Mesenteric artery reactivity was deter-
mined in organ chambers, vascular oxidative stress by dihy-
droethidine and MitoSOX staining, and expression of target
proteins by immunohistochemical staining. Aging-related
blunted NO- and EDHF-mediated responses were associated
with downregulation of endothelial NO synthase (eNOS) and
calcium-activated potassium channels (SKCa and IKCa) ex-
pression. Endothelial dysfunction was also associated with
oxidative stress and an upregulation of angiotensin II and
AT1 receptor expressions. Intake of TQ for 14 days restored
NO- and EDHF-mediated relaxations, normalized oxidative
stress, the expression level of eNOS, SKCa, IKCa, and the
components of the angiotensin system in the mesenteric artery
N. Idris-Khodja : V. Schini-Kerth (*)
CNRS UMR 7213, Laboratoire de Biophotonique et
Pharmacologie, Faculty of Pharmacy, University of Strasbourg,
74, route du Rhin, BP 60024, 67401 Illkirch, France
e-mail: valerie.schini-kerth@unistra.fr
N. Idris-Khodja
e-mail: noureddine.idris-khodja@unistra.fr
of middle-aged rats. Thus, TQ improves endothelial function
in aging, at least in part, through inhibition of oxidative stress
and normalization of the angiotensin system. TQ may
represent a novel therapeutic approach for aging-associated
vascular diseases.
Keywords Aging . Thymoquinone . Endothelium-derived
hyperpolarizing factor . Endothelium-derived nitric
oxide . Angiotensin system . Oxidative stress
Introduction
The endothelium, the inner layer of the blood vessel wall,
plays a key role in vascular homeostasis. The endothelium
controls vascular permeability and prevents thrombotic
events. The endothelium is also able to respond to a broad
variety of endogenous and exogenous stimuli with the syn-
thesis and release of several potent relaxing factors including
nitric oxide (NO), endothelium-derived hyperpolarizing factor
(EDHF), and prostacyclin (PGI2) (Feletou and Vanhoutte
2006). NO, a highly diffusible small molecule, is synthesized
by endothelial NO synthase (eNOS) from L-arginine (Rees et
al. 1989; Vanhoutte 2009). EDHF-mediated relaxations are
defined as the endothelium-dependent response that persists in
the presence of inhibitors of NO and PGI2 synthesis and
involves hyperpolarization of smooth muscle cells. In the
mesenteric artery as well as in several other types of arteries,
EDHF-mediated responses involve the activation of endothe-
lial SKCa and IKCa channels (small and intermediate conduc-
tance Ca2+-activated K+ channels, respectively) inducing
hyperpolarization of the endothelium, which is thereafter
transmitted, in part, to the underlying vascular smooth cells
via myo-endothelial gap junctions with subsequent relaxation
(Edwards et al. 2010).
45

Endothelial dysfunction is a pathological condition often
characterized by a reduced formation and/or an increased
degradation of NO associated to an enhanced formation of
vasoconstrictive factors such as thromboxane A2 and endo-
thelins (Flammer and Luscher 2010). It has been observed in
patients with hypertension, dyslipidaemia, diabetes mellitus,
obesity, hyperhomocysteinemia, and in aging. All of these
conditions are characterized by an overproduction of reac-
tive oxygen species (ROS) in the arterial wall (Vanhoutte et
al. 2009). It has been proposed that oxidative stress is one of
the major mechanisms in the development of aging-related
endothelial dysfunction, if not its major contributor (Wenzel
et al. 2008). Aging-related endothelial dysfunction is also
associated with an activation of the angiotensin system.
Indeed, both an angiotensin-converting enzyme (ACE) in-
hibitor and an AT1 receptor antagonist have been shown to
improve the aging-related endothelial dysfunction (Goto et
al. 2004; Kansui et al. 2002).
Endothelial dysfunction contributes to the pathogenesis of
cardiovascular diseases (Halcox et al. 2002; Widlansky et al.
2003; Schachinger et al. 2000), and improving this dysfunc-
tion is likely to reduce the cardiovascular risk (Widlansky et
al. 2003). Thymoquinone (TQ) is the most active constituent
of the volatile oil of Nigella sativa seeds, which has shown
potential medicinal properties and has long been used in
traditional and folk medicines (Ragheb et al. 2009). TQ has
well-documented antioxidative properties and vasodilator ef-
fect (Butt and Sultan 2010; Tesarova et al. 2011; Suddek
2010). Indeed, TQ prevented L-NAME-induced hypertension
and renal damage in rats possibly via its antioxidant activity
(Khattab and Nagi 2007) and induced relaxations of
phenylephrine-contracted rat pulmonary artery rings (Suddek
2010). On the basis of the above, we hypothesized that TQ
may attenuate oxidative stress in middle-aged rat arteries and,
hence, improve the endothelial dysfunction related to aging.
Material and methods
In vivo treatment of rats
This study conforms to the Guide of Care and the Use of
Laboratory Animals published by the US National Institutes
of Health (NIH publication no. 85-23, revised 1996). Food
and water were given ad libitum. The study was conducted
in 12 middle-aged (46 weeks) male Wistar rats divided into
two groups of six rats receiving either solvent (3 % ethanol
v/v) or TQ (10 mg/kg/day) in the drinking water for 14 days.
Six male Wistar rats (16 weeks old) were used as young
controls. Before sacrifice, rats were anaesthetized with pen-
tobarbital (50 mg/kg, intraperitoneally). After excision, the
mesenteric artery was placed in Krebs bicarbonate solution
(mM: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.18,
Naunyn-Schmiedeberg's Arch Pharmacol
CaCl2 1.25, NaHCO3 25 and D-glucose 11, pH 7.4) for the
subsequent determination of vascular reactivity using organ
chambers, immunohistochemical staining and measurement
of vascular oxidative stress.
Vascular reactivity studies
The main superior mesenteric artery was cleaned of
connective tissue and cut into rings (2–3 mm in length).
Endothelium-dependent relaxations in this type of blood ves-
sel involve both NO and EDHF (Nakashima et al. 1993). In
some preparations, the endothelium was removed by rubbing
the intimal surface of rings with a pair of forceps. Rings were
suspended in organ baths containing oxygenated (95 % O2,
5 % CO2) and heated (37 °C) Krebs bicarbonate solution for
the determination of changes in isometric tension. The rings
were stretched step by step until an optimal resting tension of
1 g was reached and then allowed to equilibrate for at least
60 min. After the equilibration period, the rings were exposed
to Krebs bicarbonate solution containing a high concentration
of potassium (80 mM) until reproducible contractile responses
were obtained. After washing with Krebs bicarbonate solu-
tion, the rings were precontracted with phenylephrine (1 μM)
to about 80 % of the maximal contraction by high K+ solution
before addition of acetylcholine (1 μM) to check the presence
of a functional endothelium. After washout and a further 30-
min equilibration period, rings were again contracted with
phenylephrine before the application of increasing concentra-
tions of either acetylcholine, sodium nitroprusside (an exoge-
nous NO donor) or levcromakalim (an ATP-sensitive K+
channel opener) to construct concentration–response curves.
Sodium nitroprusside- and levcromakalim-induced relaxa-
tions were examined in endothelial-denuded rings of mesen-
teric artery. In some experiments, rings were exposed to an
inhibitor for 30 min before contraction with phenylephrine.
The NO-mediated component of relaxation was determined in
the presence of indomethacin (10 μM) and charybdotoxin
(CTX, 100 nM) plus apamin (APA, 100 nM) to inhibit the
participation of prostanoids and EDHF, respectively. The
EDHF-mediated component of relaxation was determined in
the presence of indomethacin (10 μM) and Nω-nitro-L-argi-
nine (L-NA, 300 μM) to inhibit the formation of prostanoids
and NO, respectively. EDHF induces relaxation of the
vascular smooth muscle mostly by closing voltage-operated
calcium channels. Relaxations were expressed as percentage
of the contraction induced by phenylephrine.
Immunohistochemical determination of target proteins
in the mesenteric arterial wall
Segments of the main mesenteric artery were removed,
embedded in OCT compound (Tissue-Tek, Sakura Finetek)
and snap-frozen in liquid nitrogen. Frozen arteries were
46
Naunyn-Schmiedeberg's Arch Pharmacol
cryosectionned at 14 μm. Sections were air-dried for 15 min
and stored at -80 °C until use. Sections were first fixed with
paraformaldehyde at 4 %, washed and treated with 10 %
milk or 5 % goat serum in phosphate buffered saline (PBS)
containing 0.1 % Triton X100 for 1 h at room temperature to
block nonspecific binding. Sections were then incubated
overnight at 4 °C with an antibody directed against either
eNOS (1/100), calcium-dependent potassium channels
(SKCa, IKCa, 1/200), angiotensin II (1/500), AT1 receptor
(1/400), ACE (1/200), and nitrotyrosine (1/200). Sections
were then washed with PBS, incubated with the secondary
antibody (Alexa 488-conjugated goat anti-rabbit IgG or
Alexa 637-conjugated goat anti-rabbit) diluted (1/400) in
the same buffer for 2 h at room temperature in the dark
and washed before being mounted in Vectashield (mounting
medium for fluorescence, Vector Laboratories, Burlingame,
CA 94010) and cover-slipped. For negative controls, primary
antibodies were omitted. The samples were observed using a
confocal laser-scanning microscope (Leica SP2 UV DM
IRBE). Quantification of proteins levels was performed using
Image J 1,42q software (NIH, USA).
Determination of vascular and mitochondrial
ROS formation
The redox-sensitive fluorescent dye dihydroethidine was
used to evaluate in situ formation of ROS following the
method previously described by Sarr et al. (2006). Mesen-
teric arterial rings (3–4 mm length) were embedded in OCT
compound, and frozen in a nitrogen bath for cryostat sec-
tions. Dihydroethidine (2.5 μM, Sigma) was applied onto
25 μm unfixed cryosections of mesenteric arteries for
30 min at 37 °C in a light-protected humidified chamber to
determine the in situ formation of ROS. MitoSOX (Invitro-
gen, Carlsbad, CA, USA), a mitochondrion-specific
hydroethidine-derivative fluorescent dye, was used to assess
mitochondrial ROS formation in situ as described by Knorr et
al. (2011). Briefly, 14 μm unfixed cryosections of mesenteric
arteries were incubated with MitoSOX (5 μM at 37 °C for
60 min) in a light-protected humidified chamber. Sections
were then washed three times, mounted in Vectashield, and
cover-slipped. Images were obtained with a Leica SP2 UV DM
IRBE laser scanning confocal microscope. Quantification of
staining levels was performed using Image J 1,42q software.
Materials
Antibodies were purchased as indicated: mouse anti-eNOS
(BD Biosciences Pharmingen, San Diego, CA,USA), anti-
KCa3.1 (intermediate conductance Ca2+-activated K+ chan-
nel 4, IKCa) and anti-KCa2.3 (small conductance Ca2+-
activated K+ channel 3, SKCa; Alomone Labs, Jerusalem,
Israel), ACE antibody (Abbiotec, San Diego, CA, USA),
rabbit anti-angiotensin II (Peninsula Laboratories, San Carlos,
CA, USA), rabbit anti-AT1 receptor (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA), mouse anti-nitrotyrosine (United
States Biological, Swampscott, MA, USA), Alexa fluor 488
or 637 labelled goat anti-rabbit IgG (Invitrogen, Molecular
Probes). Apamin and charybdotoxin were obtained from
Latoxan (Valence, France) and Nω-nitro-L-arginine, indo-
methacin, acetylcholine, phenylephrine, sodium nitroprus-
side, and levcromakalim from Sigma-Aldrich.
Statistical analysis
Data are presented as mean ± SEM of n different experi-
ments. Mean values were compared using analysis of vari-
ance (ANOVA) followed by the post hoc Bonferroni test to
identify significant difference between treatments, using
GraphPad Prism (version 5 for Microsoft Windows, Graph-
Pad software, San Diego, CA, USA). Significant differences
between relaxations in different groups were determined by
two-way ANOVA, whereas differences between staining
intensity means were determined by one-way ANOVA.
The difference was considered to be significant when the
P value was less than 0.05.
Results
TQ reversed aging-related endothelial dysfunction
in the rat mesenteric artery
In mesenteric arterial rings with endothelium, acetylcholine
caused concentration-dependent NO- or EDHF-mediated
relaxations. NO-mediated relaxations were slightly but signif-
icantly shifted to the right with no change in the maximal
relaxation in middle-aged rats compared to young rats
(Fig. 1a). EDHF-mediated relaxations were markedly blunted
in middle-aged rats in comparison to young rats (Fig. 1b).
Intake of TQ (10 mg/kg/day) in the drinking water for 14 days
significantly improved NO- and EDHF-mediated relaxations
without affecting the precontraction level evoked by phenyl-
ephrine in the mesenteric artery of middle-aged rats (Fig. 1c
and d, and data not shown). In contrast, relaxations of mesen-
teric artery rings without endothelium to the exogenous donor
of NO, sodium nitroprusside, and the ATP-sensitive K+ chan-
nel opener, levcromakalim were similar in the three groups of
rats (Fig. 1e and f).
TQ improved aging-related downregulation
of the expression of eNOS, SKCa and IKCa
in the mesenteric artery of middle-aged rats
Immunohistochemical staining of eNOS in mesenteric ar-
tery sections indicated a slightly but significantly reduced
47
 Naunyn-Schmiedeberg's Arch Pharmacol

Fig. 1 TQ reversed aging-

related endothelial dysfunction
a NO-mediated relaxation b EDHF-mediated relaxation

Relaxation (% PE, 10-6 M)

in the mesenteric artery. Young 0 * * *

Relaxation (% PE, 10 M)
0
(16-week-old) and middle-aged * *

-6
(46-week-old) control rats * *
25 25 *
received solvent (ethanol, * *
3 % v/v) in the drinking water 50 50
*
and middle-aged-treated rats
received TQ (10 mg/kg/day) for 75 75
14 days. Concentration–relaxa- Young rats Young rats
tion curves to acetylcholine in 100 M iddle-aged rats 100 M iddle-aged rats
mesenteric arterial rings with
endothelium from the indicated -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5
groups of rats, a, c in the pres- Acetylcholine, Log[M] Acetylcholine, Log[M]
ence of indomethacin (10 μM)
and apamin (100 nM) plus
charybdotoxin (100 nM) and b,
c NO-mediated relaxation d EDHF-mediated relaxation
Relaxation (% PE, 10-6 M)

d in the presence of indometh- 0

Relaxation (% PE, 10 M)
0
acin (10 μM) and Nω-nitro-L-

-6
arginine (300 μM). Concentra- 25 25
tion–relaxation curves to # #
sodium nitroprusside (e) and to 50 # 50
levcromakalim (f) in mesenteric #
arterial rings without endotheli- #
75 Middle-aged rats # 75 Middle-aged rats #
um from the indicated groups #
Control # Control
of rats. Results are shown as 100 TQ # # 100
TQ
means ± S.E.M. of five to
six different rats. *P<0.05 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5
indicates a significant differ- Acetylcholine, Log[M] Acetylcholine, Log[M]
ence versus the young control
rats, and #P<0.05 versus the
e f
Relaxation (% PE, 10-6 M)

Relaxation (% PE, 10-6 M)

middle-aged rat control group 0 Young rats
0
M iddle-aged rats (control)
25 M iddle-aged rats (TQ) 25

50 50

75
75
Young rats
100 M iddle-aged rats (control)
100
M iddle-aged rats (TQ)
-10 -9 -8 -7 -6 -9 -8 -7 -6 -5
Sodium nitroprusside, Log [M] Levcromakalim, Log [M]

endothelial staining in middle-aged rats compared to young
rats, an effect which was improved by intake of TQ for
2 weeks (Fig. 2). Since blunted EDHF-mediated relaxations
have been associated with a reduced expression of SKCa
and IKCa (Dal-Ros et al. 2010), the expression level of
both SKCa and IKCa was assessed in mesenteric artery
sections by immunohistochemical staining. Strong SKCa
and IKCa fluorescence signals were observed mostly at
the luminal surface of the arterial wall in young rats
(Fig. 2). Both the SKCa and IKCa fluorescence signals
were significantly reduced in the mesenteric artery from
middle-aged rats compared to young rats (Fig. 2).
Chronic intake of TQ for 2 weeks improved the expres-
sion level of SKCa and IKCa in the mesenteric artery of
middle-aged rats (Fig. 2).
TQ reduced oxidative stress and the nitrotyrosine staining
in the mesenteric artery of middle-aged rats
Since aging is associated with increased vascular oxidative
stress, the possibility that the TQ treatment improves the
level of oxidative stress in the mesenteric artery of middle-
aged rats was assessed using the redox-sensitive fluorescent
probe dihydroethidine (DHE). The DHE fluorescence signal
was markedly increased throughout the arterial wall in
middle-aged compared to young rats (Fig. 3). Both the
superoxide dismutase mimetic MnTMPyP (100 μM) and
PEG catalase (500 UI/ml) reduced the DHE staining in the
arterial wall of middle-aged rats to 30.08± 1.99 % and
42.11±3.75 % (n03), indicating the involvement of super-
oxide anions and hydrogen peroxide. Intake of TQ for

48
Naunyn-Schmiedeberg's Arch Pharmacol

150

Fluorescence
(% of control)
100 #
eNOS *
50
100 µm
0

150

Fluorescence
(% of control)
100
#
SKCa
50
100 µm *
0

150
#

Fluorescence
(% of control)
100
IKCa
50 *
100 µm
0
Young 14-day solvent 14-day TQ Young 14-day solvent 14-day TQ
rats rats
Middle-aged rats Middle-aged rats

Fig. 2 TQ reversed aging-related downregulation of eNOS and small
and intermediate conductance Ca2+-activated K+ channel (SKCa and
IKCa, respectively) expression in the mesenteric artery. The expression
level of eNOS, SKCa and IKCa was determined using purified poly-
clonal antibodies and fluorescence-tagged secondary antibodies by
confocal microscopy. Left panels show representative immunofluores-
cent stainings, and right panels show corresponding cumulative data.
Results are shown as means ± S.E.M of four different rats. *P<0.05
indicates a significant effect versus young control rats, and #P<0.05
versus middle-aged control rats

2 weeks was associated with a significant reduction in the aged rats (Fig. 3). Moreover, the level of mitochondrial
DHE fluorescence signal in the mesenteric artery of middle- oxidative stress was assessed using the redox-sensitive dye

500
Fluorescence
(% of control)

400 *
DHE
300
*#
200
100
100 µm
0

250
*
Fluorescence
(% of control)

200
150
MitoSOX
100 #
100 µm 50
0
250
*
Fluorescence
(% of control)

200
#
Nitro - 150
tyrosine 100

100 µm 50
0
Young 14-day solvent 14-day TQ Young 14-day solvent 14-day TQ
rats rats
Middle-aged rats Middle-aged rats

Fig. 3 TQ reduced aging-related, increased vascular formation of
ROS. Oxidative stress in mesenteric artery sections was determined
using the redox-sensitive fluorescent dye dihydroethidine (DHE), the
mitochondrial redox-sensitive dye (MitoSOX) and nitrotyrosine immu-
nohistochemical staining. Left panels show representative photographs
and right panels corresponding cumulative data. Results are shown as
means ± S.E.M. of four different rats. *P<0.05 indicates a significant
effect versus young control rats, and #P<0.05 versus middle-aged
control rats

49
 Naunyn-Schmiedeberg's Arch Pharmacol

MitoSOX. As indicated in Fig. 3, an increased staining was
observed in the mesenteric artery of middle-aged rats com-
pared to young rats, and this effect was prevented by the
intake of TQ (Fig. 3). In addition, the level of oxidative
stress in the mesenteric artery was also assessed by nitro-
tyrosine staining, an indicator of the formation of peroxyni-
trite. An increased signal was observed in the mesenteric
artery of middle-aged rats compared to young rats, an effect
which was prevented by the TQ treatment for 2 weeks
(Fig. 3).
TQ normalized the aging-related alterations
of the angiotensin system
Since previous studies have suggested the involvement of
the angiotensin system in the aging-related endothelial dys-
function (Kansui et al. 2002; Goto et al. 2004), the expres-
sion level of angiotensin II, AT1 receptors, and ACE was
assessed by immunohistochemical staining in the mesenter-
ic artery of young and middle-aged rats. The expression
level of angiotensin II and AT1 receptors was predominantly
associated with the luminal surface in the mesenteric artery
of young rats (Fig. 4). Both the expression level of angio-
tensin II and AT1 receptors was significantly increased in
the mesenteric artery of middle-aged rats predominantly
throughout the arterial wall (Fig. 4). Strong ACE fluorescence
signal was observed at the luminal surface of the mesenteric
artery of young rats, whereas it was markedly reduced
in the mesenteric artery of middle-aged rats (Fig. 4). Intake
of TQ for 2 weeks was associated with expression levels of
angiotensin II and AT1 receptors in the mesenteric artery of
middle-aged rats similar to those observed in young rats
(Fig. 4). The TQ treatment improved also the ACE signal in
middle-aged rats (Fig. 4).
Discussion
The present study suggests a potential role of TQ in improv-
ing aging-related endothelial dysfunction. Indeed, TQ im-
proved aging-related blunted NO- and EDHF-mediated
relaxations. TQ which is known to be a potent antioxidant
decreased the formation of ROS and the expression of nitro-
tyrosine in the arterial wall of middle-aged rats. In addition,
the TQ treatment improved the aging-related activation of
the angiotensin system. Altogether, these findings indicate
that TQ improved aging-related endothelial dysfunction
most likely by preventing oxidative stress and normalizing
the angiotensin system.
Various studies reported that aging is associated with
endothelial dysfunction, a major indicator and precursor of
cardiovascular diseases, defined as reduced endothelium-

300
*
Fluorescence
(% of control)

200
Ang II #
100
100 µm
0

300
*
Fluorescence
(% of control)

200
AT1R #
100
100 µm

200 *#
Fluorescence
(% of control)

150
ACE
100
100 µm
50 *
Young 14-day solvent 14-day TQ
0
rats Young 14-day solvent 14-day TQ
Middle -aged rats
rats
Middle -aged rats

Fig. 4 Effect of TQ on the expression of angiotensin II (Ang II), AT1
receptors (AT1R) and angiotensin-converting enzyme (ACE) in the rat
mesenteric arterial wall. The expression level of Ang II, AT1R and
ACE was determined using a purified polyclonal antibody and a
fluorescence-tagged secondary antibody by confocal microscopy. Left
panels show representative immunofluorescence stainings, and right
panels corresponding cumulative data. Results are shown as means ±
S.E.M. of three or four different rats. *P<0.05 indicates a significant
effect versus young control rats, and #P<0.05 versus middle-aged rats

50
Naunyn-Schmiedeberg's Arch Pharmacol
dependent relaxations (Herrera et al. 2010). The
endothelium-dependent relaxations include mainly a NO
component (Furchgott and Zawadzki 1980; Palmer et al.
1987) and often also an EDHF-mediated component (Chen
et al. 1988). Previous studies have indicated that aging is
associated with blunted NO-mediated relaxations in the
mesenteric artery (Matz et al. 2000), the aorta (van der
Loo et al. 2000), the carotid artery (Hongo et al. 1988),
the coronary arterioles (Csiszar et al. 2002), and the per-
fused kidney (Long et al. 2005) of rats, and with an impair-
ment of EDHF-mediated relaxations in the mesenteric artery
of rats (Goto et al. 2000; Long et al. 2005; Dal-Ros et al.
2011) and the human gastroepiploic distal artery (Urakami-
Harasawa et al. 1997). The present findings are in good
agreement with these previous ones since they indicate
blunted NO- and EDHF-mediated relaxations in the mesen-
teric artery of middle-aged rats in comparison to young rats.
In this study, as in others (Wu et al. 2007; Csiszar et al.
2002), aging is associated with a decreased expression of
eNOS, which may explain the reduced NO-mediated relax-
ation, and a decreased SKCa and IKCa channels expression,
which may contribute to the reduced EDHF-mediated
responses. Blunted EDHF-mediated relaxations associated
with a decreased expression of SKCa and IKCa channel
proteins have also been reported in small mesenteric arteries
of angiotensin II-induced hypertensive rats (Hilgers and
Webb 2007) and in portal hypertension-induced endothelial
dysfunction (Dal-Ros et al. 2010). Deficiency or decreased
functionality of these potassium channels per se have also
been shown to promote vascular depolarization, oxidative
stress, endothelial dysfunction, and hypertension as well as
more pronounced responses to angiotensin II treatment (Oelze
et al. 2006). In contrast, endothelium-independent relaxations
to a NO donor (sodium nitroprusside) and an ATP-sensitive
K+ channel opener (levcromakalim) were unaffected, indicat-
ing that aging-related endothelial dysfunction is not due to an
impaired responsiveness of the vascular smooth muscle.
Previous studies have indicated that aging is associated to
oxidative stress in the human brachial artery (Donato et al.
2007), the aorta and carotid arteries of mice (Lesniewski et
al. 2009; Francia et al. 2004), and the rat coronary arterioles
and aorta (Zanetti et al. 2010; Csiszar et al. 2002). The
oxidative stress is due, in part, to mitochondrial dysfunction
(Wenzel et al. 2008; Ungvari et al. 2010). Moreover, there is
some evidence indicating that oxidative stress mediates the
aging-related impairment of endothelium-dependent relaxa-
tions (Lesniewski et al. 2009; Krummen et al. 2006; Modrick
et al. 2009). The present findings also indicate an increased
vascular formation of ROS and mitochondrial oxidative stress
in the vascular wall of middle-aged rats. Oxidative stress is
recognized to be a mechanism of rapid inactivation of NO (Li
and Shah 2004) and EDHF-mediated responses (Krummen et
al. 2006), leading to endothelial dysfunction. The aging-
related decreased NO component may be a consequence of
the known rapid reaction between NO and superoxide anions
resulting in the formation of peroxynitrite. This hypothesis is
supported by the fact that an increased expression of nitro-
tyrosine, a marker of peroxynitrite, is observed in the vascular
wall of middle-aged rats. An increased level of nitrotyrosine
has also been shown in the aorta of old rats (van der Loo et al.
2000) and human microvessels (Rodriguez-Manas et al.
2009). In addition, it may also be due to eNOS uncoupling
subsequent to the oxidative stress-mediated degradation of
tetrahydrobiopterin, an essential co-factor of eNOS (Idris
Khodja et al. 2012; Munzel et al. 2005).
Several studies suggest a role of the angiotensin system
in the aging-related endothelial dysfunction via vascular
oxidative stress. Indeed, an ACE inhibitor and an AT1
receptor antagonist improved endothelial dysfunction, in
aged blood vessels, in part, by decreasing vascular oxidative
stress (Atkinson et al. 1994; Goto et al. 2000; Mukai et al.
2002; Kansui et al. 2002). Moreover, old AT1 receptor-
deficient mice present normal endothelium-dependent relax-
ations in the basilar artery (Modrick et al. 2009). In addition,
other studies suggest that oxidative stress may induce an
activation of the local renin–angiotensin system as reported
in the uric acid-induced endothelial dysfunction model (Yu
et al. 2010). The present findings also support a role for the
angiotensin system in the aging-related endothelial dysfunc-
tion since aging was associated with an increased expression
level of angiotensin II and AT1 receptors. Furthermore,
oxidative stress has been suggested to activate the local
renin–angiotensin system, which, in turn, results in a posi-
tive feedback mechanism to further increase the formation
of ROS. Although an increased expression of angiotensin II
was observed, that of ACE was decreased in mesenteric
arteries of middle-aged rats, suggesting that other sources
of angiotensin II are involved. Similar findings were also
observed in the kidney of middle-aged rats (Jung et al. 1995)
most likely suggesting an adaption to high angiotensin II
levels. One potential source of angiotensin II are chymases,
which have been shown to mediate the high glucose-
induced formation of angiotensin II in rat vascular smooth
muscle cells (Lavrentyev et al. 2007).
The chronic treatment of middle-aged rats with TQ
(10 mg/kg/d in drinking water) for 14 days improved the
aging-related reduced NO- and EDHF-mediated relaxations
in the mesenteric artery of middle-aged rats. These effects
may be due, at least in part, to an improved expression of
eNOS, SKCa, and IKCa channels. The TQ treatment reduced
the aging-related increased total and mitochondrial ROS
formation and also the aging-related increased expression
of nitrotyrosine. The antioxidant effect of TQ is well docu-
mented in several studies and hence may explain the bene-
ficial effect of TQ on aging-related endothelial dysfunction.
Indeed, TQ is a potent superoxide anion and hydroxyl
51

scavenger (Badary et al. 2003; Burits and Bucar 2000). TQ
has also been shown to improve oxidative stress in
streptozotocin-induced diabetic rats (Sankaranarayanan and
Pari 2011) and to attenuate the cyclophosphamide-induced
cardiotoxicity in rats, at least in part, by decreasing oxida-
tive stress and improving the mitochondrial function (Nagi
et al. 2011). TQ attenuated also the diethylnitrosamine-
induced hepatic carcinogenesis through an antioxidant effect
(Sayed-Ahmed et al. 2010), the high cholesterol-induced
oxidative stress in the rabbit liver (Attia et al. 2010), and
the mercuric chloride-induced renal oxidative damage in
rats (Fouda et al. 2008).
The present data indicate also that intake of TQ is able to
normalize the expression level of angiotensin II, AT1 recep-
tors and ACE in the mesenteric artery of middle-aged rats to
a level similar as those observed in young rats, possibly due
to the antioxidative effect of TQ. Indeed, oxidative stress
has been shown to induce an upregulation of AT1 receptors
in rat kidney (Banday and Lokhandwala 2011, 2008). An
inhibitory effect of natural products on the angiotensin sys-
tem has also been observed previously. Indeed, natural
antioxidant products, such as tea (Camellia sinensis), oak
leaves (Quercus), and Vaccinium myrtillus, have been
shown to inhibit ACE activity and as a consequence to
reduce the formation of angiotensin II (Dong et al. 2011;
Rivas-Arreola et al. 2010; Persson et al. 2009; Actis-Goretta
et al. 2006). Furthermore, resveratrol, a potent antioxidant
polyphenol, also downregulated AT1 receptor expression in
vascular smooth muscle cells (Miyazaki et al. 2008).
Conclusion
In conclusion, aging is associated with the development of
an endothelial dysfunction in the rat mesenteric artery, char-
acterized by reduced NO- and EDHF-mediated relaxations.
The TQ treatment improved the aging-related endothelial
dysfunction most likely by normalizing oxidative stress and
the local activation of the angiotensin system.
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Synthèse des résultats de l’article 2

Dans cette étude, nous avons montré que le traitement avec la thymoquinone dans

l’eau de boisson à raison de 10 mg/kg/j pendant deux semaines est capable de corriger la

dysfonction endothéliale liée au vieillissement chez le rat âgé de 46 semaines. En effet, ce

traitement avec la thymoquinone restore les composantes NO et EDHF de relaxation chez le

rat âgé de 46 semaines à un niveau proches de celui observé chez le rat jeune de 16 semaines.

Cet effet est dû au moins à l’amélioration de l’expression de la eNOS (enzyme responsable de

formation du NO) et de SKCa et IKCa (canaux potassiques impliqués dans l’hyperpolarisation

dépendante de l’endothélium) au niveau de l’endothélium des sections d’artères

mésentériques de rats traité avec la thymoquinone. Nous avons ensuite montré que l’effet

bénéfique de la thymoquinone sur les composantes NO et EDHF de relaxation est associé à

une diminution de formation de ROS du stress mitochondriale et du marquage de la

nitrotyrosine, un marqueur du stress oxydant. Le traitement de rats âgés avec la thymoquinone

diminue également l’expression de l’angiotensine II et de son récepteur AT1 au niveau de la

paroi de l’artère mésentérique.

En conclusion, la thymoquinone améliore les composantes NO et EDHF de relaxation

des artères mésentérique de rat âgé vraisemblablement en diminuant le stress oxydant

vasculaire. La thymoquinone augmente l’expression de la eNOS, de SKCa et de IKCa, et

diminue l’activation du système angiotensine locale.

55
Article 3 :

Noureddine Idris Khodja, Cyril Auger, Egon Koch and Valérie B Schini-Kerth. Crataegus special
extract WS1442 prevents aging-related COX-mediated endothelium-dependent contractions.
(Phytomedicine 2012, sous presse)

Dans les études 1 et 2, nous nous sommes intéressés aux effets curatifs des

polyphénols du vin rouge et de la thymoquinone contre la dysfonction endothéliale liée au

vieillissement. L’objectif de notre troisième étude est d’étudier l’effet d’un traitement

préventif avec les polyphénols sur la dysfonction endothéliale liée au vieillissement. Pour

cette étude, nous avons utilisé une source riche en polyphénols qui est l’extrait d’aubépine

(WS®1442). C’est un extrait sec standardisé obtenu à partir de feuilles et de fleurs, ajusté à

18,75 % d’oligomères de procyanidines (solvant d’extraction : 45% d’éthanol). Des études

précédentes ont montré que cet extrait augmente le débit coronaire par augmentation de

formation endothéliale de NO (Koch & Malek 2011) et induit des relaxations médiées par le

NO et l’EDHF dans les anneaux d’artère coronaire de porc (Brixius et al. 2006). La formation

du NO est due à une activation de la eNOS par phosphorylation de la serine 1177 (Brixius et

al. 2006) par une voie redox-sensible dépendante de l’activation de Src/PI3-kinase/Akt

(Anselm et al. 2009).

La dysfonction endothéliale liée au vieillissement est caractérisée par une diminution

des relaxations dépendantes de l’endothélium mais souvent aussi par une augmentation de

réponses contractiles dépendantes de l’endothélium.

Cette étude a évalué l’effet d’un traitement préventif de rat Wistar avec l’extrait

d’aubépine à raison 100 mg/kg/j ou de 300 mg/kg/j dans l’alimentation sur la fonction

endothéliale en évaluant les composantes relaxantes et contractiles dépendantes de

l’endothélium au niveau de l’artère mésentérique. Les rats sont traités durant 40 semaines à

partir de l’âge de 25 semaines et comparé à des animaux jeunes âgés de 16 semaines.

56
 Nous avons évalué également l’effet du traitement sur le stress oxydant au niveau des

sections des artères mésentériques et nous nous sommes intéressés à la nature et aux sources

enzymatiques du stress oxydant vasculaire observé lors du vieillissement.

Enfin, nous avons étudié à l’aide de technique d’immunomarquage le niveau

d’expression de cyclooxygénases, protéines impliquées dans la synthèse des prostanoïdes

responsables des réponses contractiles dépendantes de l’endothélium.

57
Nous avons évalué également l’effet du traitement sur le stress oxydant au niveau des sections

des artères mésentériques et nous nous sommes intéressés à la nature et aux sources

enzymatiques du stress oxydant vasculaire observé lors du vieillissement.

Enfin, nous avons étudié à l’aide de technique d’immunomarquage le niveau

d’expression de cyclooxygénases, protéines impliquées dans la synthèse des prostanoïdes

responsables des réponses contractiles dépendantes de l’endothélium.

57
 ARTICLE IN PRESS
G Model
PHYMED-51217; No. of Pages 8

Phytomedicine xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Phytomedicine
journal homepage: www.elsevier.de/phymed

Crataegus special extract WS® 1442 prevents aging-related endothelial

dysfunction
N. Idris-Khodja a , C. Auger a , E. Koch b , V.B. Schini-Kerth a,∗
a
CNRS UMR 7213, Faculty of Pharmacy, University of Strasbourg, Illkirch, France
b
Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

a r t i c l e i n f o a b s t r a c t

Keywords: Aging is associated with a markedly increased incidence of cardiovascular diseases due, in part, to the
Aging development of vascular endothelial dysfunction. The present study has evaluated whether the Crataegus
Endothelial dysfunction special extract WS® 1442 prevents the development of aging-related endothelial dysfunction in rats, and,
Crataegus
if so, to determine the underlying mechanisms. Wistar rats received either a control diet or the same
Polyphenols
diet containing 100 or 300 mg/kg/day of WS® 1442 from week 25 until week 65. Vascular reactivity was
Endothelium-derived hyperpolarizing
factor assessed in mesenteric artery rings using organ chambers, oxidative stress by dihydroethidine staining
Endothelium-derived nitric oxide and cyclooxygenase-1 (COX-1) and -2 (COX-2) expression by immunohistochemistry. Acetylcholine-
Endothelium-derived contracting factors induced endothelium-dependent relaxations in mesenteric artery rings were blunted in 65-week-old rats
Oxidative stress compared to 16-week-old rats. This effect was associated with a marked reduction of the endothelium-
derived hyperpolarizing factor (EDHF) component whereas the nitric oxide (NO) component was not
affected. Aging was also associated with the induction of endothelium-dependent contractile responses
to acetylcholine. Both aging-related impairment of endothelium-dependent relaxations and the induc-
tion of endothelium-dependent contractile responses were improved by the Crataegus treatment and by
COX inhibitors. An excessive vascular oxidative stress and an upregulation of COX-1 and COX-2 were
observed in the mesenteric artery of old rats compared to young rats, and these effects were improved by
the Crataegus treatment. In conclusion, chronic intake of Crataegus prevented aging-related endothelial
dysfunction by reducing the prostanoid-mediated contractile responses, most likely by improving the
increased oxidative stress and the overexpression of COX-1 and COX-2.
© 2012 Published by Elsevier GmbH.

Introduction formation and/or increased degradation of EDRFs and/or to an

Aging is associated with a markedly increased incidence of
cardiovascular diseases due, in part, to the development of vas-
cular endothelial dysfunction (Lakatta and Levy 2003). Indeed,
the endothelium has a major role in the regulation of vascu-
lar homeostasis through the release of several potent relaxing
and contracting factors. The endothelium-derived relaxing fac-
tors (EDRFs), which are predominant in healthy blood vessels and
promote vascular protection, include nitric oxide (NO, Furchgott
and Zawadzki 1980), endothelium-derived hyperpolarizing fac-
tor (EDHF, Chen et al. 1988) and prostacyclin (Moncada and
Vane 1978). The endothelium-derived contracting factors (EDCFs),
which appear frequently in diseased blood vessels, include
endothelins, angiotensin II, cyclooxygenase-derived prostanoids
and reactive oxygen species (ROS, Vanhoutte and Tang 2008). The
unbalance between EDRFs and EDCFs subsequent to a reduced
∗ Corresponding author.
E-mail address: valerie.schini-kerth@unistra.fr (V.B. Schini-Kerth).
over-production of EDCFs contributes to endothelial dysfunction,
which accompanies major vascular diseases. Such an endothelial
dysfunction is also observed during physiological aging, which
is characterized by a progressive decrease in NO bioavailabil-
ity (Imaoka et al. 1999), a reduced EDHF component (Goto
et al. 2000) and often also an increased formation of EDCFs, in
particularly, ROS (Hamilton et al. 2001) as well as cyclooxygenase-
derived prostanoids (Vanhoutte and Tang 2008). Aging-related
endothelial dysfunction has been observed in the human brachial
artery (Celermajer et al. 1994), in the aorta (Kung and Luscher
1995) and the perfused mesenteric bed (Atkinson et al. 1994)
of rats.
Chronic intake of polyphenol-rich food and beverages has
been shown to improve endothelial function and to reduce
the incidence of cardiovascular diseases (Michalska et al.
2010). Hawthorn extract (Crataegus ssp.) is a rich source of
polyphenols, which has been shown to have cardiotonic and car-
dioprotective properties. The Crataegus special extract WS® 1442
contains predominantly two major groups of polyphenolic com-
pounds, a mixture of monomeric flavonoids and oligomeric

0944-7113/$ – see front matter © 2012 Published by Elsevier GmbH.

http://dx.doi.org/10.1016/j.phymed.2012.04.005

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procyanidins. Previous studies have shown that the Crataegus
extract is a potent inducer of NO- and EDHF-mediated relax-
ations of coronary artery rings (Brixius et al. 2006). The aim of
the present study was to determine whether chronic intake of
the Crataegus extract is able to prevent the endothelial dysfunc-
tion occurring during aging, and if so, to investigate the underlying
mechanisms.
Materials and methods
Crataegus special extract WS® 1442
Crataegus WS® 1442 is a special extract obtained from leaves
with flowers of selected hawthorn species (Crataegus oxyacan-
tha/Crataegus monogyna) by extraction with 45% (w/w) aqueous
ethanol (drug/extract ratio 4–6.6:1). The extract is adjusted to a
content of 17.3–20.1% oligomeric procyanidins.
In vivo treatment of rats
This study conforms to the Guide of Care and the Use of
Laboratory Animals published by the US National Institutes of
Health (NIH publication no. 85-23, revised 1996). Water was
given ad libitum in a controlled environment (room tempera-
ture 21–22 ◦ C, room humidity 50 ± 5%). Male Wistar rats received
either a control diet (9 rats) or the same diet containing either
100 (9 rats) or 300 mg/kg/day (9 rats) of the Crataegus spe-
cial extract from week 25 until week 65. 16-Week-old rats (6
rats) were used as young control rats. Rats were anaesthetized
with pentobarbital (50 mg/kg, intraperitoneally) and after exci-
sion, the mesenteric artery was placed in Krebs bicarbonate
solution for the subsequent determination of vascular reactivity
using organ chambers, immunohistochemistry and measurement
of ROS.
Vascular reactivity studies
For the determination of changes in isometric tension, mesen-
teric artery rings were suspended in organ baths as described
previously (Dal-Ros et al. 2011). Rings were contracted with
1 ␮M of phenylephrine (PE) before the application of increas-
ing concentrations of acetylcholine (Ach), sodium nitroprusside
(an exogenous NO donor) or levcromakalim (an ATP-sensitive
K+ channel opener) to construct concentration–response curves.
Sodium nitroprusside- and levcromakalim-induced relaxations
were examined in endothelium-denuded rings of mesenteric arter-
ies. In some experiments, rings were exposed to an inhibitor for
30 min before contraction with PE. The NO-mediated component
of relaxation was determined in the presence of indomethacin
(10 ␮M) and charybdotoxin (100 nM) plus apamin (100 nM) to
inhibit the formation of prostanoids and EDHF-mediated responses,
respectively. The EDHF-mediated component of the relaxation was
determined in the presence of indomethacin (10 ␮M) and Nω -nitro-
l-arginine (l-NA, 300 ␮M) to inhibit the formation of prostanoids
and NO, respectively. Endothelium-dependent contractions were
assessed in the presence of l-NA to prevent the endothelial forma-
tion of NO, and charybdotoxin + apamin to inhibit EDHF-mediated
responses. Rings were contracted with PE (10–100 nM) to about
25% of the maximal contraction obtained by the Krebs bicarbonate
solution containing a high K+ concentration (80 mM). There-
after increasing concentrations of ACh were added to the organ
chamber.
The basal endothelial formation of NO was assessed indirectly
by determining the endothelium-dependent depression of PE-
induced contractile responses (Martin et al. 1986). For this purpose,
contractions to increasing concentrations of PE were determined
in endothelial-intact and endothelial-denuded rings of mesenteric
artery.
Immunohistochemical determination of cyclooxygenase-1 and -2
expression in the mesenteric arterial wall
Segments of main mesenteric arteries were removed, embed-
ded in OCT compound (Tissue-Tek, Sakura Finetek) and snap-frozen
in liquid nitrogen. Frozen arteries were cryosectioned at 14 ␮m.
Sections were air-dried for 15 min and stored at −80 ◦ C until use.
The slides were fixed with paraformaldehyde (4%), washed and
treated with 10% milk containing 0.1% Triton-X 100 for 1 h at
room temperature to block non-specific binding. The sections were
then incubated overnight at 4 ◦ C with an antibody directed against
COX-1 or -2 (1/200). Sections were then washed with PBS, incu-
bated with the secondary antibody (Alexa 637-conjugated goat
anti-rabbit) diluted (1/200) in the same buffer for 2 h at room
temperature in the dark, and washed before being mounted in Vec-
tashield (mounting medium for fluorescence, Vector Laboratories,
Inc., Burlingame, CA 94010) and cover-slipped. For negative con-
trols, primary antibodies were omitted. The samples were observed
using a confocal laser-scanning microscope (Leica SP2 UV DM IRBE).
Quantification of protein levels was performed using Image J 1.42q
software (National Institutes of Health, USA).
Determination of vascular reactive oxygen species (ROS)
formation
The oxidative fluorescent dye dihydroethidine was used to
evaluate the in situ formation of ROS. Mesenteric arterial rings
(3–4 mm length) were embedded in OCT compound (Tissue-
Tek) and snap-frozen in liquid nitrogen. Frozen arteries were
cryosectioned at 25 ␮m. Sections were air-dried for 15 min and
stored at −80 ◦ C until use. Dihydroethidine (2.5 ␮M, Sigma) was
applied onto unfixed cryosections of mesenteric arteries for 30 min
at 37 ◦ C in a light-protected humidified chamber to determine
the in situ formation of ROS. To determine the nature and the
source of ROS, sections were incubated with either, superoxide
dismutase (500 UI/ml), MnTMPyP (membrane-permeant super-
oxide dismutase mimetic, 100 ␮M), polyethylene glycol-catalase
(membrane-permeant analog of catalase, 500 UI/ml), catalase
(500 UI/ml), l-NA (NO synthase inhibitor, 300 ␮M), indomethacin
(cyclooxygenase inhibitor, 10 ␮M) for 30 min at 37 ◦ C before addi-
tion of dihydroethidine. Images were obtained with a confocal
microscope and analyzed with Image J 1.42q software.
Materials
Antibodies were purchased as indicated: COX-1 monoclonal
antibody, COX-2 polyclonal antibody (Cayman Chemical Company,
Michigan, USA), 637 labeled goat anti-mouse IgG or 637 labeled
goat anti-rabbit IgG (Invitrogen, Molecular Probes). Apamin and
charybdotoxin were obtained from Latoxan (Valence, France). l-
NA, indomethacin, ACh, PE, sodium nitroprusside, levcromakalim
were obtained from Sigma–Aldrich.
Statistical analysis
Data are presented as mean ± S.E.M. of n different experiments.
Mean values are compared using analysis of variance followed
by Bonferroni post hoc test (comparison of selected pairs), using
GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA).
The difference was considered to be significant when p value was
less than 0.05.

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Fig. 1. Crataegus treatment prevents aging-related blunted endothelium-dependent relaxations and increased endothelium-dependent contractile responses without affect-
ing NO and EDHF components of relaxations. Wistar rats were fed for 40 weeks with either a standard diet or the same food enriched with Crataegus extract (100 mg/kg/day
or 300 mg/kg/day) from week 25 until week 65. 16-Week-old rats were used as young control. Mesenteric artery rings with endothelium from young and old rats were
contracted with 1 ␮M of PE, before a concentration–relaxation curve to ACh was constructed (A) in absence of inhibitors, (C) in the presence of indomethacin (10 ␮M) and
apamin (100 nM) plus charybdotoxin (100 nM) to inhibit the participation of prostanoids and EDHF, respectively or (D) in the presence of indomethacin (10 ␮M) and l-NA
(300 ␮M) to rule out the participation of prostanoids and NO, respectively. (B) Concentration–contraction curve to ACh assessed in mesenteric artery rings with endothelium
contracted with 10–100 nM of PE, in the presence of l-NA (300 ␮M) to prevent the endothelial formation of NO, and apamin (100 nM) plus charybdotoxin (100 nM) to inhibit
EDHF-mediated responses. (E) Concentration–relaxation curves to sodium nitroprusside and (F) levcromakalim in mesenteric artery rings without endothelium. Results are
shown as mean ± S.E.M. of 5–6 different rats. *p < 0.05 indicates a significant difference versus young control rats and # p < 0.05 versus 65-week-old control rats.

Results rats. The ACh-induced NO-mediated relaxations were similar in

Crataegus treatment prevents the aging-related endothelial
dysfunction in the mesenteric artery of 65-week-old rats
In mesenteric artery rings with endothelium, acetylcholine
(Ach) caused concentration-dependent relaxations (Fig. 1A), which
were significantly reduced in 65-week-old rats compared to young
mesenteric artery rings from young rats and 65-week-old rats
(Fig. 1C), whereas, the EDHF-mediated relaxations were markedly
reduced in mesenteric artery rings of 65-week-old rats com-
pared to 16-week-old rats (Fig. 1D). ACh was also able to evoke
concentration-dependent contractile responses, which were sig-
nificantly increased in 65-week-old rats compared to young rats
(Fig. 1B). To determine whether aging is also related to an

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A Young control rats

B Old control rats

3 3
With Endothelium * With Endothelium
Without Endothelium
* Without Endothelium
*

Contraction (g)
Contraction (g)

2 2

1 1

0 0

-9 -8 -7 -6 -5 -9 -8 -7 -6 -5
Phenylephrine, Log [M] Phenylephrine, Log [M]

C Old rats / Crataegus (100 mg/Kg/j)

D Old rats / Crataegus (300 mg/Kg/j)

3 3
With Endothelium With Endothelium *
Without Endothelium
*
Without Endothelium

Contraction (g)
Contraction (g)

2 2

1 1

0 0

-9 -8 -7 -6 -5 -9 -8 -7 -6 -5
Phenylephrine, Log [M] Phenylephrine, Log [M]

Fig. 2. Crataegus treatment restores the ability of the endothelium to depress PE-induced contractile responses in old mesenteric arteries. Concentration–contraction to PE
in mesenteric artery rings with and without endothelium from (A) control young rats (16-week-old) and old rats (65-week-old) fed for 40 weeks with either (B) a standard
diet or the same food enriched with Crataegus extract (C and D). Data are shown as means ± S.E.M. of 6 experiments. *p < 0.05 indicates a significant difference versus
endothelial-intact rings.

impaired basal formation of NO, the ability of the endothelium
to depress contractile responses was evaluated. As expected, con-
tractile responses to PE were slightly but significantly reduced in
mesenteric artery rings with endothelium compared to those with-
out endothelium in young control rats (Fig. 2A). In contrast to young
rats, PE induced similar contractile responses in rings with and
without endothelium of 65-week-old rats (Fig. 2B) indicating that
the endothelium is no longer able to depress PE-induced contractile
responses.
The chronic oral ingestion of either 100 or 300 mg/kg/day of
the Crataegus extract from week 25 until week 65 significantly
improved the aging-related blunted ACh-induced endothelium-
dependent relaxations (Fig. 1A) and increased endothelium-
dependent contractile responses (Fig. 1B), and restored also the
ability of the endothelium to depress the PE-induced contractile
responses (Fig. 2C and D). However, the Crataegus treatment did
affect neither the NO nor the EDHF components (Fig. 1C and D). In
addition, the Crataegus treatment did not affect relaxations evoked
by either the exogenous donor of NO (sodium nitroprusside) or the
ATP-sensitive K+ channel opener (levcromakalim) in mesenteric
artery rings without endothelium (Fig. 1E and F).
COX inhibitors and a TP receptor antagonist improve the
aging-related endothelial dysfunction
The aging-related blunted ACh-induced endothelium-
dependent relaxations were improved by treatment of rings
with either the COX inhibitor (indomethacin), the COX-1 inhibitor
(SC-560), the COX-2 inhibitor (NS-398) or the TP receptor antag-
onist (GR32191B) (Fig. 3A). In addition, indomethacin abolished
the aging-related increased ACh-induced endothelium-dependent
contractile responses (Fig. 3B).
Crataegus treatment prevents the aging-related increased
vascular formation of ROS
ROS formation was markedly increased throughout the mesen-
teric arterial wall of 65-week-old rats compared to young rats.
Chronic ingestion of 300 mg/kg/day of the Crataegus extract was
associated with a significant reduced ROS level in the mesen-
teric arterial wall of 65-week-old rats, whereas a reduction was
also observed with the dose of 100 mg/kg/day however this effect
did not reach a statistically significant level (Fig. 4A). In order to
determine the nature and sources of ROS, arterial sections from
65-week-old rats were treated with different inhibitors and the for-
mation of ROS was assessed by fluorescence. The increased vascular
ROS formation was markedly reduced by membrane-permeant
analogs of superoxide dismutase (MnTMPyP) and catalase (PEG-
catalase) but not by native superoxide dismutase and catalase
indicating involvement predominantly of an intracellular forma-
tion of superoxide anions and hydrogen peroxide (Fig. 4B). It was
also blunted by l-NA (an NO synthase inhibitor) and indomethacin
demonstrating the involvement of uncoupled eNOS and cyclooxy-
genases (Fig. 4B).

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A Old control rats B Old control rats

1.2 Control
0
Relaxation (% PE, 10-6 M) Indo methacin (10-5 M )

Contraction (g)
25 0.8

50
Control 0.4
Indomethacin
75 *
SC-560 * * * 0.0 * *
NS-398 * * *
100
GR32191B
-9 -8 -7 -6 -5 -8 -7 -6 -5
Acetylcholine, Log [M] Acetylcholine, Log [M]

Fig. 3. Role of arachidonic acid-derived prostanoids in the aging-related blunted endothelium-dependent relaxations and increased endothelium-dependent contractile
responses. (A) Effects of the COX-1 inhibitor (SC-560, 0.1 ␮M), the COX-2 inhibitor (NS-398, 1 ␮M), the COX inhibitor (indomethacin, 10 ␮M) and the TP receptor antagonist
(GR32191B, 1 ␮M) on relaxations to ACh in mesenteric artery rings with endothelium contracted with PE (1 ␮M) from control old rats (65-week-old). (B) Effects of the COX
inhibitor (indomethacin, 10 ␮M) on concentration–contraction curves to ACh in mesenteric artery rings with endothelium contracted with PE (10–100 nM) from control old
rats (65-week-old), in the presence of l-NA (300 ␮M) to prevent the endothelial formation of NO and apamin (100 nM) plus charybdotoxin (100 nM) to inhibit EDHF-mediated
responses. Data are represented as means ± S.E.M. of 6 experiments. *p < 0.05 indicates a significant difference versus control.

Crataegus treatment normalizes the aging-related vascular
up-regulation of COX-1 and COX-2 expression
Since COXs are involved in the aging-related endothelial dys-
function and vascular oxidative stress, the vascular expression level
of COX-1 and COX-2 was assessed by immunohistochemistry. The
expression level of COX-1 and COX-2 was significantly increased
throughout the arterial wall in mesenteric arteries from 65-week-
old rats compared to young rats (Fig. 5). The chronic ingestion of
300 mg/kg/day of Crataegus extract normalized the expression of
these proteins in mesenteric arteries of old rats to level similar as
that seen in young rats, whereas the lower dose (100 mg/kg/day)
reduced also COX-1 and COX-2 expression level but this effect did
not reach significance (Fig. 5).
Discussion
The major findings of the present study is that chronic intake of
the Crataegus special extract WS® 1442 from week 25 until week
65 prevented the aging-related blunted endothelium-dependent
relaxations and the increased endothelium-dependent contractile
responses in the mesenteric artery. The protective effect of the
Crataegus extract most likely involves its ability to normalize the
aging-related vascular oxidative stress and COX-1- and COX-2-
mediated endothelium-dependent contractile responses.
Advancing age is a major risk factor for the development of
cardiovascular diseases which is attributable, at least in part, to
the development of vascular endothelial dysfunction (Seals et al.
2011). The present study indicates that ACh-induced endothelium-
dependent relaxations are strongly reduced in the mesenteric
artery of 65-week-old rats in comparison to young rats. These
results are in line with several previous studies indicating that
aging is associated with impaired endothelium-dependent relax-
ations as observed in various type of arteries such as the human
brachial artery (Celermajer et al. 1994), the rat aorta (Kung and
Luscher 1995), the rat carotid artery (Hongo et al. 1988) and
the rat perfused mesenteric bed (Atkinson et al. 1994). In the
rat mesenteric artery, endothelium-dependent relaxations to ACh
involve mainly a NO component (Furchgott and Zawadzki 1980)
and an EDHF component (Chen et al. 1988). Therefore, experi-
ments have been performed to determine whether aging affects
both components. The findings indicate that the ACh-induced
EDHF-mediated relaxations are markedly reduced, whereas the
NO-mediated relaxations are not affected. Endothelial dysfunc-
tion affecting predominantly the EDHF component with no or
little effect on the NO component has also been observed previ-
ously in the mesenteric artery of hypertensive rats (Hilgers and
Webb 2007), type 2 diabetic rats (Young et al. 2008) and aged
rats (Goto et al. 2000). However, some studies have also observed
that aging is associated with blunted NO-mediated relaxations,
such as in the second and the third branch of the superior mesen-
teric artery (Matz et al. 2000), the aorta (van der Loo et al. 2000),
the carotid artery (Hongo et al. 1988) and the coronary arteriole
(Csiszar et al. 2002) of rats. In addition, experiments have been
also performed to determine whether aging affects basal NO for-
mation in the mesenteric artery as assessed indirectly by the ability
of endothelial cells to depress contractile responses (Martin et al.
1986). Although phenylephrine-induced contractile responses are
depressed in mesenteric artery rings with endothelium from young
rats compared to those without endothelium, no such effect is
observed in mesenteric artery rings from 65-week-old rats indi-
cating that aging is associated with reduced basal NO formation.
The fact that endothelium-dependent relaxations in mesen-
teric artery rings of old rats were improved by indomethacin (a
non-selective COX inhibitor), SC-560 (a selective COX 1 inhibitor),
NS-398 (a selective COX 2 inhibitor) and by GR32191B (an antag-
onist of TP receptors) indicates that the endothelial dysfunction
involves cyclooxygenase-derived prostanoids, which contract the
vascular smooth muscle through activation of TP receptors, thereby
counteracting endothelium-dependent relaxations. Similar obser-
vations have also been made in the aorta (Heymes et al. 2000) and
the mesenteric artery (Matz et al. 2000) of old rats. These find-
ings are in good agreement with the fact that pretreatment of rings
with indomethacin abolished the aging-associated increased ACh-
induced endothelium-dependent contractile responses
Several studies have indicated that vascular oxidative stress
plays a major role in aging-related endothelial dysfunction (Csiszar
et al. 2002). In agreement with these data, the present findings indi-
cate that oxidative stress was significantly increased in the arterial
wall of 65-week-old rats compared to young rats, and involved an
increased intracellular formation of superoxide anions and hydro-
gen peroxide. Since, the aging-related increased formation of ROS
is reduced in the presence of inhibitors of either cyclooxyge-
nases or eNOS, it implies the involvement of cyclooxygenases and
uncoupled eNOS. COX-2 has also been involved in the excessive for-
mation of ROS associated with the endothelial dysfunction in the

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Fig. 4. Crataegus treatment prevents aging-related increased vascular formation of ROS in rat mesenteric arteries: involvement of several sources. (A) Mesenteric arterial
sections from the indicated groups were exposed to the redox-sensitive fluorescent dye dihydroethidine (DHE) for 30 min at 37 ◦ C. Thereafter, ethidium fluorescence was
determined by confocal microscopy. Upper panel represents ethidium staining and lower panel corresponding cumulative data. (B) Mesenteric artery sections from old
rats were exposed to either superoxide dismutase, MnTMPyP (membrane-permeant superoxide dismutase mimetic), catalase, PEG-catalase (membrane-permeant analog of
catalase), l-NA (NO synthase inhibitor) or indomethacin for 30 min before DHE staining. Thereafter, ethidium fluorescence was determined by confocal microscopy. Results
are shown as mean ± S.E.M. of 4 different rats. *p < 0.05 indicates a significant effect versus corresponding control and # p < 0.05 versus old control rats.

mesenteric artery of a rat model of endotoxic shock (Actis-Goretta
et al. 2006). The possibility that aging-related COX-mediated con-
tractile responses and vascular oxidative stress are associated
with changes in the vascular expression of COX-1 and COX-2 was
evaluated by immunohistochemistry. Both the expression level
of COX-1 and COX-2 were increased in 65-week-old rats com-
pared to young rats. In addition, previous studies have indicated
that oxidative stress plays a key role in the prostanoid-derived
increased endothelium-dependent contractile responses in differ-
ent cardiovascular diseases (Tian et al. 2011). Indeed, ROS have been
suggested to activate endothelial COX-2 leading to the production
PGF2␣, the most likely EDCF to be involved in the endothelial dys-
function in renal arteries of renovascular hypertensive rats (Tian
et al. 2011). Moreover, ROS have also been shown to activate
COX-1 leading to the production of endoperoxides, which acti-
vate TP receptors, thereby accounting for endothelium-dependent
contractile responses to ACh in the aorta of spontaneously hyper-
tensive rats (Yang et al. 2002). Furthermore, angiotensin II caused
a NADPH oxidase-mediated formation of ROS, which is able to
stimulate COX-1 activity leading to the production of contracting
prostanoids in small mesenteric arteries of mice (Virdis et al. 2007).
Altogether, these findings indicate that oxidative stress activates
cyclooxygenases, which, in turn, results in an increased formation
of vasocontracting prostanoids but most likely also to an increased
formation of ROS, which perpetuates the endothelial dysfunction.
The present findings indicate that chronic ingestion of either 100
or 300 mg/kg/day of a Crataegus extract for 40 weeks prevented
the aging-related blunted endothelium-dependent relaxations in
rats. Previous studies have also shown that oral supplementation
with polyphenol-rich sources improves endothelial function in var-
ious animal models of cardiovascular pathologies associated with
endothelial dysfunction. Indeed, red wine poyphenols improved
the endothelial function in L-NA-induced hypertension (Pechanova
et al. 2004), angiotensin II-induced hypertension (Sarr et al. 2006)
and in middle-aged rats (Dal-Ros et al. 2011). The characterization
of the protective effect of the Crataegus treatment indicated that
neither the ACh-induced NO- nor the EDHF-mediated relaxations
were improved despite a consistent improvement of endothelium-
dependent relaxations. The findings also indicate that the Crataegus
treatment prevented the aging-related blunted basal endothelial

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Fig. 5. Crataegus treatment prevents aging-related COX-1 and COX-2 up-regulation in rat mesenteric arteries. The expression level of COX-1 and COX-2 was determined
using a purified polyclonal antibody and a fluorescence-tagged secondary antibody by confocal microscopy. Upper panels show representative immunofluorescence staining
and lower panels corresponding cumulative data. Results are shown as mean ± S.E.M. of 4 different rats. *p < 0.05 indicates a significant effect versus young control rats and
#
p < 0.05 versus old control rats.
formation of NO as assessed indirectly by the depression of the
contractile responses at the dose of 300 mg/kg/day. In addition,
the aging-related increased ACh-induced endothelium-dependent
contractions are abolished by the Crataegus treatment. The benefi-
cial effect of the Crataegus treatment on aging-related endothelium
dependent contractile responses is most likely explained by the
normalization of the vascular expression of COX-1 and COX-2 and
vascular oxidative stress. Previous studies have also shown that
5-day oral treatment with Crataegus pinnatifida prevents inflam-
matory responses induced by lipopolysaccharide in rats, at least
in part, through inhibition of the hepatic expression of COX-2,
TNF-␣, IL-1␤, and IL-6 (Li and Wang 2011). Moreover, Cratae-
gus oxycantha reduced myocardial oxidative stress in a model of
ischemia-reperfusion injury (Swaminathan et al. 2010).
Conclusion
Aging is associated with the development of an endothelial dys-
function in the mesenteric artery of rats, which is most likely due
to a reduced EDHF-mediated endothelium-dependent relaxation,
reduced basal NO formation and the appearance of endothelium-
derived contractile responses involving cyclooxygenase-derived
prostanoids acting on TP receptors to contract the vascular
smooth muscle. Chronic intake of the Crataegus extract effectively
prevented the development of aging-related endothelial dysfunc-
tion as indicated by normalized endothelium-derived contractile
responses and basal NO formation; these effects are most likely
the consequences of a reduced expression of COX-1 and COX-2 and
formation of ROS.
Conflict of interest
This work was supported, in part, by a research grant by Dr. Will-
mar Schwabe GmbH & Co. KG, Karlsruhe, Germany. The funders have
participated in the study design and redaction of manuscript, but
they did not contribute to data collection and analysis. No addi-
tional external funding was received for this study.
Please cite this article in press as: Idris-Khodja, N., et al., Crataegus special extract WS® 1442 prevents aging-related endothelial dysfunction.
Phytomedicine (2012), http://dx.doi.org/10.1016/j.phymed.2012.04.005
7
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65
Synthèse des résultats de l’article 3 :

Nous avons montré dans cette étude que le traitement préventif des rats Wistar avec un

l’extrait d’aubépine, pendant 40 semaines, à partir de l’âge de 25 semaines améliore la

fonction endothéliale au niveau de l’artère mésentérique de rat. Le traitement préventif avec

extrait d’aubépine prévient la réduction des relaxations dépendantes de l’endothélium ainsi

que l’augmentation des réponses contractiles dépendante de l’endothélium. Cet effet est

observé avec les deux doses utilisées (100 mg/kg/j et 300 mg/kg/j). Il est bien connu que les

relaxations dépendantes de l’endothélium au niveau de l’artère mésentérique sont constituées

essentiellement de deux composantes de relaxation qui sont le NO et l’EDHF. La

caractérisation des composantes de relaxation dépendantes de l’endothélium révèle que le

traitement préventif avec l’extrait de polyphénols améliore ni la composante NO ni la

composante EDHF de relaxation. De plus, le traitement des anneaux d’artère mésentériques

de rats âgés avec l’indométacine (inhibiteur non sélectif de cyclooxygénases), SC-560

(inhibiteur sélectif de COX-1), NS-398 (inhibiteur sélectif de COX-2) ou le GR32191B

(antagoniste des récepteurs TP) restore les relaxations dépendantes de l’endothélium. Les

réponses contractiles observées dans l’artère mésentérique de rats âgés sont abolies par

l’indométacine. Le traitement préventif des rats avec l’extrait de Crataegus prévient

l’augmentation de la formation des ROS observée chez les rats âgés de 65 semaines.

L’analyse de la nature et des sources des ROS indique qu’il s’agit essentiellement d’anions

superoxydes et de peroxyde d’hydrogène qui sont formés par plusieurs sources enzymatiques

impliquant la eNOS découplée et les cyclooxygénases. De plus, le traitement préventif avec

l’extrait d’aubépine prévient la diminution de la formation du NO basale chez les rats âgés et

cet effet est observé seulement avec la dose de 300 mg/kg/j. La formation du NO basale a été

évaluée par mesure de la capacité de l’endothélium à diminuer les réponses contractiles en

mesurant les contractions des anneaux d’artères mésentérique avec et sans endothélium en

66
Synthèse des résultats de l’article 3 :

Nous avons montré dans cette étude que le traitement préventif des rats Wistar avec un

l’extrait d’aubépine, pendant 40 semaines, à partir de l’âge de 25 semaines améliore la

fonction endothéliale au niveau de l’artère mésentérique de rat. Le traitement préventif avec

extrait d’aubépine prévient la réduction des relaxations dépendantes de l’endothélium ainsi

que l’augmentation des réponses contractiles dépendante de l’endothélium. Cet effet est

observé avec les deux doses utilisées (100 mg/kg/j et 300 mg/kg/j). Il est bien connu que les

relaxations dépendantes de l’endothélium au niveau de l’artère mésentérique sont constituées

essentiellement de deux composantes de relaxation qui sont le NO et l’EDHF. La

caractérisation des composantes de relaxation dépendantes de l’endothélium révèle que le

traitement préventif avec l’extrait de polyphénols améliore ni la composante NO ni la

composante EDHF de relaxation. De plus, le traitement des anneaux d’artère mésentériques

de rats âgés avec l’indométacine (inhibiteur non sélectif de cyclooxygénases), SC-560

(inhibiteur sélectif de COX-1), NS-398 (inhibiteur sélectif de COX-2) ou le GR32191B

(antagoniste des récepteurs TP) restore les relaxations dépendantes de l’endothélium. Les

réponses contractiles observées dans l’artère mésentérique de rats âgés sont abolies par

l’indométacine. Le traitement préventif des rats avec l’extrait de Crataegus prévient

l’augmentation de la formation des ROS observée chez les rats âgés de 65 semaines.

L’analyse de la nature et des sources des ROS indique qu’il s’agit essentiellement d’anions

superoxydes et de peroxyde d’hydrogène qui sont formés par plusieurs sources enzymatiques

impliquant la eNOS découplée et les cyclooxygénases. De plus, le traitement préventif avec

l’extrait d’aubépine prévient la diminution de la formation du NO basale chez les rats âgés et

cet effet est observé seulement avec la dose de 300 mg/kg/j. La formation du NO basale a été

évaluée par mesure de la capacité de l’endothélium à diminuer les réponses contractiles en

66
mesurant les contractions des anneaux d’artères mésentérique avec et sans endothélium en

réponse à la phénylephrine.

Enfin, le traitement préventif de rat avec l’extrait d’aubépine prévient la surexpression

des cyclooxygénases 1 et 2 observée au cours du vieillissement. Cet effet pourrait être à

l’origine de la diminution des réponses contractiles induites par les prostanoïdes dérivés de

l’endothélium observée chez le rat âgé traité avec l’aubépine.

En conclusion, le traitement préventif des rats avec l’extrait d’aubépine pendant 40

semaines à partir de l’âge de 25 semaines prévient la dysfonction endothéliale liée au

vieillissement caractérisée par une exacerbation des réponses contractiles médiées par les

prostanoïdes dérivés de l’endothélium, par un stress oxydant et par une augmentation de

l’expression de cyclooxygénases 1 et 2 dans la paroi de l’artère mésentérique.

67
Article 4 :

Noureddine Idris Khodja, Marouane Kheloufi and Valérie B. Schini-Kerth. Crataegus special extract
WS1442 delays replicative senescence in coronary endothelial cells. (en préparation, 2012)

Le vieillissement est associé à une incidence élevée des maladies cardiovasculaires qui sont

dues en partie à une dysfonction endothéliale. An niveau vasculaire, la dysfonction

endothéliale liée au vieillissement est considérée comme une diminution des réponses

vasorelaxantes et anti-thrombotiques de l’endothélium. Au niveau cellulaire, le vieillissement

des cellules endothéliales mène à un état irréversible d’arrêt du cycle cellulaire appelé

sénescence. Ces cellules sénescentes présentent des changements spécifiques au niveau de

leur morphologie et de l’expression génique, menant à leur dysfonction. La sénescence dite

réplicative est déclenchée par le raccourcissement des télomères survenant à chaque division

cellulaire, mais elle peut également être induite prématurément par le stress oxydant (SIPS).

L’objectif principal de cette étude est de caractériser la sénescence réplicative de cellules

endothéliale d’artères coronaires de porc et d’étudier le potentiel effet protecteur d’un

traitement avec un extrait d’aubépine riche en procyanidines sur la sénescence. Nous avons

isolé et cultivé des cellules endothéliales provenant d’artères coronaires de porc. Les cellules

ont subi des passages successifs jusqu’à l’induction de la sénescence, en présence ou en

absence de l’extrait d’aubépine. La sénescence des cellules endothéliales est associée à une

augmentation de l’activité de la β-galactosidase à pH=6. Nous avons utilisé deux techniques

de mesure de l’activité de la β-galactosidase : la méthode cytochimique et la détection par

cytométrie en flux. La méthode cytochimique est basée sur clivage d’un substrat

chromogénique (5-bromo-4-chloro-3-indolyl β-galactopyranoside) par la β-galactosidase

donnant lieu à une coloration bleue des cellules. La sénescence est plus importante quand le

pourcentage des cellules bleues est plus important. La seconde méthode est basée sur le

68
clivage du C12FDG par la β-galactosidase donnant lieu à substrat fluorescent dont la quantité

qui est évaluée par cytométrie en flux. La détection cytochimique de l’activité la de β-

galactosidase nécessite moins de temps et d’équipements que les tests basés sur la

fluorescence. L'inconvénient est que le pourcentage des cellules bleues qui est fait par

comptage manuel est moins sensible et subjectif. La mesure de l’activité de la β-

galacatosidase par fluorescence est probablement la meilleure solution pour la quantification.

Nous nous sommes également intéressés au niveau d’expression de la protéine

suppresseur de tumeurs p53 et des protéines inhibitrices de cycle cellulaire p16 et p21, ainsi

que la eNOS au cours de la sénescence et l’impact d’un traitement avec les polyphénols sur

leurs niveaux d’expression.

69
 Crataegus special extract WS 1442 delays replicative senescence

in coronary artery endothelial cells : Role of eNOS-derived NO

Idris Khodja N, Kheloufi M and Schini-Kerth VB

CNRS UMR 7213, Faculty of Pharmacy, University of Strasbourg, Illkirch, France

Key words: senescence, endothelial dysfunction, eNOS, polyphenols, oxidative stress

ABSTRACT

Endothelial cell senescence promotes endothelial dysfunction, which has been suggested to

have a critical role in the initiation and/or progression of atherosclerosis, and also to

contribute to the pathogenesis of age-associated vascular disorders. Endothelial senescence is

characterized by an irreversible cell cycle arrest, which involves an increased activity of p53

and its downstream effector p21. Endothelial senescence is also associated with a decrease in

the expression of endothelial nitric oxide synthase (eNOS). The present study has evaluated

whether the Crataegus special extract WS1442, a rich source of polyphenols and a potent

activator of eNOS, prevents replicative senescence of porcine coronary artery endothelial

cells, and, if so, to elucidate the underlying mechanism. Replicative senescence was induced

by sequential passaging of primary endothelial cells up to the fourth passage (P4). Changes of

endothelial senescence were determined by measuring senescence-associated β-galactosidase

(SA-β-gal) activity. Western blot was used to analyze the protein expression of p53, p21 and

eNOS. Compared to P1, the SA-β-gal activity was increased in cells at P3 and P4, and this

effect was associated with an increased expression of p53, p21 and a decreased expression of

eNOS. Treatment of P3 cells with the p53 inhibitor (pifithrin) reduced SA-β-gal activity

indicating a role of p53 activity in replicative senescence. Treatment of endothelial cells with

the Crataegus extract reduced the SA-β-gal activity, improved eNOS expression and

prevented the up-regulation of p21 in cells from P3 and P4 without affecting the expression of
70
p53. The inhibitor of eNOS, L-NAME promoted the induction of endothelial senescence at P1

and reduced the inhibitory effect of the Crataegus extract on SA-β-gal activity at P3.

In conclusion, the present findings indicate that the Crataegus extract

delays endothelial cell replicative senescence most likely by preventing the downregulation of

eNOS expression and activity and the upregulation of p53/p21 pathway.

71
INTRODUCTION

Aging-related endothelial dysfunction is associated with endothelial senescence and both have

been suggested to play a key role in the initiation and/or progression of atherosclerosis

(Herrera et al. 2010). Endothelial cell senescence is characterized by an irreversible cell cycle

arrest with enlarged, flattened cell morphology and increased granularity and vacuolization

(Foreman & Tang 2003). Nevertheless, the cells remain viable and metabolically active

(Minamino & Komuro 2007). Senescence can be induced in vitro either by serially passaging

endothelial cells (replicative senescence) (Kalashnik et al. 2000), by a mechanism involving

telomere shortening (Foreman & Tang 2003), or prematurely by cellular stress such as

oxidative stress, radiations and DNA damage (stress-induced premature senescence)

(Erusalimsky & Skene 2009; Goligorsky et al. 2009). Ultimately, almost all of these signals

promoting senescence lead to the activation of either or both the p53/p21 and the

p16/retinoblastoma protein pathways (Chen et al. 2006; Erusalimsky 2009).

The vascular cell senescence contributes to the pathogenesis of age-associated vascular

diseases such as atherosclerosis. Indeed, an increased number of senescent endothelial cells

has been observed in human atherosclerotic coronary arteries of patients with ischemic heart

diseases (Minamino et al. 2002). In addition, the overexpression of p53 in the rat aorta

induced endothelial dysfunction and stimulated plasminogen activator inhibitor 1 (Kumar et

al. 2011), a marker and necessary factor of cell senescence (Kortlever et al. 2006).

Polyphenol-rich food and beverages have been shown to improve endothelial function and to

reduce the incidence of cardiovascular diseases in human (Michalska et al. 2010). Hawthorn

extract (Crataegus ssp.) is a rich source of polyphenols containing monomeric flavonoids and

oligomeric procyanidins, which has been shown to have cardiotonic and cardioprotective

properties. Previous studies have shown that the Crataegus special extract WS1442 is a

72
potent inducer of nitric oxide (NO)- and endothelium-derived relaxing factor (EDHF)-

mediated relaxations of coronary artery rings (Brixius et al. 2006) and that is also prevents

aging-related endothelial dysfunction (Idris-Khodja et al. 2012). The aim of the present study

was to determine whether the Crataegus extract is able to delay endothelial replicative

senescence, and, if so, to investigate the underlying mechanism.

73
METHODS

Crataegus special extract WS 1442

Crataegus WS1442 is a special extract obtained from leaves with flowers of selected

hawthorn species (Crataegus spp.) by extraction with 45% (w/w) aqueous ethanol

(drug/extract ratio 4–6.6:1). The extract is adjusted to a content of 17.3–20.1% oligomeric

procyanidins.

Isolation and culture of coronary artery endothelial cells

Porcine coronary artery segments were flushed with phosphate-buffered saline (PBS) without

calcium to remove remaining blood. Thereafter, endothelial cells were isolated by collagenase

treatment (type I, Worthington, 1 mg/mL for 12 min at 37 °C), cultured in culture dishes

containing medium MCDB 131 (Invitrogen) and 15% fetal calf serum supplemented with

penicillin (100 U/mL), streptomycin (100 U/mL), fungizone (250 mg/mL), and L-glutamine

(2 mM) (all from Cambrex), and grown for 48–72 h (passage 0). To prepare cultures for

passage 1 to 4, cells were detached with trypsin-EDTA (Gibco-BRL, Grand Island, N.Y.,

USA) and further passaged at a ratio of 1:3 at regular intervals until senescence was reached.

The medium was changed every 48 h.

Cytochemical detection of senescence-associated β-galactosidase (SA-β-gal) activity

The cytochemical detection of SA-β-gal activity used the chromogenic substrate 5-bromo-4-

chloro-3-indoyl β-D-galactopyranoside (X-gal), which yields an insoluble blue compound

when cleaved by β-galactosidase. Endothelial cells were fixed for 10 min in 2%

formaldehyde, 0.2% glutaraldehyde in PBS, and incubated for 12 h at 37°C without CO2 with

fresh staining solution: 1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium

ferricyanide, and 2 mM MgCl2, pH 6.0. The cells were counterstained with 4-6-

74
diamidinophenylindole (DAPI; 0.1 mg/ml in PBS) for 10 min to count the total cell number.

After the staining, the cells are washed with phosphate-buffered saline (PBS) and viewed by

bright field microscopy. The percentage of SA-β-gal positive cells was determined by

counting the number of blue cells under bright field illumination, and then the total number of

cells in the same field under phase contrast.

Fluorescence detection of SA-β-gal activity

To measure SA-β-gal activity by flow cytometry, we used the fluorogenic substrate C12FDG.

This compound is a membrane permeable, non-fluorescent substrate of β-galactosidase, which

after hydrolysis of the galactosyl residues emits green fluorescence and remains confined

within the cell. Endothelial cells were pretreated with chloroquine (300 µM) for 1 h to

increase the internal pH of lysosomes. Chloroquine is a weak base that concentrates in

lysosomes, raising the pH to approximately 6. C12FDG (33 µM) was then added to the

pretreatment medium (without phenol red) and the incubation was continued for 1 h. At the

end of the incubation, cultures were washed with ice-cold PBS, resuspended following

trypsinization, and analysed immediately using a FAC Scan flow cytometer (FACScan, BD

Biosciences, San Jose, CA, USA). Data were acquired and analyzed with Cellquest software

(Becton Dickinson). Light scatter parameters were used to eliminate dead cells and

subcellular debris. The C12-fluorescein signal was measured on the FL1 detector and SA-β-gal

activity was estimated using the median fluorescence intensity (MFI) of the population.

Autofluorescence was assessed in parallel in cells not exposed to C12FDG. In all cases these

values were found to be negligible when compared to the fluorescence levels of equivalent

samples incubated with C12FDG, and therefore were not taken into consideration for the final

estimation of SA-β-gal activity.

75
Western blot analysis

After treatment, cells were washed twice with PBS and then lysed in extraction buffer

[composition in mM: Tris/HCl 20 (pH 7.5; QBiogene), NaCl 150, Na3VO4 1, sodium

pyrophosphate 10, NaF 20, okadaic acid 0.01 (Sigma), a tablet of protease inhibitor (Roche),

and 1% Triton X-100 (QBiogene)]. Total proteins (10 or 30 mg) were separated on 8 or 12 %

sodium dodecyl sulfate–polyacrylamide (Sigma) gels at 100 V for 2 h and transferred

electrophoretically onto polyvinylidene difluoride membranes (Amersham) at 100 V for 120

min. Membranes were blocked with blocking buffer [Tris-buffered saline solution (Bio-Rad)

and 0.1% Tween 20 (Sigma)] containing 3% bovine serum albumin for 1 h. For detection of

proteins, membranes were incubated with the respective primary antibody (eNOS, p16, p21

and p53) overnight at 4 °C. After washing, membranes were incubated with the secondary

antibody (peroxidase-labeled anti-rabbit and anti-mouse immunoglobulin G, dilution of

1:5000; Cell Signaling Technology) at room temperature for 60 min. Prestained markers

(Invitrogen) were used for molecular mass determinations. Immunoreactive bands were

detected by enhanced chemiluminescence (Amersham). The amount of protein in each lane

was normalized to the housekeeping protein, β-actin, before the analysis was performed.

76
RESULTS

Crataegus treatment delays replicative endothelial senescence

To evaluate the effect of Crataegus special extract WS1442 on replicative senescence, we

subjected primary porcine coronary endothelial cells to serial passaging. Endothelial cells

after P4 stoped division and did not reach confluence even after 1 week under standard culture

conditions (data not shown). Cytochemical detection of SA-β-gal activity showed a

significant increase of the number of SA-β-gal positive cells at P4 compared to P1. Treatment

of endothelial cells with 10 µg/ml of Crataegus extract since P1 markedly reduced the number

of positive cells at P4 (Figure 1). Flow cytometry detection of C12-fluorescein fluorescence,

which is a better quantitative method to detect SA-β-gal activity, showed a gradual increase in

SA-β-gal activity upon passages. Treatment of cells with Crataegus extract since P1 with 10

µg/ml reduced endothelial senescence significantly at P2, P3 and P4 and also with 1 µg/ml at

P3 and P4 (Figure 2). Furthermore, a 48-h treatment period with the Crataegus extract

concentration-dependently decreased endothelial senescence with a 50 % reduction observed

at concentrations of or greater than 30 µg/ml in cells from P2 and P3 (Figure 3).

Crataegus treatment does not affect replicative senescence-induced p53 expression

p53 is a key regulator in cellular senescence (Bond et al. 1996). Therefore, we investigated

the effect of Crataegus extract on p53 expression in endothelial cells. Increasing the passage

number was associated with an upregulation of p53, which was not affected by the Crataegus

treatment (Figure 4A). Treatment of endothelial cells at P3 with the p53 inhibitor pifithrin

concentration-dependently decreased SA-β-gal activity (Figure 4B), mimicking the effect of

the Crataegus treatment (Figure 3). Thus, these findings suggest that the Crataegus extract

inhibits the activity of p53 rather than its expression.

77
Crataegus treatment prevents replicative senescence-induced p21 and p16 expression

To investigate the effect of the Crataegus treatment on cell cycle regulatory proteins, the

levels of p21 and p16 were assessed by Western blot analysis. Serial passaging of endothelial

cells increased p21 levels at P3 and P4 compared to P1, and treatment with the crataegus

extract significantly reduced the senescence-induced p21 upregulation (Figure 5A). A

preliminary experiment indicated that p16 expression was upregulated by increasing passage

number, and that this effect seems to be prevented by the Crataegus treatment (Figure 5B).

Role of eNOS-dervied NO in the protective effect of the Crataegus extract on endothelial

senescence

Since eNOS plays a critical role in endothelial function, we investigated the effect of the

Crateagus treatment on eNOS levels in senescent endothelial cells. Serial passaging of

endothelial cells resulted in a remarkably reduced eNOS level at P3 and P4 compared to P1.

Treatment of endothelial cells with the Crataegus extract significantly reduced the replicative

senescence-induced downregulation of eNOS expression at P3 and P4 (Figure 6A).

Furthermore, treatment of P1 cells with the eNOS inhibitor L-NAME increased SA-β-gal

activity by about 2 fold, suggesting the induction of a premature senescence-like phenotype,

such an effect was prevented by the Crataegus treatment (Figure 6B). A preliminary data

suggest that the beneficial effect of the Crataegus treatment on endothelial senescence at P3

seems to be reduced by L-NAME indicating the involvement of NO (Figure 6C).

78
DISCUSSION

The major findings indicate that the Crataegus extract delays replicative endothelial

senescence of porcine coronary artery endothelial cells. The protective effect of the Crataegus

extract is associated with a reduced p21 and p16 expression and the prevention of the

downregulation of eNOS expression.

Consistent with previous studies, primary cultured coronary artery endothelial cells undergo

replicative senescence upon successive passaging (Lee et al. 2010). The present study

provides evidence that Crataegus polyphenols protect endothelial cells against serial

passaging-induced replicative senescence. These findings are consistent with a previous study

showing that red wine extract reduced oxidative stress-induced endothelial cell senescence

(Botden et al. 2012). In addition, resveratrol prevented H2O2-induced senescence of human

umbilical vein endothelial cells (Kao et al. 2010) and reduced endothelial progenitor cell

senescence through an increase of the telomerase activity (Xia et al. 2008). Furthermore, the

antioxidant treatment with N-acetylcysteine delayed endothelial cell replicative senescence by

preventing ROS-induced telomerase export (Haendeler et al. 2004).

p53 is a tumor suppressor gene involved in replicative senescence (Deng et al. 2008).

Endothelial cells subjected to serial passaging showed an increase in p53 expression, further

supporting the onset of senescence. This observation is consistent with a previous study

showing an inverse relationship between telomere lengths and levels of p53 in bovine corneal

endothelial cells during aging (Whikehart et al. 2000). Senescence of endothelial cells is also

associated with an overexpression of p21, a DNA damage-related cyclin-dependent kinase

inhibitor and an important mediator of endothelial cell senescence (Freedman & Folkman

2005). This is in agreement with previous findings suggesting that accumulation of cell cycle

inhibitors underlies the onset of replicative senescence (Wagner et al. 2001). The Crataegus

79
treatment did not affect p53 expression, suggesting that it may possibly act on p53 activity.

Consistent with such hypothesis, polyphenols are known to activate Sirt1, a histone

deacetylase that negatively regulates p53 activity by deacetylation (Chung et al. 2010).

Crataegus treatment clearly prevented replicative senescence-induced p21 upregulation

supporting the hypothesis that the Crataegus extract may inhibit p53 activity. Furthermore

SA-β-gal activity was reduced in senescent cells treated with the p53 inhibitor, pifithrin.

In addition, preliminary findings indicate that serial passaging of endothelial cells increases

the expression of p16, a member of the Ink4 family of CDK inhibitors and one of the

principal factors involved in senescence (Romagosa et al. 2011). In endothelial cells, p16 has

been reported to contribute to the induction of premature senescence since HUVECs

transfected with p16-EGFP showed an increased proportion of senescent cells (Chen et al.

2006). The effect of Crateagus treatment on replicative senescence-induced p16 expression is

unclear and needs further experiments.

The present findings indicate that eNOS expression decreases progressively by passaging

endothelial cells. Such an effect is in accordance with previous findings with cultured

endothelial cells (Lee et al. 2010) and also with an in vivo study indicating that eNOS

expression is reduced in the mesenteric arteries of aged rats (Idris Khodja et al. 2012).

Increasing evidence suggests that endothelial cell senescence is directly linked to endothelial

dysfunction. Indeed, it has been reported that cells subjected to replicative senescence showed

a decreased eNOS expression and NO formation (Matsushita et al. 2001; Minamino et al.

2002), which were rescued by stable hTERT expression (Matsushita et al. 2001).

Furthermore, NO appears to delay endothelial senescence by modulating telomerase activity

(Vasa et al. 2000), and has been reported to mediate the protective effect of bradykinin

against oxidative stress-induced senescence (Oeseburg et al. 2009), implying a positive

regulatory loop between telomerase and eNOS-derived NO. Such an effect may also explain

80
the protective effect of the Crataegus treatment on replicative senescence, since the Crataegus

extract is a potent activator of eNOS in endothelial cells (Anselm et al. 2009). Consistent with

such hypothesis, treatment of endothelial cells with the eNOS inhibitor L-NAME partially

inhibited the protective effect of the Crataegus extract on endothelial senescence. Recent

findings suggest a role of p53 in the mechanism underlying the downregulation of eNOS.

Indeed, p53 has been shown to downregulate KLF2 expression, a positive transcriptional

regulator of eNOS expression (Atkins & Jain 2007), either directly by transcriptional

repression (Kumar et al. 2011), or indirectly by upregulation of p66shc (Kim et al. 2008;

Kumar et al. 2009), since oxidative stress is implicated in replicative senescence (von

Zglinicki 2000), it remains to determine whether the antioxidant properties of the Crataegus

extract mediate the beneficial effect on endothelial senescence.

In conclusion, the Crataegus treatment delays replicative endothelial senescence by

mechanisms involving the prevention of the downregulation of eNOS and the increased

expression of p21.

81
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83
Legends

Figure 1: Crataegus treatment decreases the cytochemical detection of SA-β-gal activity

in serially passaged porcine coronary artery endothelial cells. Porcine coronary artery

endothelial cells were serially passaged from P1 to P4, and treated with either vehicle or

Crataegus extract (3 or 10 µg/ml) from P1 until P4. Upper panels show representative images,

and lower panels corresponding cumulative data. Results are shown as mean ± S.E.M. of 3

different experiments. *P < 0.05 indicates a significant effect versus P1 and #P < 0.05 versus

control group.

Figure 2: Crataegus treatment decreases the detection of SA-β-gal activity by flow

cytometry in serially passaged porcine coronary artery endothelial cells. (A) Flow

cytometry histograms of C12-fluorescein fluorescence in serially passaged primary endothelial

cell; cultures were incubated with C12FDG for 1 h at 37°C in the presence of 300 µM de

chloroquine prior to analysis. (B) C12-fluorescein median fluorescence intensity (MFI) of

serially passaged endothelial cells from P1 to P4. (C) C12-fluorescein MFI of serially passaged

endothelial cells from P1 to P4 treated with either vehicle or Crataegus extract (1 or 10

µg/ml). *P < 0.05 indicates a significant effect versus control group.

Figure 3: Concentration-dependent effects of Crataegus extract on SA-β-gal activity (3

days of exposure) of cultured cells at passage 2 and 3. C12-fluorescein median fluorescence

intensity (MFI) of serially passaged endothelial cells from P2 to P3 treated with increasing

concentrations of Crataegus extract (3-70 µg/ml). *P < 0.05 indicates a significant effect

versus control group.

Figure 4: Expression of p53 in serially passaged endothelial cells with or without

Crataegus treatment and effect of p53 inhibition on endothelial cell senescence. (A)

Representative Western blots showing immunodetectable p53 in serially passaged endothelial

84
cells treated or not with the Crataegus extract. (B) Densitometric analysis of results. The

quantification of proteins was normalized to that of the housekeeping protein, GAPDH, for

equal loading. *P < 0.05 indicates a significant effect versus P1. (C) C12-fluorescein median

fluorescence intensity (MFI) of endothelial cells at P3 treated with increasing concentrations

of the p53 inhibitor pifithrin (0.3-10 µM). *P < 0.05 indicates a significant effect versus

control group.

Figure 5: Crataegus treatment prevents replicative senescence-induced p21 and p16

overexpression in serially passaged porcine coronary endothelial cells. (A, C)

Representative immunoblots of p21 and p16. (B, D) Densitometric analysis of results. The

quantification of proteins was normalized to that of the housekeeping protein, GAPDH, for

equal loading. *P < 0.05 indicates a significant effect versus P1, and #P < 0.05 versus control

group.

Figure 6: eNOS expression in serially passaged endothelial cells treated or not with the

Crataegus extract and the effect of eNOS inhibition. (A) Representative immunoblots of

eNOS and densitometric analysis. The quantification of proteins was normalized to that of the

housekeeping protein, α-actin, for equal loading. *P < 0.05 indicates a significant effect

versus P1 and #P < 0.05 versus control group. (B) C12-fluorescein median fluorescence

intensity (MFI) of endothelial cells at P1 treated with 10 µg/ml of Crataegus, 1 mM of L-

NAME or both. *P < 0.05 indicates a significant effect versus P1, control and #P < 0.05

versus L-NAME. (C) C12-fluorescein MFI of endothelial cells at P3 treated with increasing

concentrations of the Crataegus extract in the presence of 1 mM of L-NAME.

85
Figure1

Treatement started after replication of primary coronary endothelial cells

P1 P2 P3 P4

Control

Crataegus
(10 µg/ml)

100
*
% of positive cells

80 *
Control
60 Crataegus (1 µg/ml)
40 Crataegus (10 µg/ml) *#
20

0
P1 P2 P3 P4

Figure 2

A B
C12FDG fluorescence (% of P1)

P1
Events (% of max)

30

P2 *
P3 *
20 *
P4
10

0
P1 P2 P3 P4

C12FDG fluorescence

C
Treatement started after replication of primary coronary endothelial cells
C12FDG fluorescence (% of control)

P1 P2 P3 P4
150 150 150 150

100 100 100 100

*
* *
50 50 50 *# 50 *

0 0 0 0

Control 1 10 Control 1 10 Control 1 10 Control 1 10

Crataegus (µg/ml) Crataegus (µg/ml) Crataegus (µg/ml) Crataegus (µg/ml)

86
Figure 3

A B
Treatment started at P2 treatment started at P3
C12FDG fluorescence (% of P2)

C12FDG fluorescence (% of P3)

150 150

100 100

* *
* * * * *
50 50 *

0 0
3 10 30 50 70
Control Control 3 10 30 50 70
Crataegus (µg/ml) Crataegus (µg/ml)

Figure 4

p53

GAPDH
B
250
Control 150
*
p53 protein level
(% of P1 control)

C12FDG fluorescence

200 Crataegus (10 µg/ml)

(% of P3 control)

150
* 100
*
* *
100 *
50
50

0 0
P1 P2 P3 P4 0,3 1 3 10
Control
Pifithrin (µM)

87
Figure 5

A B

p21 p16

GAPDH GAPDH

200 400
*

p16 protein level

*

(% of P1 control)
p21 protein level
(% of P1 control)

150 300
#
#
100 200
#

50 100

0 0
P1 P2 P3 P4 P1 P2 P3 P4

Control
Crataegus (10 µg/ml)

Figure 6

eNOS

α-actin
150
eNOS protein level
(% of P1 control)

Control
100
# Crataegus (10 µg/ml)

50 * #
*
0
P1 P2 P3 P4

B 250 C 150
Control
C12FDG fluorescence

C12FDG fluorescence

200 L-NAM E (1mM )

(% of control P1)

(% of P3 control)

*
100
150
#
100
50
50

0 0
ControlCrataegus L-NAME Crataegus 3 10 30 50 70
Control
(10 µg/ml) (1 mM) + L-NA
Crataegus (µg/ml)

88
Synthèse des résultats de l’article 4
Dans ce travail, nous avons montré que le passage successif de cultures primaires de

cellules endothéliales de coronaire de porc induit une sénescence réplicative au bout du 4ème

passage. La culture de cellule endothéliale au delà de ce passage n’était pas possible du fait

que les cellules s’arrêtent définitivement de se diviser. Le test cytochimique a révélé que la

plupart des cellules du passage P1 sont négatif pour l’activité de la β-galactosidase, tandis que

dans la plupart des cellules du passage P4 étaient positifs. La proportion de cellules positives

augmente progressivement de 3-6 % dans les cultures en P1 à 80-85 % dans les cellules

sénescentes réplicatives à P4. L'examen des cultures à différents passages par cytométrie de

flux indique une augmentation de l’activité de la β-galactosidase dans les cellules de passage

P2, P3 et P4 par rapport à P1. Le traitement avec l’extrait d’aubépine retarde l’apparition de la

sénescence réplicative de cellules endothéliale. En effet, le traitement avec l'extrait d’aubépine

(10 µg/ml) à partir de P1 jusqu’au P4 diminue significativement l'activité de la β-

galactosidase dans la culture en P2, P3 et P4. Le traitement avec l'extrait de Crataegus ou un

inhibiteur de p53 (protéine suppresseur de tumeur) de cellules endothéliales en P3 diminue

également l'activité de la β-galactosidase. le niveau d’expression de p53 et de p21(protéine

inhibitrice du cycle cellulaire) augmente avec les passages et le traitement avec le Crataegus

diminue la surexpression de p21 sans affecter l’expression de p53. Nous avons montré

également que le niveau d’expression de la eNOS diminue progressivement avec les passages

et que le traitement avec l’extrait d’aubépine augmente l’expression de la eNOS dans les

cellules sénescentes. L’inhibiteur de la eNOS accélère la sénescence des cellules endothéliales

en P1 et bloque l’effet bénéfique d’extrait d’aubépine sur des cellules en P3. En conclusion,

l’extrait d’aubépine retarde la sénescence réplicative des cellules endothéliales

vraisemblablement en diminuant l’activité de p53 et en augmentant et l’expression et

l’activité de la eNOS.

89
 4. DISCUSSION GENERALE ET PERSPECTIVES

Le vieillissement est défini comme un déclin graduel des fonctions biologiques. Le

vieillissement est associé à une augmentation de l’incidence des maladies cardiovasculaires

qui constitue la première cause de mortalité chez le sujet âgé. Selon l’organisation mondiale

de la santé, les pays occidentaux connaissent un vieillissement croissant de leur population et

le nombre de sujets âgés de plus de 60 ans passera de 605 millions en 2000 à environ deux

milliards en 2050. Les études épidémiologiques révèlent que le vieillissement, à lui seul et en

absence des autres facteurs de risque (e.g. diabète, hypertension, hyperhomocystéinémie,

hypercholestérolémie), augmente la morbidité et la mortalité cardiovasculaire. Le

vieillissement vasculaire est associé à une dysfonction endothéliale qui est considérée comme

un facteur de risque pour le développement des pathologies cardiovasculaires. Le traitement

ou la prévention de la dysfonction endothéliale liée au vieillissement serait une cible de

prévention des pathologies cardiovasculaires.

Ce travail de thèse a porté sur les effets protecteurs des substances antioxydantes

d’origine naturelle (polyphénols et thymoquinone) contre la dysfonction endothéliale liée au

vieillissement chez le rat. Nous avons montré que les polyphénols sont capables aussi bien de

prévenir l’installation d’une dysfonction endothéliale que de corriger une dysfonction

endothéliale établie mais aussi de retarder l’apparition d’une sénescence réplicative des

cellules endothéliale. La thymoquinone était aussi capable de corriger la dysfonction

endothéliale liée au vieillissement.

90
 Young rats (16 w) M ature-adult rats (25 w) M iddle-aged rats (46 w)

Old rats (65 w) Very old rats (96 w)

A NO-mediated relaxation B EDHF-mediated relaxation

Relaxation (% PE, 10-6 M)

Relaxation (% PE, 10-6 M)

0 0
* * * * * * *
* *
25 * 25
* * * * *
* *
50 * * 50
*
*
* *
75 75

-9 -8 -7 -6 -5 -9 -8 -7 -6 -5
Acetylcholine, Log [M] Acetylcholine, Log [M]

Figure 8 : Diminiution des composantes NO et EDHF de relaxation au cours du vieillissement

chez le rat Wistar. La composante NO de relaxation est caractérisée dans les anneaux d’artère
mésentérique précontracté à la phényléphrine (1 µM) en présence de l’indométacine (10 µM) et la
charybdotoxine (CTX, 100 nM) plus apamine (APA, 100 nM) pour bloquer la participation de
prostanoïdes et de l’EDHF respectivement (A, C). La composante EDHF de relaxations est enregistrée
en présence de indométacine (10 µM) et de Nω-nitro-L-arginine (L-NA, 300 µM) pour inhiber la
participation de prostanoïdes et du NO, respectivement (B, D). Les résultats sont représentés par la
moyenne ± SEM de 5 à 6 différentes expériences. *P < 0.05 indique une différence significative
versus le groupe contrôle.

Au cours de ces travaux de thèse et pour différents projets de recherches, nous avons

pu évaluer la fonction endothéliale de l’artère mésentérique chez le rat Wistar à différents âge.

(16, 25, 46, 65 et 96 semaines, Figure 8). Nos résultats permettent de caractériser la cinétique

d’induction de de la dysfonction endothéliale chez le rat Wistar. En effet, la composante NO

est légèrement diminuée au cours du vieillissement par rapport à la composante EDHF qui est

fortement diminuée. Pour la composante NO, la courbe de relaxation est déplacée vers la

droite d’un demi-log sans diminution de l’effet max chez les rats âgés de 46 semaines par

rapport aux rats âgés de 16 semaines. Une diminution de l’effet max de la courbe de

relaxation de NO est observée chez les rats très âgés où il passe de 96 % chez les rats jeunes à

82 % chez les rats âgés de 96 semaines. La composante EDHF de relaxation apparait très

91
sensible au processus physiologique du vieillissement. Déjà, à l’âge de 25 semaines où la

composante NO est peu modifiée, la courbe de relaxation de l’EDHF est déplacée d’un log et

l’effet max passe à 80 % par rapport à une relaxation maximum de 94 % chez les rats jeunes.

A 46 semaines, la courbe est déplacée d’un log et demi et le max de relaxation est de 38 %. A

partir de 65 semaines, la courbe de relaxation de l’EDHF est fortement diminuée avec un max

de 30 % à 65 semaines et de 19 % à 96 semaines. De plus, la composante EDCF de

contraction est absente chez les rats âgés de 46 semaines et elle apparait chez les rats âgés de

65 semaines et 96 semaines. Des expériences faites avec des rats Wistar issue d’une

production locale au niveau dans notre laboratoire ont montré une cinétique d’induction de

dysfonction endothéliale plus rapide et une diminution plus importante de la composante NO

de relaxation (Dal-Ros et al. 2011; Dal-Ros et al. 2012). Divers articles ont rapporté

l’hétérogénéité de l’impact du vieillissement sur les composantes de relaxation en fonction de

l’âge, du lit vasculaire, de l’espèce (Koga et al. 1988; Matz et al. 2000b) et du sexe

vraisemblablement à cause de l’effet des œstrogènes (Jensen-Urstad & Johansson 2001).

Cette hétérogénéité peut être due également aux conditions d’élevage vu les différences entre

les résultats obtenus dans ce travail de thèse utilisant des rat Wistar du commerce (Janvier) et

ceux obtenu avec des rats Wistar issus d’un élevage local.

L’importance des composantes NO et EDHF dans les relaxations dépendantes de

l’endothélium diffère en fonction du lit vasculaire. En effet, la composante NO est majoritaire

dans la régulation du tonus vasculaire des artères de conductance et diminue dans les artères

de résistance. Alors que la composante EDHF est absente dans l’aorte et augmente dans les

artères de résistances où elle est majoritaire (Shimokawa et al. 1996). Cette composante

EDHF de relaxation diminue fortement avec l’âge et peut contribuer au développement de

pathologies cardiovasculaires liées au vieillissement. De plus, la composante EDHF est très

92
affecté dans l’hypertension artérielle (Hilgers & Webb 2007; Dal-Ros et al. 2009). La

composante EDHF est fortement diminuée dans les artères de résistance du pénis chez le sujet

diabétique et serait responsable de l’échec du traitement des sujets diabétiques présentant une

dysfonction érectile avec le sildénafil (inhibiteur de PDE5 qui augmente la disponibilité du

NO) (Angulo et al. 2003). Il est possible qu’au cours du vieillissement, la diminution de la

composante EDHF de relaxation dans les artères de résistance pouvait entrainer une

augmentation du tonus des vaisseaux de résistance et ainsi favoriser l’hypertension artérielle.

Nos résultats indiquent que le traitement curatif des rats âgés de 46 semaines avec les

polyphénols du vin pendant 4 semaines améliore les composantes NO et EDHF de relaxation.

Alors que le même traitement n’a pas d’effet chez les rats âgés de 96 semaines (Figure 9. A-

B). Des résultats similaires ont été rapportés par le groupe de Thorin qui montre que le

traitement de souris C57Bl/6 âgées de 9 mois avec la catéchine (30 mg/kg/j) pendant 3 mois

améliore la fonction endothéliale (Gendron & Thorin 2007), alors que le traitement de souris

hApoB(+/+) âgées de 9 mois avec une athérosclérose établie aggrave la dysfonction

endothéliale (Gendron et al. 2010). De plus, l’aspirine à faible dose (3-4 μg/Kg/j pendant 8

semaines) corrige la dysfonction endothéliale et le stress oxydant liés au vieillissement chez

les souris C57B/J6 âgées de 60 semaines, et est sans effets chez les souris de 96 semaines

(Bulckaen et al. 2008). De même, les polyphénols du thé (epigallocatechin gallate) induisent

des contractions de l’aorte de rats Wistar-Kyoto âgés qui sont exacerbées chez le rat âgé

spontanément hypertendu (Li et al. 2011), alors que ces mêmes polyphénols induisent des

relaxations de l’aorte de rats Wistar-Kyoto jeunes (Alvarez et al. 2006). L’ensemble de ces

données suggère que le traitement curatif de la dysfonction endothéliale liée au vieillissement

est efficace chez les animaux adultes matures vers la moitié de leur espérance de vie où la

93
dysfonction endothéliale est a priori réversible. Par contre, la dysfonction endothéliale semble

être difficilement réversible chez les animaux très âgés vers la fin de leur espérance de vie.

A NO-mediated relaxation B EDHF-mediated relaxation

Relaxation (% PE, 10 M)
0 0
Relaxation (% PE, 10 M)

-6
-6

25 25

50 50

75 75
Control Control
100 100
RWPs RWPs

-9 -8 -7 -6 -5 -9 -8 -7 -6 -5
Acetylcholine, Log [M] Acetylcholine, Log [M]

C NO-mediated relaxation D EDHF-mediated relaxation

0
Relaxation (% PE, 10 M)

Relaxation (% PE, 10 M)

0
-6

-6

25 25
* *
* *
50 * 50
* 75
75
Control Control
100 100
losartan losartan

-9 -8 -7 -6 -5 -9 -8 -7 -6 -5
Acetylcholine, Log [M] Acetylcholine, Log [M]

Figure 9 : Le traitement avec du losartan améliore la dysfonction endothéliale dans l’artère

mésentérique de rats très âgés contrairement aux polyphénols du vin. Des rats âgés de 96
semaines sont traités pendant 4 semaines avec un extrait de polyphénols du vin rouge (red wine
polyphenols, RWPs) à raison de 100 mg/kg/j ou par l’apocynine (inhibiteur de NADPH oxydase) à
raison de 10 mg/kg/j ou par le véhicule. La composante NO de relaxation est caractérisée dans les
anneaux d’artère mésentérique précontracté à la phényléphrine (1 µM) en présence de l’indométacine
(10 µM) et la charybdotoxine (CTX, 100 nM) plus apamine (APA, 100 nM) pour bloquer la
participation de prostanoïdes et de l’EDHF respectivement (A, C). La composante EDHF de
relaxations est enregistrée en présence de indométacine (10 µM) et de Nω-nitro-L-arginine (L-NA, 300
µM) pour inhiber la participation de prostanoïdes et du NO, respectivement (B, D). Les résultats sont
représentés par la moyenne ± SEM de 5 à 6 différentes expériences. *P < 0.05 indique une différence
significative versus le groupe contrôle.

Dans notre troisième étude portant sur les effets protecteurs d’aubépines, on a vu que

le traitement préventif avec l’extrait d’aubépine n’améliore pas les composantes NO et EDHF

de relaxation mais diminue les réponses contractiles dépendantes de l’endothélium. De même,

94
l’effet préventif d’un traitement de souris âgées de 12 mois avec de la catéchine est observé

quand le traitement est instauré à l’âge de 9 mois alors que si le traitement est commencé à

l’âge de 3 mois jusqu’au 12 mois, aucun effet bénéfique sur la relaxation n’est observé

(Gendron et al. 2012). Les auteurs suggèrent que ceci est expliqué par le fait qu’une

exposition précoce des cellules endothéliales, durant leur phase de maturation, à de faibles

concentrations de radicaux libres, a des effets bénéfiques et qui est en accord avec le principe

hormesis selon lequel une exposition à un faible stress oxydant protège contre un stress

oxydant plus fort qui se produit plus tard (Thorin-Trescases et al. 2010). Par contre, un

traitement préventif des rats avec les polyphénols du vin de l’âge de 16 semaines jusqu’à 40

semaines protège contre l’altération des composantes NO et EDHF de relaxation (Dal-Ros et

al. 2011). Néanmoins, la comparaison entre les différentes études n’est pas aisée du fait de la

différence entre les durées du traitement, les doses d’extrait de polyphénols administrées et la

différence entre le pouvoir antioxidant des polyphénols. Des études complémentaires doivent

être faites pour caractériser les paramètres influençant l’effet préventif des polyphénols à

savoir, l’âge de l’animal, la durée de traitement ainsi que la dose.

Le traitement de rats âgés de 51 semaines avec les polyphénols du vin pendant 4

semaines améliore la dysfonction endothéliale (Dal-Ros et al. 2012). Dans notre travail, nous

avons établie la cinétique d’apparition de cet effet protecteur. On a vu que l’effet protecteur

est déjà significatif après une semaine traitement et qui atteint l’effet maximum après deux

semaines de traitement. Le traitement de rats âgés avec les poyphénols du vin pendant trois ou

quatre semaines n’a pas d’effet bénéfique supplémentaire sur la fonction endothéliale. Dans

une étude préliminaire on vu que le traitement de rats âgé pendant 5 jours avec les

polyphénols du vin a tendance à améliorer la dysfonction endothéliale et que le traitement

pendant 3 jours était sans effet (données non publiées).

95
 Nous avons également évalué la cinétique de disparition de l’effet protecteur des

polyphénols. En effet, l’originalité principale de notre première étude porte sur l’effet soutenu

dans le temps d’un traitement avec les polyphénols. A notre savoir, c’est le premier travail qui

montre qu’un traitement in vivo des animaux avec un extrait polyphénolique entrainent des

effets bénéfiques qui sont observés même après deux semaines d’arrêt de traitements. Cette

observation soulève des questions concernant la biodisponibilité des polyphénols et leur

accumulation dans les tissus. Plusieurs travaux ont montré une accumulation des polyphénols

dans le plasma et les tissus après ingestion des produits riches en polyphénols chez le rat et le

porc (de Boer et al. 2005; Bieger et al. 2008; Ishisaka et al. 2011). Il est bien connu qu’un

traitement, avec les polyphénols, présente des effets bénéfiques contre l’athérosclérose chez

l’homme (Chiva-Blanch et al. 2012) et dans plusieurs modèles animaux pathologiques

(Frederiksen et al. 2007; Katsuda et al. 2009; Loke et al. 2010). Des scientifiques japonais on

réussi à synthétiser des anticorps spécifiques contre les la quercetin-3-O-β-D-glucoronide et

l’epicatechin-gallate (Kawai 2011) et ils ont montré que ces polyphénols s’accumulent

spécifiquement dans les macrophages des artères sclérotiques (Kawai et al. 2008; Kawai

2011). D’autres études ont montré une interaction chimique covalente entre les polyphénols et

les protéines ce qui peut expliquer l’accumulation des polyphénols et leur effet soutenu dans

le temps. En effet, les polyphénols du thé, (-)-epigallocatechin-3-gallate, interagissent avec les

groupements thiol des résidus cystéine de la GAPDH par auto-oxydation entrainant son

inhibition (Ishii et al. 2008). La quercétine interagit également avec les groupements

sulfhydryles de la protéine alpha-actine (Ishii et al. 2009). Ces données suggèrent que le

traitement de rats par les polyphénols du vin entraine une accumulation des polyphénols dans

les tissus où ils interagissent d’une manière covalente et exercent leurs effets bénéfiques

durant au moins deux semaines après l’arrêt du traitement. Néanmoins, on a vu que le

96
traitement des rats âgés modifie l’expression des protéines cibles. Le traitement de deux

semaines avec les polyphénols du vin augmente l’expression de la eNOS et des canaux

potassiques dépendant de calcium responsables des composantes NO et EDHF de relaxation

et diminue l’expression de la NADPH oxydase responsable en partie du stress oxydant. Ces

effets sur l’expression des protéines cibles sont maintenus même après deux semaines d’arrêt

de traitement suggérant que l’effet rémanent des polyphénols serait dû à une modification

d’expression de protéines qui est maintenue dans le temps.

Nous avons montré que la dysfonction endothéliale liée au vieillissement est associée

à un stress oxydant dû à une formation accrue des espèces réactives d’oxygène par différentes

sources enzymatiques incluant essentiellement la NADPH oxydase, la eNOS découplée, la

cyclooxygénase et la chaine respiratoire mitochondriale. Ceci en accord avec les données de

la littérature montrant l’implication de la NADPH oxydase (Hamilton et al. 2001), la chaine

respiratoire mitochondriale (van der Loo et al. 2000), la xanthine oxydase (Aranda et al.

2007) et la NO synthase découplée (Cosentino et al. 2001) et la cyclooxygenase-2 (Armstead

2003). Dans une étude récente, les auteurs montrent clairement que le stress oxydant est à

l’origine de la dysfonction endothéliale liée au vieillissement au niveau de l’aorte de souris,

vu qu’elle était corrigée par 3 semaines de traitement avec un antioxydant comme le tempol

(Fleenor et al. 2012). Les polyphénols sont connus pour leurs effets antioxydants. Il a été déjà

montré que les polyphénols du vin rouge ainsi que ceux du thé retardent le développement de

l’athérosclérose dans l’aorte de souris apoE -/- via leur effet antioxydant (Hayek et al. 1997;

Miura et al. 2001). Les polyphénols du vin rouge empêchent le développement de

l’athérosclérose et la dysfonction endothéliale dans le modèle du lapin

hypercholestérolémique (Wang et al. 2005). Le traitement avec la catéchine de souris

empêche la dysfonction endothéliale liée au vieillissement par diminution de la libération de

97
thromboxane A2 et la diminution du stress oxydant vasculaire (Gendron & Thorin 2007). De

plus, le resvératrol améliore l’état de santé et l’espérance de vie de souris âgées (Baur et al.

2006). Le traitement avec les polyphénols d’artichaut sauvage corrige la dysfonction

endothéliale chez les rats âgés (Rossoni et al. 2005).

La dysfonction endothéliale et le stress oxydant liés au vieillissement peut être aussi

corrigé par des inhibiteurs des sources enzymatiques de ROS telles que de la NADPH

oxydase (Trott et al. 2011; Dal-Ros et al. 2012), les cyclooxygénases (Bulckaen et al. 2008),

par un donneur exogène de BH4 responsable du recouplage de la eNOS (Sindler et al. 2011) et

par l’inhibition de NF-κB (Pierce et al. 2009). Ces résultats suggèrent que les effets

protecteurs des polyphénols ne sont pas du à leur effet antioxydant direct permettant de

neutraliser les espèces réactive de l’oxygène mais plutôt à leur effet sur les enzymes pro-

oxydantes et/ou antioxydantes. En effet, les polyphénols du thé diminuent l’expression de la

NADPH oxydase augmentent l’expression de la catalase (Ying et al. 2003), de la superoxyde

dismutase et de la glutathion peroxydase (Bruckner et al. 2012). Les polyphénols du vin rouge

diminuent également l’activité de la NADPH oxydase (Laurent et al. 2012). D’autant plus que

l’on voit une diminution de l’expression des sous-unités de la NADPH oxydase après le

traitement de rats âgés avec les polyphénols du vin rouge. Par contre, d’autres études mettent

en avance l’effet antioxydant direct des polyphénols dans les effets protecteurs contre la

dysfonction endothéliale liée au vieillissement. En effet, une étude clinique a montré qu’un

traitement aigu avec un mélange d’antioxydants (vitamine C + vitamine E + acide lipoique

alpha) corrige la diminution des relaxations dépendante de l’endothélium chez les sujets âgés

(Wray et al. 2012). De plus, un traitement aigu avec le résveratrol d’aortes de rats âgés

diminue le stress oxydant et améliore la formation du NO (Rajapakse et al. 2011).

98
 Nous avons montré que l’effet protecteur des polyphénols est associé à une

diminution d’expression de l’angiotensine II et son récepteur AT1. Ces résultats sont en

accord avec les données de la littérature indiquant que le traitement de rats âgés avec un

inhibiteur de l’enzyme de conversion de l’angiotensine II (énalapril à raison de 20 mg/kg/j,

Goto et al. 2000) ou d’un antagoniste des récepteurs AT1 (candesartan 3,5 mg/kg/j, Kansui et

al. 2002) améliore la composante EDHF de relaxation de l’artère mésentérique altérée par le

vieillissement. Une autre étude a montré que le traitement avec inhibiteur de l’enzyme de

conversion ou un antagoniste des récepteurs AT1 restore les relaxations dépendante de

l’endothélium au niveau de l’aorte et ce en supprimant les réponses contractiles dépendantes

de l’endothélium et induites par les dérives prostanoïdes et que cet effet est associé à une

diminution du stress oxydant vasculaire (Mukai et al. 2002). Cette étude suggère que le stress

oxydant vasculaire est dû, au moins, en partie à l’activation du système angiotensine II. De

plus les souris transgéniques n’exprimant pas les récepteurs AT1 ne présentent pas de

dysfonction endothéliale liée au vieillissement (Modrick et al. 2009). Des études ont montré

que les polyphénols peuvent agir comme inhibiteurs de l’enzyme de conversion de

l’angiotensine (Actis-Goretta et al. 2003; Persson et al. 2009; Dong et al. 2011) ce qui nous

permet de suggérer que l’effet bénéfique du traitement avec les polyphénols est dû, en partie,

à une inhibition directe de l’enzyme de conversion de l’angiotensine. De plus, la

thymoquinone diminue aussi l’activation du système angiotensine locale. Il est bien connu

que le stress oxydant active le système angiotensine locale (Wong et al. 2010; Yu et al. 2010)

et que l’angiotensine II induit une dysfonction endothéliale (Rajagopalan & Harrison 1996;

Sarr et al. 2006; Dal-Ros et al. 2009). Il est plus vraisemblable que les polyphénols et la

thymoquinone diminuent l’activation du système angiotensine locale via leur effet

antioxydant.

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Le stress oxydant semble jouer un rôle important dans le vieillissement cellulaire et la

sénescence des cellules endothéliales. En effet, le traitement de cellules endothéliale en

culture avec de l’angiotensine II augmente le stress oxydant qui accélère la sénescence

probablement via l’activation de la NADPH oxydase (Imanishi et al. 2005). A priori, la

sénescence endothéliale joue un rôle important dans le développement de la dysfonction

endothéliale liée au vieillissement. En effet, la surexpression de la p53, protéine suppresseur

de tumeurs et fortement impliquée dans la sénescence endothéliale, est associée à une

diminution des relaxations dépendantes de l’endothélium (Kumar et al. 2011). La p66Shc qui

est une protéine adaptatrice qui contrôle la réponse cellulaire au stress oxydant a été

impliquée dans la dysfonction endothéliale observé dans le diabète (Camici et al. 2007) et du

vieillissement (Francia et al. 2004; Camici et al. 2008) mais aussi dans la sénescence

(Afanas'ev 2010). Au niveau vasculaire, la dysfonction endothéliale est due à une diminution

de la formation du NO (Tschudi et al. 1996). De même, au niveau cellulaire, la sénescence

réplicative endothéliale est associée à une diminution de formation du NO (Yang et al.

2009a). Le traitement de cellules endothéliales avec un donneur de NO retarde le

développement de la sénescence réplicative (Vasa et al. 2000). On peut suggérer que la

sénescence réplicative serait en partie responsable de la dysfonction endothéliale liée au

vieillissement et que la prévention de la sénescence réplicative endothéliale améliorait la

dysfonction endothéliale liée au vieillissement. Dans la quatrième étude nous avons montré

que les polyphénols d’aubépine retardent le développement de la sénescence endothéliale

vraisemblablement via une augmentation de l’expression et de l’activité de la eNOS. Ces

résultats peuvent suggérer que l’effet bénéfique que l’on obtient avec le traitement de rats

âgés avec les polyphénols est dû en partie à une diminution de la sénescence endothéliale in

vivo. Des expériences préliminaires nous ont permis de voir une augmentation de l’expression

100
de p53 dans l’endothélium d’aorte de rats âgés. Des expériences sont au cours pour

caractériser l’effet protecteur des polyphénols contre la sénescence endothéliale in vivo.

En conclusion, ces études ont permis de mettre en évidence des effets protecteurs des

polyphénols et de la thymoquinone contre la dysfonction endothéliale liée au vieillissement.

Cette amélioration de la fonction endothéliale est caractérisée ex vivo par une augmentation

des composantes NO et EDHF de relaxation, une diminution du stress oxydant vasculaire et

une amélioration de l’expression des protéines cibles comme celles impliquées dans le

système angiotensine. Cependant un grand nombre d’interrogations subsiste sur l’impact

d’une telle amélioration de la fonction endothéliale sur l’état de santé général chez l’animal et

chez l’homme. Une amélioration de la fonction endothéliale devrait améliorer l’irrigation

sanguine des différents organes permettant ainsi d’améliorer leur fonctionnement. Il a été déjà

montré qu’un traitement chronique avec les polyphénols améliore la capacité physique à

l’exercice chez le rat âgé vraisembablement suite à une amélioration de fonction endothéliale

(Dal-Ros et al. 2011). Il serait intéressant, dans un avenir proche de caractériser l’impact de

l’amélioration de la fonction endothéliale par un traitement avec les polyphénols sur la

fonction cardiaque, l’apprentissage et la fonction cognitive et sur la fonction érectile.

101
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