Professor M D Delmas

Professor M.D. STEPHAN Isabelle Lartaud

Professor C Marchand

Dr Malak Abbas

Dr Thaise Proto

Khorsheed Sherzad

Waseem Rasheed

Ikram Ashraf

Ullah

Chabert Philippe

Antonio José

Léon Gonzaléz

Sonia Khemais

Said Amissi

Phuong Nguyen

Nga

Alain N'guessan Yao

Zahid Rasul

Iveta Najmanová

Amissi Said

Henri Die-Kacou

Philippe Chabert

Cyril Auger

Valérieschini-Kerth
email: valerie.schini-kerth@unistra.fr

PhD. Valérie B Schini-Kerth

Analysis of Variance ARAs Angiotensin Receptor Antagonists ARBs Angiotensin Receptor Blockers AT1R Angiotensin II Type 1 Receptor AT2R Angiotensin II Type 2 Receptor ATP Adenosine Tri-Phosphate BH4 Tetrahydrobiopterin BP Blood Pressure BW Bodyweight CAD Coronary Artery Disease cAMP Cyclic Adenosine-3',5'-Mono-Phosphate CBDL Common Bile Duct Ligation cGMP Cyclic Guanosine 3'-5' Monophosphate CHD Coronary Heart Disease CHF Congestive Heart Failure COX Cyclooxygenases CRP C-reactive protein CVD Cardiovascular Disease Cx37 Connexin 37 CYP Cytochrome DHA Docosahexaenoic Acid DM Diabetes Mellitus DPA Docosapentaenoic Acid EC Endothelial Cell EDCF Endothelium-Derived Contracting Factor EDH Endothelium-Dependent Hyperpolarization EDHF Endothelium-Derived Hyperpolarizing Factor EDRF Endothelium-Derived Relaxing Factor EETs Epoxyeicosatrienoic Acids EFA Essential Fatty Acids eNOS Endothelial Nitric Oxide

Keywords: Phyllanthusamarus, DOCA-salt, hypertension, endothelial dysfunction

indomethacin and N ω -nitro-L-arginine) relaxation to acetylcholine were significantly reduced in the DOCA-salt group compared to the control group. Fluorescence study showed that the endothelial dysfunction was associated with a reduced expression level of Cx37, an increased expression of eNOS and the formation of ROS in main mesenteric artery. The antihypertensive effect of AEPA was related to an improvement of the blunted NO-and EDH-mediated relaxation, and an increased vascular oxidative stress and modulation of the expression levels of target proteins in DOCA-salt rats.

Conclusion:

Altogether, our study shows that AEPA is able to act as an antihypertensive agent, and to prevent endothelial dysfunction in DOCA-salt hypertensive rats, in part, by preventing vascular oxidative stress.

List of publications 1. Publications:

Rasul Zahid Niazi, Silva G.C, Porto Ribeiro T, Zgheel F, Auger C, Schini-Kerth V. Choronic oral Intake of the omega 3 formulations EPA:DHA 6:1 prevents the angiotensin II-induced hypertension and endothelial dysfunction in rats (submitted to the British Journal of Pharmacology).

Mohamad Kassem, Zahid Rasul Niazi , Malak Abbas , Ali El Habhab, Guillaume Kreutter , Sonia Khemais-Benkhiat, Cyril Auger, Maria-Cristina Antal, Valerie B. Schini-Kerth, Florence Toti, Laurence Kessler.The Endocrine Pancreas, an Early Sensor of Senescence in Middle-aged Rats with Still Normal Vascular Function. (Under revision)

Alain N'guessan Yao, Zahid Rasul Niazi, Iveta Najmanová, Mamadou Kamagaté, Amissi Said, Henri Die-Kacou, Cyril Auger, Philippe Chabert, Valérie Schini-Kerth. Chronic treatment with an aqueous extract of Phyllanthus amarus prevents hypertension and endothelial dysfunction in Docasalt rats. (paper in preparation) Amissi Said, Zahid Rasul Niazi, Mélanie Burban, Romain Kessler, Mathieu Canuet, Florence Toti, Laurent Monassier, Nelly Boehm, Cyril Auger, Ferhat Meziani and Valérie B. Schini-Kerth. The omega-3 EPA:DHA 6:1 superior formulation prevents the monocrotaline-induced pulmonary hypertension, endothelial dysfunction and vascular remodeling, and right ventricular failure in rats. (paper in preparation)

Poster Communications:

1) Niazi Z, Silva G.C, Porto Ribeiro T, Zgheel F, Auger C, Schini-Kerth V. Choronic oral Intake of the omega 3 formulation EPA:DHA 6:1 prevents the angiotensin II-induced hypertension and endothelial dysfunction in rats, JCI (Campus Illkirch day) 13-14 April Illkirch France 2015.

2) Niazi Z, Silva G.C, Porto Ribeiro T, Zgheel F, Auger C, Schini-Kerth V. Choronic oral Intake of the omega 3 formulation EPA:DHA 6:1 prevents the angiotensin II-induced hypertension and endothelial dysfunction in rats, CPBI (Congrès de la société de physiologie et de biologie intégrative) 4-6 May Strasbourg-France 2015.

3) Niazi Z, Silva G.C, Porto Ribeiro T, Zgheel F, Auger C, Schini-Kerth V. Choronic oral Intake of the omega 3 formulation EPA:DHA 6:1 prevents the angiotensin II-induced hypertension and endothelial dysfunction in rats, FMTS (Fédération de médecine translationnelle de Strasbourg) 16-17 April, Strasbourg-France 2015. 4) Kassem M, Niazi Z, Abbas M, Samama B, Auger C, Constantinescu A, Vauchelles R, Lessinger JM, BOEHM N, Schini-kerth V, Toti F, Kessler L. Morphological and functional abnormalities of the endocrine pancreas in middle age rat: Impact for transplantation, CPBI (Congrès de la Société de Physiologie et de Biologie Intégrative) 4-6 may, Strasbourg-France. La résumé est publié au journal Acta Physiologica Volume 214, Issue Supplement S700, Pages 1-92, May 2015. Table 1. Structure and taxonomy of fatty acids (Simopoulos 1998) 2. Structure of different unbranched fatty acids with a methyl end and a carboxyl (acidic) end.--

List of Figures and Tables

Abstract

Cardiovascular diseases are the leading cause of death worldwide, both in developed and developing countries. The incidence of cardiovascular diseases is linked to several risk factors that are either non-modifiable (age, gender, genetic background) or modifiable. The modifiable risk factors include lifestyle (western diet, tobacco use, alcohol abuse, physical inactivity) and treatable diseases such as hypertension, dyslipidemia, obesity, and diabetes. Many preclinical and clinical studies have shown that development of cardiovascular diseases and risk factors are associated early with an endothelial dysfunction.

The endothelium, the monocellular layer lining all blood vessels, represents the largest cell compartment in contact with blood flow. Endothelial cells contribute to the vascular tone and constitute a protective surface with anti-thrombotic properties, mainly through the release of potent vasoprotective factors such as nitric oxide (NO). In cardiovascular diseases and also during physiological ageing, the endothelial dysfunction is characterized by a decreased formation of vasoprotective factors and an increased formation of vasoconstricting factors, resulting in an imbalance leading towards the accelerated development of vascular pathologies. Endothelial dysfunction is involved in atherosclerotic lesion formation by the promotion of both the early and late mechanisms of atherosclerosis including up-regulation of adhesion molecules, increased chemokine secretion and leukocyte adherence, increased cell permeability, enhanced low-density lipoprotein oxidation, platelet activation, cytokine elaboration, and vascular smooth muscle cell proliferation and migration.

In addition, several studies have demonstrated that endothelial dysfunction and cardiovascular diseases development are also associated with an increased vascular oxidative stress and an upregulation of the local angiotensin system. Angiotensin II (Ang II) contributes to the pathophysiology of atherosclerosis and vascular diseases not only via its role in hypertension but also via its direct effects on vascular cell growth and migration. Ang II also contributes to the development of cardiovascular diseases through the induction of oxidative stress by up-regulating NADPH oxidase, the main producer of reactive oxygen species (ROS) in the vascular wall.

Diets could play a role in the development of cardiovascular diseases. Indeed, while western diet rich in saturated fatty acids has been associated with an increased risk of cardiovascular diseases, other diets such as the Mediterranean diet rich in unsaturated fatty acids have been associated with a reduced incidence of cardiovascular diseases up to 30 %. Several epidemiological studies and clinical trials have shown that dietary intake of fish, fish oil or omega-3 polyunsaturated fatty acids (n-3 PUFAs) have beneficial effects against coronary heart disease, stroke, and hypertension.

Moreover, the dietary consumption of the major n-3 PUFAs, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has been related to a reduced risk of cardiovascular disease morbidity/mortality. The exact mechanism by which n-3 PUFAs inhibit atherosclerosis is still unclear, but it may relate to the modulation of lipid metabolism, decrease in pro-inflammatory cytokine production, and inhibition of inflammatory processes.

It has been demonstrated in endothelial cells that EPA stimulates the endothelial nitric oxide synthase (eNOS) activation by inducing its detachment from the inhibitory supportive protein caveolin, while DHA stimulates eNOS activity by increasing the interaction between eNOS and heat shock protein 90 (HSP-90) which activates PKB/Akt pathway resulting in eNOS phosphorylation and activation. In conditions like hypertension or renal failure, n-3 PUFA could reduce the increased level of asymmetric circulating dimethylarginine (ADMA, an endogenous inhibitor of eNOS) resulting in an increase of eNOS activity.

Moreover, our research team recently demonstrated that the stimulation of the endothelial function by n-3 PUFAs is dependent on both the purity and the ratio of EPA and DHA. Indeed, an optimized EPA:DHA 6:1 formulation is a potent stimulator of the endothelial formation of NO, and to a lesser extent, of an increased endothelium-dependent hyperpolarisation (EDH) response. The induction of the endothelial formation of NO by omega-3 fatty acids is mediated by redox-sensitive activation of the Src/PI3-kinase/Akt and MAPKs pathways leading to eNOS activation, which is dependent on the ratio and amount of the EPA:DHA in the formulation.

In humans, a direct vasodilatory effect has been demonstrated following intake of DHA, which inhibits the vasoconstrictor response produced by angiotensin and norepinephrine. A large body of studies demonstrated that n-3 PUFA are able to reduce systemic blood pressure and a recent meta-analysis confirmed that a consumption of more than 2 g/d of EPA + DHA can reduce systolic and diastolic blood pressure in humans.

The aim of the present study was to determine whether chronic oral intake of the optimized EPA:DHA 6:1 formulation is able to prevent the hypertension and endothelial dysfunction induced by Ang II in rats.

Male Wistar rats received by daily gavage 500 mg/kg BW of either corn oil (control) or the EPA:DHA 6:1 formulation. After one week, the rats underwent either sham surgery or implantation of an osmotic mini-pump infusing 0.4 mg/kg/day of angiotensin II. Systolic blood pressure was measured twice weekly using the tail cuff sphingomanometry method. After 4 weeks of gavage, the animals were euthanized and the organ was collected. The secondary branch of mesenteric artery were used for vascular reactivity studies using a wire myograph, for immunofluorescence and fluorescence histochemistry studies on frozen section, and for western blot analysis of protein expression.

The major results of our study show that infusion of Ang II (0.4 mg/kg/day) caused a significant increased in systolic blood pressure, which was reaching 194±5.9 mmHg compared to 120±5.6 mmHg in control rats. Oral intake of EPA:DHA 6:1 (500 mg/kg/day) significantly prevented the Ang II-induced hypertension (147±5.9 mmHg), while having no effect on basal systolic blood pressure (110±3.9 mmHg).

The chronic intake of the optimized EPA:DHA 6:1 formulation is associated with significantly increased plasmatic presence in omega-3 fatty acids, mainly as EPA, DHA and the intermediate elongated metabolite of EPA, the docosapentaenoic acid (DPA), resulting in a decreased omega-6/omega-3 ratio. The reduction of this ratio have been associated with a shift towards beneficial health effects of omega-3, including reduced cardiovascular and cancer risk, whereas increased ratios such as the Western diet has been associated with increased prevalence of cardiovascular and chronic diseases Vascular reactivity studies in the secondary branch of mesenteric artery indicate that Ang II induced an endothelial dysfunction characterized by reduced relaxations in response to acetylcholine affecting both the NO-and EDH-mediated component, and increased formation of endothelium-derived contractile factors (EDCFs) in response to acetylcholine. The chronic intake of EPA:DHA 6:1 normalized both the NO, EDH and EDCF responses in secondary branch of mesenteric artery [START_REF] Shin | Remnant Lipoprotein Particles Induce Apoptosis in Endothelial Cells by NAD (P) H Oxidase-Mediated Production of Superoxide and Cytokines via Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Activation Prevention by Cilostazol[END_REF].

To better characterize the molecular mechanisms involved in the protective effects of EPA:DHA 6:1 intake, we performed quantitative analysis of protein expression in the secondary branch of the mesenteric artery by immunofluorescence.

Firstly, we studied the expression of eNOS, arginase-1, SKCa and Cx37 (EDH component of relaxation), and cyclooxygenases (COXs, involved in EDCFs). Compared to the controls, Ang II significantly up-regulate the expression of eNOS, arginase 1, COX-1 and COX-2, the inducible isoform of COXs, while down-regulating the expression of SKCa and Cx37. The intake of EPA:DHA 6:1 also normalized the expression levels of eNOS, arginase 1, SKCa, Cx37, COX-1 and COX-2.

As endothelial dysfunction is associated with a vascular oxidative stress, we measured the level of oxidative stress in the vascular wall of the secondary branch mesenteric artery using the redoxsensitive fluorescent probe dihydroethidium (DHE). Ang II induced a significant increases of DHE fluorescence throughout the vascular wall as compared to control rats, which was significantly prevented by the EPA:DHA 6:1 intake. As the increased vascular oxidative stress in Ang IIinduced hypertension has been attributed, at least in part, to the up-regulation of NADPH oxidase expression through the activation of the Ang II type 1 receptor (AT1R), we then determined that expression levels of both AT1R and NADPH oxidase sub-units p22 phox and p47 phox . Compared to control rats, the secondary branch of the mesenteric artery of rats infused with Ang II exhibit a significantly increased expression level of AT1R, p22 phox and p47 phox . The EPA:DHA 6:1 treatment significantly improves the Ang II-induced vascular oxidative stress, up-regulation of AT1R and NADPH oxidase.

To confirm the results obtained by immunofluorescence in the secondary branch of the mesenteric artery, we performed Western blot analysis of the expression levels of eNOS, COX-2, and the NADPH oxidase subunit p22 phox in the main mesenteric artery. The Ang II group presented a significantly increased expression of eNOS, COX-2, and the NADPH oxidase subunit p22 phox , that was prevented by the chronic oral intake of EPA:DHA 6:1.

Altogether, the present findings indicate that chronic intake of the optimized EPA:DHA 6:1 formulation prevented the development of hypertension and endothelial dysfunction induced by the infusion of Ang II in rats. The Ang II-induced endothelial dysfunction is associated to an upregulation of the local angiotensin system and an increased vascular oxidative stress. The beneficial effect of EPA:DHA 6:1 is mediated by an improvement of both the NO-and the EDHmediated relaxations and a reduction of endothelium-dependent contractile response, most likely by preventing the oxidative stress induced by the up-regulation of the local angiotensin system.

Résumé

Les maladies cardiovasculaires représentent la première cause de mortalité dans le monde, que cela soit dans les pays développés ou ceux en cours de développement. L'incidence des maladies cardiovasculaires est associée à de nombreux facteurs de risques pouvant être non-modifiables (âge, sexe, patrimoine génétique …) ou modifiables. Parmi ces derniers, on trouve des facteurs liés au style de vie (régime occidental, tabagisme, alcoolisme, sédentarité) ou des pathologies pouvant être traitées comme l'hypertension, les dyslipidémies, l'obésité ou les diabètes. De plus, de nombreuses études précliniques et cliniques ont montré que les maladies cardiovasculaires sont précocement associées à une dysfonction endothéliale.

L'endothélium, la monocouche cellulaire tapissant l'intérieur des vaisseaux sanguins, est le plus grand organe en contact direct avec le flux sanguin. Les cellules endothéliales ont un rôle clé dans le maintien du tonus vasculaire et constituent une couche protective exerçant des effets antithrombotiques, principalement grâce à la formation et libération de puissants facteurs vasoprotecteurs tels que le monoxyde d'azote (NO). Dans les maladies cardiovasculaires ou au cours du vieillissement physiologique, apparait une dysfonction endothéliale caractérisée par une diminution de la formation des facteurs protecteurs et une augmentation de la formation des facteurs vasoconstricteurs, le tout engendrant un déséquilibre menant au développement accéléré des pathologies vasculaires. La dysfonction endothéliale est impliquée dans la formation de lésions athéromateuses en favorisant les mécanismes précoces et tardifs du développement de l'athérosclérose dont l'augmentation de l'expression des molécules d'adhésion, de la sécrétion de chimiokines, de l'adhésion des leucocytes, de l'oxydation des LDL, de l'activation plaquettaires, PUFAs réduit la pression artérielle systolique chez l'homme, et une récente méta-analyses a confirmé que la consommation de EPA plus DHA supérieure à 2 g/j pouvait réduire les pressions artérielles systolique et diastolique.

L'objectif de la présente étude est de déterminer si la consommation chronique de la formulation optimisée EPA:DHA 6:1 est capable de prévenir l'hypertension et la dysfonction endothéliale induites par l'Ang II chez le rat.

Des rats Wistar males ont reçu quotidiennement par gavage 500 mg/kg soit d'huile de maïs (contrôle) soit de la formulation EPA:DHA 6:1. Après une semaine, les rats subissent soit une procédure simulée, soit l'implantation d'une mini-pompe osmotique infusant 0,4 mg/kg/j d'Ang II. La pression artérielle est mesurée deux fois par semaine pendant l'ensemble de la procédure expérimentale à l'aide de la méthode de sphyngomanométrie par brassard caudal. Après 4 semaines de gavage, les animaux sont euthanasiés et les organes sont prélevés. Les branches secondaires de l'artère mésentérique sont utilisées pour l'étude de la réactivité vasculaire à l'aide d'un myographe à fil, pour des études en immunofluorescence et histochimie fluorescente sur coupes congelées, et pour des analyses en Western blot de l'expression de protéines.

Les principaux résultats de notre étude indiquent que l'infusion d'Ang II (0,4 mg/kg/j) à des rats induit une augmentation significative de la pression artérielle systolique, qui atteint 194±5,9 mmHg par rapport au 120±5,6 mmHg chez les rats contrôles. La consommation orale de EPA:DHA 6:1 (500 mg/kg/j) prévient significativement l'hypertension induite par l'Ang II (147±5,9 mmHg) mais n'a aucun effet sur la tension artérielle normale (110±3,9 mmHg). 1 Chapter 1

Les

Physiology of the endothelium

Cardiovascular diseases

Cardiovascular diseases are a group of several pathologies including coronary heart diseases (CHD) such as myocardial infarction, cerebrovascular diseases (stroke), hypertension, peripheral artery diseases, rheumatic heart diseases, congenital heart diseases and heart failure [START_REF] Cheng | Role of prostacyclin in the cardiovascular response to thromboxane A2[END_REF]. Cardiovascular diseases are the leading cause of mortality and morbidity worldwide, with an estimated 17 million annual deaths (WHO 2011) [START_REF] Towfighi | Stroke declines from third to fourth leading cause of death in the United States historical perspective and challenges ahead[END_REF]. Amongst cardiovascular diseases, CHD accounts for 7.2 million deaths and 5.7 million are due to stroke (Lloyd-Jones, Adams et al. 2009). It is now well established that endothelial dysfunction is an early hallmark of major cardiovascular and other diseases (Figure 1), which is thought to contribute to the initiation and the development of these diseases [START_REF] Austin | Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease[END_REF].

Vascular endothelium

All blood vessels including arteries, arterioles, capillaries, veins and venules are the part of the circulatory system. The wall of the blood vessels can be divided into three distinguished layers which are the intima, the media and the adventitia [START_REF] Pugsley | The vascular system: An overview of structure and function[END_REF] (Figure 2). The intima (tunica interna/intima) is the innermost monocellular layer called endothelium which is supported by connective tissue and the internal elastic lamina [START_REF] Krstic | Human microscopic anatomy: an atlas for students of medicine and biology[END_REF]). The endothelium is located at the interface between the blood flow and the vessel wall. The media (tunica media) is mainly buildup of smooth muscle cells, collagen fibers and elastic lamina. [START_REF] Severs | A. The Vascular Smooth Muscle Cell as a Specialized[END_REF].

The adventitia (tunica externa or tunica adventitia) is the outermost layer comprising of collagen, elastin, fibroblasts, macrophages, vasa vasorum, nerve endings and fibers for the protection of the blood vessels [START_REF] Mulvany | Structure and function of small arteries in hypertension[END_REF]). The importance of each layer depends on the size and location of the arteries. In large conducting arteries, a high number of elastic fibers are present in the media. Muscular arteries contain more smooth muscles cells while only few smooth muscle cells along the internal elastic lamina are found in arterioles. Capillaries only contain endothelial cell and the basement membrane with connective tissues [START_REF] Pais | Characteristics of Blood Vessels[END_REF]).

Endothelial cells line the whole circulatory system starting from large arteries arising from the heart to the capillaries and veins. They regulate the flow of nutrient substances and blood cells [START_REF] Mangge | Antioxidants, inflammation and cardiovascular disease[END_REF]) and act as a selective barrier between the lumen of blood vessel and surrounding tissues [START_REF] Galvão | An application of subagging for the improvement of prediction accuracy of multivariate calibration models[END_REF]. Covering a largest surface area, the endothelium plays an important role in the regulation of blood flow and is a chief regulator of body homeostasis through the synthesis and secretion of various active molecules, including vasodilatating factors and vasoconstricting factors finely controlling vascular tone. Endothelial cells also regulate smooth muscle cells (SMC) proliferation, exchanges of molecules between the plasma and the interstitial fluid. They also play a vital role in the balance between pro-and anticoagulant mechanisms and in immunity [START_REF] Klein | Cunningham's textbook of veterinary physiology[END_REF].

Endothelial regulation of vascular tone

The vascular endothelium plays a major role in the regulation of vascular tone through a variety of mechanisms. Several mediators which can modify vascular tone are derived from the endothelium [START_REF] Schalkwijk | Vascular complications in diabetes mellitus: the role of endothelial dysfunction[END_REF][START_REF] Klein | Cunningham's textbook of veterinary physiology[END_REF]). In 1980, Furchgott and Zawadzki demonstrated the phenomenon of endothelium-dependent arterial relaxation. Acetyle choline induces relaxation in arterial rings by releasing endothelium-derived relaxing factor (EDRF) which stimulates soluble guanylyl cyclase responsible for the conversion of GTP to cyclic GMP. Later on, EDRF was identified as the radical gas nitric oxide (NO) [START_REF] Arnal | Endothelium-derived nitric oxide and vascular physiology and pathology[END_REF]. NO diffuses from the endothelium to the underlying smooth muscle where it activates soluble guanylyl cyclase to cause a rise in intracellular cyclic GMP and relaxation of the vessel wall [START_REF] Gryglewski | Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor[END_REF][START_REF] Rubanyi | Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor[END_REF].

The endothelium has the capacity to regulate the local vascular homeostasis by maintaining the balance between vasodilation and vasoconstriction, by controlling vascular smooth muscle cell (VSMC) proliferation and migration, and by acting on thrombosis and fibrinolysis via the release of various factors [START_REF] Davignon | Role of endothelial dysfunction in atherosclerosis[END_REF]. Imbalance of these different mechanisms promotes endothelial dysfunction, which may lead to serious cardiovascular diseases such as hypertension, a major CVD risk factors.

In response to physical and chemical stimuli such as changes in pressure, shear stress, and pH as well as to substances released by autonomic and sensory nerves and circulating hormones, autacoids, and cytokines, the vascular endothelium synthesizes relaxing and contractile factors responsible for the modulation of VSMC tone. Relaxation factors include nitric oxide (NO), prostacyclin (PGI2), endothelium-derived hyperpolarization (EDH), and contractile factors includes thromboxane A2, isoprostanes, superoxide anions (ROS), endothelin-1, and angiotensin-II factors (Figure 3) [START_REF] Mombouli | Endothelial dysfunction: from physiology to therapy[END_REF][START_REF] Feletou | Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)[END_REF].

-25 -

Nitric oxide (NO)

NO is a key cellular signaling molecule involved in a number of physiological and pathological processes. In the beginning, it was identified as a factor capable of activating soluble guanylyl cyclase responsible for the relaxation of vascular smooth muscle cells [START_REF] Katsuki | Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine[END_REF]. In 1980, Furchgott and Zawadski revealed that the endothelium causes vasorelaxation by the production of endothelium-derived relaxing factor (EDRF) which was later identified as NO (Furchgott and Zawadzki 1980;[START_REF] Palmer | Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor[END_REF][START_REF] Palmer | Vascular endothelial cells synthesize nitric oxide from L-arginine[END_REF][START_REF] Palmer | A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells[END_REF]. Besides its role in regulation of vascular tone, NO inhibits leukocytes adhesion, platelet aggregation, and has anti-apoptotic and antithrombotic effects. Moreover, NO is an important factor of endothelium viability, longevity and cardiovascular health [START_REF] Morello | Phosphoinositide 3-kinase signalling in the vascular system[END_REF].

vasorelexants vasoconstrictors

•NO •PGF2 •EDHF •Ang-II •ET •TxA2/PGH2

Endothelium

The endothelium-derived NO is produced by the endothelial NO synthase (eNOS) from L-arginine and plays a critical role in normal vascular biology and pathophysiology (Cai and Harrison 2000) (Figure 4). There are three different isoforms of NO synthase; the neuronal NOS (NOS1 or nNOS), the inducible NOS (NOS2 or iNOS) and the endothelial NOS (NOS3 or eNOS) [START_REF] Weiming | The role of nitric oxide in cancer[END_REF]. iNOS is an inducible isoform whereas nNOS and eNOS are being consititutively expressed [START_REF] Mombouli | Endothelial dysfunction: from physiology to therapy[END_REF][START_REF] Stuehr | Mammalian nitric oxide synthases[END_REF]. Under normal conditions, the majority of eNOS is bound to the protein caveolin-1, which inactivates eNOS, and this complex is located in micro domains in the cell membrane named caveolae [START_REF] Michel | Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin[END_REF][START_REF] Bucci | In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation[END_REF]. eNOS can be activated by Ca 2+ dependent and independent pathways. eNOS can be activated by substituting caveolin-1 by Ca 2+ /CaM in response to Ca 2+ -mobilizing agonists. When intracellular Ca 2+ levels increase, calmodulin detaches eNOS from caveolin-1 thus permitting the enzyme to become active. Furthermore, eNOS has been shown to be regulated by the interaction with positive and negative protein modulators such as heat shock protein 90 [START_REF] Jha | Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity[END_REF][START_REF] Garcia-Cardena | Dynamic activation of endothelial nitric oxide synthase by Hsp90[END_REF][START_REF] Pritchard | Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase[END_REF]) (Figure 5). molecules and tissue factor [START_REF] Tsao | Fluid flow inhibits endothelial adhesiveness. Nitric oxide and transcriptional regulation of VCAM-1[END_REF][START_REF] Dimmeler | Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32like proteases[END_REF][START_REF] Hermann | Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase[END_REF]. (Figure 6) eNOS can also be regulated in endothelial cells at a post-translational level primarily through multisite phosphorylations and protein/protein interactions. Residues Ser1177 and Ser 615 are the activation sites and the residues Thr495 and Ser114 are the inhibition sites on eNOS [START_REF] Dimmeler | Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32like proteases[END_REF][START_REF] Bohm | Combined endothelin receptor blockade evokes enhanced vasodilatation in patients with atherosclerosis[END_REF][START_REF] Bauer | Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase[END_REF][START_REF] Fleming | Molecular mechanisms underlying the activation of eNOS[END_REF]) (Figure 7). In response to several physiological stimuli, phosphorylation of eNOS across key regulatory sites plays an important a role in the regulation of the enzymatic activity [START_REF] Jha | Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity[END_REF][START_REF] Newby | ERK 1/2 Plays a Role in eNOS Phosphorylation and Cortisol Biosynthesis in the Ovine Fetal Adrenal[END_REF]. Phosphorylation of eNOS at Ser1177 is associated with an increased enzyme activity [START_REF] Gallis | Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002[END_REF][START_REF] Mccabe | Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation[END_REF]. Akt, one of the major regulatory targets of PI3-kinase, has been shown to directly phosphorylate eNOS at Ser117 and activate the enzyme in response to vascular endothelial growth factor (VEGF), sphingosine-1-phosphate, and estrogen [START_REF] Dimmeler | Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32like proteases[END_REF][START_REF] Fulton | Regulation of endothelium-derived nitric oxide production by the protein kinase Akt[END_REF]). Furthermore, eNOS can be also activated by phosphorylation on Ser1177 by AMP-activated protein kinase, protein kinase A (PKA), and protein kinase G (PKG) [START_REF] Busse | EDHF: bringing the concepts together[END_REF][START_REF] Flemming | The biofilm matrix[END_REF]. There are various assumed phosphorylation sites, but the most extensively studied eNOS residues, are serine residue in the reductase domain (human eNOS sequence: Ser1177; bovine sequence Ser1179), which positively regulates NO production, and a threonine residue within the CaMbinding domain (human eNOS sequence: Thr495; bovine sequence Thr497) [START_REF] Boo | Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism[END_REF]. Ischemia-reperfusion injury is another eNOS regulator which leads to the eNOS phosphorylation at Ser1177 and Ser 633 through the activation of PKA pathway [START_REF] Li | Tongxinluo reduces myocardial no-reflow and ischemiareperfusion injury by stimulating the phosphorylation of eNOS via the PKA pathway[END_REF]. Furthermore, there are numerous kinases reported to be involved in the phosphorylation of eNOS following cell activation by different stimuli such as shear stress, vascular endothelial growth factor [START_REF] Butt | Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases[END_REF], hypoxia [START_REF] Michell | The Akt kinase signals directly to endothelial nitric oxide synthase[END_REF][START_REF] Chen | Tannin 1-alpha-O-galloylpunicalagin induces the calciumdependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3kinase/Akt pathway in endothelial cells[END_REF], including extracellular signal-regulated kinase 1/2 which alters eNOS protein expression and activity [START_REF] Ramasamy | Regulation of endothelial nitric oxide synthase gene expression by oxidized linoleic acid[END_REF]. eNOS can be phosphorylated on serine, tyrosine, and threonine residues leading to eNOS activation or inactivation (Figure 8). (1) At rest, the eNOS is coupled to cav-1 (caveolin-1, a structural protein of caveolae) that decreases its activity. ( 2) eNOS is constitutively phosphorylated at Thr 495 preventing its activation by the Ca 2+ /CaM. (3) eNOS may be inhibited in response to oxidative stress by tyrosine phosphorylation by PYK2 (proline-rich tyrosine kinase). ( 4) eNOS can be activated by both Ca 2+ /CaM (calcium / calmodulin) and phosphorylation of Ser1177. Hsp90 (heat shock protein) facilitates the recruitment of Akt responsible for the phosphorylation of eNOS [START_REF] Fleming | Molecular mechanisms underlying the activation of eNOS[END_REF].

Inactive active P P

Inhibitory site Stimulatory site

Nucleus Nucleus

PECAM-1 CAV-1 GPCR

Endothelium-derived hyperpolarization (EDH)

EDH is defined as a hyperpolarization of endothelial origin that is transmitted to the vascular smooth muscle leading to its relaxation. The EDH was formerly known as the Endothelium-Derived Hyperpolarizing Factor (EDHF). Beside NO and prostacyclin, the EDH mediated component of relaxation plays a major role in endothelium-dependent relaxation in most of the medium to small calibre resistance arteries, small arteries and arterioles such as second and third-branch mesenteric artery as well as in coronary arteries [START_REF] Feletou | Endothelium-derived hyperpolarizing factor[END_REF][START_REF] Shimokawa | The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation[END_REF]. The role of the EDH component of relaxation is more important in resistance blood vessels as compare to that of NO and prostacyclin, including in humans [START_REF] Nakashima | Endothelium-dependent hyperpolarization caused by bradykinin in human coronary arteries[END_REF][START_REF] Shimokawa | The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation[END_REF].

The EDH component of the relaxation is evaluated in the presence of the combination of inhibitors of eNOS like L-NAME and of COXs like indomethacin [START_REF] Gerber | Enhanced acetylcholine induced relaxation in small mesenteric arteries from pregnant rats: an important role for endothelium derived hyperpolarizing factor (EDHF)[END_REF]). In the EDH mediated response, SKCa and IKCa are activated so that potassium ions move from the intracellular compartment to the extracellular space of endothelial cells, which leads to their hyperpolarization (Figure 9). This higher concentrations of potassium ions in the extracellular space can activate inwardly rectifying K + (KIR) channels and Na + /K + -ATPase to cause potassium ions efflux from VSMC leading to hyperpolarization and hence, relaxation [START_REF] Edgley | Slow Pressor'Hypertension From Low Dose Chronic Angiotensin II Infusion[END_REF][START_REF] Félétou | Endothelial dysfunction: a multifaceted disorder (the Wiggers Award Lecture)[END_REF]). In 1998, Edwards et al reported that hyperpolarization can also be transferred from endothelial cells to VSMC via myo-endothelial gap junctions. Myo-endothelial gap junctions are intracellular channels which can transfer signals from the endothelial cells to the underling vascular smooth muscle cells (Sandoo, van Zanten et al. 2010). Hyperpolarization of VSMC leads to the reduction in cytosolic calcium concentration following closure of voltage-activated calcium channels leading to relaxation. Endothelial hyperpolarization can also be mediated by hydrogen peroxide (H2O2) [START_REF] Matoba | Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries[END_REF] or arachidonic acid-derived metabolites including epoxyeicosatrienoic acids [START_REF] Quilley | Is EDHF an epoxyeicosatrienoic acid?[END_REF].

Prostacyclin

Prostacyclin (PGI2) is the major product of cyclooxygenase (COX) catalyzed metabolism of arachidonic acid in the endothelium [START_REF] Cheng | Role of prostacyclin in the cardiovascular response to thromboxane A2[END_REF]. Prostacyclin is produced from prostaglandin under the action of the enzyme prostacyclin synthase [START_REF] Dogne | New developments on thromboxane and prostacyclin modulators part I: thromboxane modulators[END_REF]).

PGI2, PGG2 and PGH2 are major products of vascular cyclooxygenase (COX). There are two isoforms of COX encoded by two separate genes. COX-1 is constitutively expressed and is present in many tissues, including endothelial cells [START_REF] Gryglewski | Bradykinin as a major endogenous regulator of endothelial function[END_REF]. COX-2 is not constitutively expressed, but can be induced rapidly and transiently in many cells, including vascular endothelial cells and smooth muscle cells, under the effect of physical stimuli and proinflammatory agents. PGI2 stimulates smooth muscle relaxation by stimulating adenylyl cyclase and formation of cyclic adenosine -3', 5'-monophosphate (cAMP) that activates protein kinase A, which reduces intracellular Ca 2+ by decreasing Ca 2+ release from the endoplasmic reticulum and by stimulating its uptake by it. The vasodilator activity of PGI2 is determined by the expression of specific receptors which are prostaglandin I2 receptors of the G-protein coupled receptor family in vascular smooth muscle cells. PGI2 is a potent vasodilator, and an effective endogenous inhibitor of platelet aggregation [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF]). In addition, PGI2 facilitates the release of NO by endothelial cells [START_REF] Shimokawa | Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig[END_REF]) and in turn, the action of PGI2 in vascular smooth muscle cells and platelets is potentiated by NO [START_REF] Delpy | Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3[END_REF]. PGI2 synthase preferentially couples with COX-2 rather than COX-1 in coexpression systems (Figure 10) [START_REF] Ueno | Coupling between cyclooxygenases and terminal prostanoid synthases[END_REF].

1.3.2

The endothelium-derived vasocontracting factors 1.

Angiotensin II

The octapeptide Angiotensin II (Ang II) is a potent vasoconstrictor hormone of the reninangiotensin system (RAS) that is formed following the conversion of Ang I into Ang II by Angiotensin I Converting Enzyme (ACE) [START_REF] Baker | Renin-angiotensin system involvement in pressureoverload cardiac hypertrophy in rats[END_REF]). Angiotensin II (Ang II) is a multifunctional peptide hormone that regulates blood pressure (BP), plasma volume, as well as cardiac, renal and neuronal function, and controls thirst responses. This peptide is of central importance in hypertension and myocardial remodeling, and is the main effector of the reninangiotensin system (RAS) [START_REF] Weber | Pathological hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system[END_REF]. Taken as a whole, the RAS is involved in different cardiovascular pathologies such as left ventricular hypertrophy, post-infarct remodeling, or neointima formation [START_REF] Li | Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling[END_REF]. The classical effects of Ang II on its target organs are mostly mediated by two membrane receptors, the Ang II type 1 receptors (AT1R) and type 2 receptors (AT2R), which mediate tissue-specific functions [START_REF] Horiuchi | Recent progress in angiotensin II type 2 receptor research in the cardiovascular system[END_REF].

AT1R and AT2R are a G-protein coupled receptors involved in the regulation of vascular cell proliferation and cell death [START_REF] Kaschina | Angiotensin AT1/AT2 receptors: regulation, signalling and function[END_REF]. AT1R are expressed in all organs, including heart, kidney, liver, adrenal glands, brain, lung and in all cells of the cardiovascular system, namely endothelial cells, smooth muscle cells, fibroblasts, monocytes, macrophages and cardiac myocytes and, thus, is important in cardiovascular pathobiology. While AT2R is highly expressed in fetal heart and fetal aorta, lung and liver [START_REF] Dasgupta | Angiotensin II receptors and drug discovery in cardiovascular disease[END_REF], AT2R expression declines fast after birth, but can be induced later in adult life under pathological conditions (Figure 11). [START_REF] Greco | Analysis of Cardiovascular Responses to Major Products of Angiotensinconverting Enzyme and Angiotensin-converting Enzyme[END_REF]). In addition, Ang II stimulation of AT1R activates the extracellular-signalregulated kinase (ERK) cascade, platelet-derived growth factor, epidermal growth factor receptor (EGFR), insulin receptor pathways and non-receptor tyrosine kinases belonging to the c-Src family, proline-rich tyrosine kinase 2, focal adhesion kinase and janus kinases (JAKs) [START_REF] Berry | Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide[END_REF].

Unlike AT1R, the AT2R contributes to the maintenance of blood pressure by controlling the vascular tone through vasodilatation [START_REF] Dasgupta | Angiotensin II receptors and drug discovery in cardiovascular disease[END_REF]. AT2R stimulation by Ang II leads to an increase in cGMP levels through a mechanism involving bradykinin B2 receptor, causing endothelial formation of NO [START_REF] Abadir | Angiotensin II type 2 receptor-bradykinin B2 receptor functional heterodimerization[END_REF]. Although AT2R expression decreases after birth, it can increase again in some pathophysiological conditions. Stimulation of de novo AT2R expression may inhibit neointima formation, cell proliferation, and inflammation in vascular injury, myocardial infarction and ischemic diseases, suggesting its protective role [START_REF] Ichiki | Downregulation of angiotensin II type 1 receptor by hydrophobic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in vascular smooth muscle cells[END_REF].

Endothelin-1

Endothelin (ET)-1 is a potent vasoconstrictor peptide originally isolated from endothelial cells. There are three structurally different ET isoforms (i.e. ET-1, ET-2, ET-3) as well as a vasoactive intestinal constrictor [START_REF] Böhm | The importance of endothelin-1 for vascular dysfunction in cardiovascular disease[END_REF]. Amongst the three ET isopeptides, the 21-amino acid peptide ET-1 is regarded as the most prominent isoform in the cardiovascular system, accounting for the majority of pathological effects exerted by ETs [START_REF] Barton | Endothelin, hypercholesterolemia and atherosclerosis[END_REF].

Under physiological conditions, ET-1 is produced in small amounts mainly in endothelial cells, primarily acting as an autocrine/paracrine mediator [START_REF] Pernow | New perspectives on endothelin-1 in atherosclerosis and diabetes mellitus[END_REF]. Under pathophysiological conditions, however, the production is stimulated in a large number of different cell types, including endothelial cells, vascular smooth muscle cells, cardiac myocytes, and inflammatory cells such as macrophages and leukocytes [START_REF] Grieve | Role of oxidative stress in cardiac remodelling after myocardial infarction[END_REF]).

The biological effects of ET-1 are transduced by two distinguishable receptor subtypes, ETA and ETB receptors, respectively [START_REF] Hunley | Update on endothelins-biology and clinical implications[END_REF]. In the vasculature, the ETA receptor is mainly located on vascular smooth muscle cells and mediates potent vasoconstriction. ET-1 may also induce indirect vasoconstrictor effects due to the generation of endothelium-derived thromboxane A2 [START_REF] Marasciulo | Endothelin-1: the yin and yang on vascular function[END_REF]. The ETB receptor is primarily located on endothelial cells, but may also be present on vascular smooth muscle cells [START_REF] Schneider | Contrasting actions of endothelin ETA and ETB receptors in cardiovascular disease[END_REF]. Stimulation of the endothelial ETB receptor results in release of NO and prostacyclin which cause vasodilatation, whereas stimulation of the vascular smooth muscle cell ETB receptor results in vasoconstriction [START_REF] Seo | Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels[END_REF]). Thus, the net effect produced by ET-1 is determined on the receptor localization and the balance between ETA and ETB receptors [START_REF] Davie | ETA and ETB receptors modulate the proliferation of human pulmonary artery smooth muscle cells[END_REF]) (Figure 12). In healthy arteries the production of ET-1 is small and the bioavailability of NO is preserved. In endothelial dysfunction there is increased expression of ET-1 in smooth muscle cells and macrophages (MØ). Both the ETA and the ETB receptor on smooth muscle cells may mediate formation of superoxide (O2 -). Collectively the balance of effects is shifted towards more vasoconstriction, inflammation and oxidative stress in endothelial dysfunction [START_REF] Böhm | The importance of endothelin-1 for vascular dysfunction in cardiovascular disease[END_REF].

Thromboxane A2 & Prostacyclin I2 (TxA2 & PGI2)

The prostanoids prostacyclin (PGI2) and thromboxane A2 (TXA2) play an essential role in the maintenance of vascular homeostasis. PGI2 is a vasodilator and an inhibitor of platelet aggregation, whereas TXA2 is a vasoconstrictor and a promoter of platelet aggregation [START_REF] Gamble | Roles of Cyclooxygenase (COX)-1 and COX-2[END_REF].

PGI2 and TXA2 are products of arachidonic acid (AA) metabolism by cyclooxygenase (COX), followed by metabolism of the COX product, PGH2, by the terminal synthase enzymes, prostacyclin or TX synthase, respectively [START_REF] Ruan | Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease[END_REF]. Two isoforms of COX have been identified: COX-1 is expressed constitutively in most cell types, whereas COX-2 is induced by inflammatory stimuli such as bacterial endotoxin and cytokines [START_REF] Caughey | Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2[END_REF]. It is considered that PGI2 is the main prostanoid synthesized by vascular endothelium and TXA2 is the main prostanoid produced by platelets [START_REF] Smith | The eicosanoids: cyclooxygenase, lipoxygenase, and epoxygenase pathways[END_REF]. However, the endothelium has been reported to synthesize TXA2 in addition to PGI2, and both COXs isoforms have been observed, with only COX-1 being detectable in unstimulated cells [START_REF] Morteau | Prostaglandins and inflammation: the cyclooxygenase controversy[END_REF]. Endothelial COX-2 can be up-regulated in vitro by inflammatory stimuli and shear stress [START_REF] Brown | COX-2: a molecular target for colorectal cancer prevention[END_REF]. Because the balance between PGI2 and TXA2 production is central in the maintenance of vascular tone and platelet aggregation [START_REF] Konturek | Physiology and pharmacology of prostaglandins[END_REF][START_REF] Sobrino | Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-α[END_REF], determination of the roles of endothelial COX isozymes, particularly with regard to the contribution of COX-2 in the regulation of prostanoids biosynthesis by the endothelium, is important (Figure 13) [START_REF] Caughey | Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2[END_REF].

Oxidative stress and reactive oxygen species (ROS)

Reactive oxygen species (ROS) are recognized as important signaling molecules in the cardiovascular system and are released by vascular cells during pathophysiological conditions like hypertension, diabetes mellitus, atherosclerosis and in acute and chronic inflammatory diseases [START_REF] Eisenberg | Platelet-dependent and procoagulant mechanisms in arterial thrombosis[END_REF]. NO, (O2 [START_REF] Félétou | Endothelial dysfunction: a multifaceted disorder (the Wiggers Award Lecture)[END_REF]. ROS and in particular superoxide anions can also act directly or indirectly as potent contracting agents via the reduction of the NO bioavailability or by activating COXS in vascular smooth muscle cells [START_REF] Hibino | Oxygen-derived free radical-induced vasoconstriction by thromboxane A2 in aorta of the spontaneously hypertensive rat[END_REF], leading to attenuated endothelium-dependent relaxations [START_REF] Aubin | Role of probucol on endothelial dysfunction of epicardial coronary arteries associated with left ventricular hypertrophy[END_REF][START_REF] Liu | Heart disease and stroke statistics-2009 update a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee[END_REF]). Moreover, ROS can also impair EDH-mediated endotheliumdependent relaxations through the reduction of calcium-activated potassium channels activity [START_REF] Kusama | Reduced hyperpolarization in endothelial cells of rabbit aortic valve following chronic nitroglycerine administration[END_REF] or by modifying the transmission of the hyperpolarization from endothelial cells to the underlying smooth muscle cells through myoendothelial gap junctions [START_REF] Griffith | 5-Methyltetrahydrofolate and tetrahydrobiopterin can modulate electrotonically mediated endothelium-dependent vascular relaxation[END_REF]. Several studies have shown the beneficial effects of antioxidants on the deleterious effect of oxidative stress on the endothelial function [START_REF] Kanani | Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans[END_REF][START_REF] Aubin | Role of probucol on endothelial dysfunction of epicardial coronary arteries associated with left ventricular hypertrophy[END_REF][START_REF] Liu | Heart disease and stroke statistics-2009 update a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee[END_REF]).

Endothelial dysfunction

Endothelial dysfunction is a broad term which implies dysregulation of endothelial cell functions, including impairment of the barrier functions of endothelial cells, vasodilation, disturbances in proliferative capacities, migratory as well as tube formation properties, angiogenic properties, attenuation of synthetic function, and deterrence of white blood cells from adhesion and diapedesis. Several factors contribute to endothelial dysfunction including smoking, high blood pressure, diabetes, high cholesterol levels, obesity, hyperglycemia, advance glycation end products (AGEs), and genetic factors. Endothelial dysfunction has been associated with an impairment of endothelium-dependent relaxations involving a reduced bioavailability of NO in major CV diseases such as hypertension, atherosclerosis, chronic renal failure, and diabetes [START_REF] Griendling | Oxidative stress and cardiovascular injury: Part II: animal and human studies[END_REF][START_REF] Rush | Vascular nitric oxide and oxidative stress: determinants of endothelial adaptations to cardiovascular disease and to physical activity[END_REF]. The mechanism underlying endothelial dysfunction has been linked to increased oxidative stress which is associated with a reduced NO bioavailability and the formation of inflammatory mediators such as vascular cell adhesion molecule-1 (VCAM-1) expression (Figure 14) [START_REF] Khan | Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells[END_REF][START_REF] Libby | Inflammation in atherosclerosis[END_REF]. In addition, different enzymes have been involved in the arterial oxidative stress involving NADPH oxidases, xanthine oxidases, COX-1 and COX-2, cytochrome P450 monooxygenases, enzymes of the mitochondrial respiratory chain, and uncoupled eNOS. Superoxide anion can react with NO to form the radical peroxynitrite [START_REF] Koppenol | Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide[END_REF], leading to the oxidation of the eNOS cofactor tetrahydrobiopterin (BH4) and the subsequent uncoupling of eNOS, thereby further promoting oxidative stress (Cai and Harrison 2000).

Endothelial dysfunction and hypertension

Hypertension is associated with endothelial dysfunction, where the delicate balance between vasodilators and vasoconstrictors produced by the endothelium is disrupted, with disturbance in the NO pathway leading to predominance of vasoconstrictors like ET-1, which contribute to high blood pressure [START_REF] Sandoo | The endothelium and its role in regulating vascular tone[END_REF]. Even though it is still unclear whether endothelial dysfunction is the cause or the consequence of elevated blood pressure [START_REF] Program | Report of the national high blood pressure education program working group on high blood pressure in pregnancy[END_REF], it appears to be an essential factor in hypertension. Studies in humans have reported a significant impairment of the vasodilator response of small resistance vessels to acetylcholine, but not to sodium nitroprusside (SNP), in hypertensive patients [START_REF] Endemann | Endothelial dysfunction[END_REF]. Importantly, investigations have indicated a larger incidence of cardiovascular events in hypertensive patients with more severe endothelial dysfunction compared to hypertensive patients with less severe endothelial dysfunction, and therefore it is suggested as a marker for future cardiovascular events in hypertensive patients [START_REF] Calhoun | Resistant hypertension: diagnosis, evaluation, and treatment a scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research[END_REF]. Treatment with angiotensin-converting enzyme (ACE) inhibitors has been shown to improve endothelial function [START_REF] Mancini | Angiotensin-Converting Enzyme Inhibition With Quinapril Improves Endothelial Vasomotor Dysfunction in Patients With Coronary Artery Disease The TREND (Trial on Reversing ENdothelial Dysfunction) Study[END_REF]. ACE inhibitors reduce oxidative stress and stimulate bradykinin to help increase NO bioavailability [START_REF] Hornig | Comparative effect of ACE inhibition and angiotensin II type 1 receptor antagonism on bioavailability of nitric oxide in patients with coronary artery disease role of superoxide dismutase[END_REF]. Products blocking Ang II type 1 receptor (AT1R), known as Ang II receptor blockers (ARBs), are successful primarily in the therapy of hypertension, but may also be beneficial in patients with intolerance to angiotensin-converting enzyme (ACE)

inhibitors for the treatment of several cardiovascular diseases, such as stable coronary heart disease and heart failure [START_REF] Dézsi | A review of clinical studies on angiotensin II receptor blockers and risk of cancer[END_REF]. Moreover, the renin-angiotensin-aldosterone system (RAAS), as well as AT1R and AT2R, play an important role in the regulation of cell proliferation and neoplastic progression [START_REF] Ager | The renin-angiotensin system and malignancy[END_REF].

In particular, endothelial dysfunction leading to diminished NO bioavailability impairs endothelium-dependent vasodilation in patients with essential hypertension and may also lead to premature development of atherosclerosis (Figure 15) [START_REF] Vallance | Endothelial function and nitric oxide: clinical relevance[END_REF]. Different mechanisms of reduced NO bioavailability have been shown both in hypertensive states and several cardiovascular diseases, and endothelial dysfunction is likely to occur prior to vascular dysfunction (Cai and Harrison 2000). Thus, the strategies currently used to improve endothelial dysfunction may result in the improved outcome for hypertensive patients [START_REF] Quyyumi | Endothelial function in health and disease: new insights into the genesis of cardiovascular disease[END_REF].

Models of Hypertension

The Spontaneously Hypertensive Rat (SHR) is the most commonly used model of cardiovascular disease, with over 4000 Medline references in the last 10 years. The male SHR is usually used as a model of established human hypertension, for example to define hypertension-induced changes in signaling mechanisms and to test new antihypertensive drugs. One of the major advantages of the SHR is lack of inter-individual variation, with the limitation that the SHR can only model one of many possible causes of human hypertension. The SHR is a useful model as compounds that are able to lower blood pressure in SHR are likely to also be effective in hypertensive humans [START_REF] Ching | Pathways in Adrenal Development: Identification of Novel Genes and the Role of Sonic Hedgehog[END_REF].

Different experimental protocols have been described to induce DOCA-salt hypertension in the literature, including subcutaneous implantation of DOCA pellets [START_REF] Churchill | Genetic susceptibility to hypertension-induced renal damage in the rat. Evidence based on kidney-specific genome transfer[END_REF].

DOCA administration (synthetic mineralocorticoid derivative), in combination with salt loading in the diet, to young adult Wistar rats followed by surgical removal of one kidney is associated with hypertension with cardiovascular remodeling, hypertrophy, fibrosis, conduction abnormalities and endothelial dysfunction. Similar cardiovascular remodeling occurs in patients with hypertension and heart failure but these patients are usually not young, nor on a high salt diet, nor taking saltretaining compounds nor functioning with a single kidney (Iyer, Chan et al. 2010). The DOCAsalt model markedly depressed renin-angiotensin system and hence has been extensively used in hypertension research as an angiotensin-independent model [START_REF] Schenk | The pathogenesis of DOCA-salt hypertension[END_REF].

NO synthesis can be blocked by inhibitors such as L-NAME (N ω -nitro-L-arginine methyl ester) and nitro-L-arginine [START_REF] Roche | A nitric oxide synthesis inhibitor (L-NAME) reduces licking behavior and Fos-labeling in the spinal cord of rats during formalin-induced inflammation[END_REF]. Chronic administration of L-NAME increased systolic blood pressure and heart weight, and decreased renal function. Chronic administration of L-NAME to rats during gestation induces the development of a pre-eclamptic syndrome similar to humans [START_REF] Hropot | Ramipril prevents the detrimental sequels of chronic NO synthase inhibition in rats: hypertension, cardiac hypertrophy and renal insufficiency[END_REF]. The role of reduced NO production in human hypertension is still unclear; therefore it is too early to decide whether NO synthase inhibition is an appropriate model.

However, this model deserves more attention as it is technically easy with low mortality rate [START_REF] Richer | Lack of beneficial effects of the NO donor, molsidomine, in the L NAME induced pre eclamptic syndrome in pregnant rats[END_REF].

Ang II induced hypertension is one of the most widely used pharmacological model of hypertension in rats. Blockade of the renin-angiotensin system (RAS) with ACE inhibitors or angiotensin II type 1 receptor (AT1R) antagonists has become one of the most successful therapeutic approaches in medicine. This has led to the concept of combined RAS blockade. RAS is a complex system which plays a major role in the maintenance of hemodynamics by regulation of arterial pressure and water and electrolyte balance. Ang II increases the risk of cardiovascular event by increasing arterial blood pressure and directly acts on cardiac and renal tissues [START_REF] Schmieder | Renin-angiotensin system and cardiovascular risk[END_REF]). It induces endothelial dysfunction and stimulates inflammatory, proliferative, fibrotic and thrombotic processes in the vasculature, and is a key regulator of vascular remodeling and inflammation. Ang II increase vascular tone, constricts smooth muscle cells, regulates vascular cell growth, apoptosis, fibrosis, matrix metalloproteinase production and degradation of extracellular matrix [START_REF] Schiffrin | Correction of arterial structure and endothelial dysfunction in human essential hypertension by the angiotensin receptor antagonist losartan[END_REF][START_REF] Touyz | Intracellular mechanisms involved in vascular remodelling of resistance arteries in hypertension: role of angiotensin II[END_REF]). These effects are observed often in arterial hypertension and atherosclerosis (Kane, Etienne-Selloum et al. 2010). Ang II may also affect blood pressure by its effects on kidney, brain and sympathetic nervous system (Reid 1992).

Excessive Reactive Oxygen Species Production in Hypertension

ROS play a major role as intracellular signaling molecules to regulate normal biological cellular responses [START_REF] Griendling | NAD (P) H oxidase role in cardiovascular biology and disease[END_REF]. In pathological conditions, loss of redox homeostasis contributes to vascular oxidative damage [START_REF] Gao | Vascular NAD (P) H oxidase activation in diabetes: a doubleedged sword in redox signalling[END_REF]. Multiple sources of oxidative stress have been implicated in the pathogenesis of hypertension-related endothelial dysfunction.

Recently, evidences have indicated that specific enzymes, the NOX family of NADPH oxidases, have an important function in generating ROS in a highly regulated fashion in physiological conditions, and that in disease states, hyper activation of NOXs contributes to oxidative stress and consequent cardiovascular diseases [START_REF] Figueira | Mitochondria as a source of reactive oxygen and nitrogen species: from molecular mechanisms to human health[END_REF]. Investigations have gone further to demonstrate the potential mechanisms controlling two important sources of hypertensionassociated oxidative stress, NADPH oxidase and mitochondria [START_REF] Montezano | Molecular mechanisms of hypertension-reactive oxygen species and antioxidants: a basic science update for the clinician[END_REF].

Taken together with recent reports demonstrating the coordinated formation of reactive oxygen species (ROS) from NADPH oxidase and mitochondria in hypertensive states, a model of hypertension-induced ROS originating from coordinated mitochondrial sources and NADPH oxidase appears to be promising [START_REF] Dikalov | Role of mitochondrial oxidative stress in hypertension[END_REF].

Inflammatory Regulation of Hypertension-Associated Endothelial Dysfunction

Inflammatory mechanisms appear to play a significant role in some types of pulmonary hypertension (PH), including monocrotaline-induced PH in rats and pulmonary arterial hypertension of various origins in humans, such as connective tissue diseases (Sadoughi, Zhang et al. 2011). Inflammation in adipose tissue is associated with impaired endothelial function in obese patients. Adipose tissue also plays a major role in regulating metabolism and inflammation through the production of both pro-inflammatory and anti-inflammatory adipokines [START_REF] Fantuzzi | Adipose tissue, adipokines, and inflammation[END_REF].

Though most investigations relating the process of adipose inflammation to vascular endothelial function concentrate on insulin resistance and obesity, recent studies have evaluated the effect of perivascular adipose tissue on vascular homeostasis in hypertension. Adipose tissue from hypertensive rats applied to thoracic aorta segments failed to suppress phenylephrine-induced vasoconstriction, in contrast to adipose tissue from normotensive animals (Baranowska-Kuczko,

Kozłowska et al. 2016).

Recent data also define novel roles for elements of both innate and adaptive immune responses in regulating endothelial function under hypertensive conditions [START_REF] Pauletto | Inflammation and hypertension: the search for a link[END_REF].

Activation of innate immunity's complement pathway may negatively impact vascular endothelial function in hypertension, whereas increased anti-inflammatory interleukin-10 expression from the adaptive immune response blunts the adverse effects of angiotensin II-associated hypertension on endothelial function [START_REF] Ferri | C-reactive protein: interaction with the vascular endothelium and possible role in human atherosclerosis[END_REF]. Circulating endothelial progenitor cells (EPCs), derived from myeloid pluripotent stem cells that also give rise to mature mononuclear cells, also play significant roles in maintaining endothelial homeostasis through their regenerative and repair mechanisms [START_REF] Urbich | Endothelial progenitor cells characterization and role in vascular biology[END_REF]. Overall, these newer data suggest that hypertensionassociated vascular endothelial dysfunction relates to local vascular inflammation as well as to systemic inflammation [START_REF] Cottone | Oxidative stress, inflammation and endothelial biomarkers in arterial hypertension[END_REF].

Animal studies have shown that oxidative stress and renal tubulointerstitial inflammation are associated with, and have major roles in, the pathogenesis of hypertension [START_REF] Vaziri | Causal link between oxidative stress, inflammation, and hypertension[END_REF]. This relation is supported by the observations that increase level of oxidative stress and renal tubulointerstitial inflammation increase arterial pressure in animal models [START_REF] Vaziri | Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension[END_REF]. Conversely, hypertension has been shown to cause oxidative stress and inflammation in renal and cardiovascular tissues in experimental animals. All together, these observations indicate that oxidative stress, inflammation and arterial hypertension participate in a selfperpetuating cycle which can lead to progressive cardiovascular disease [START_REF] Brasier | Vascular inflammation and the renin-angiotensin system[END_REF])

Antihypertensive treatments

In clinical settings today, a variety of antihypertensive medicines are being used alone and in combination with other drugs to reach target goal of blood pressure.

Calcium channel blockers

Calcium channel blockers inhibits the entrance of calcium ions via voltage-operated calcium channel in cells of the heart and blood vessel walls, resulting in lower blood pressure [START_REF] Epstein | Mechanism of action of calcium-channel-blocking agents[END_REF] and hence are also called calcium antagonists. They relax and dilate blood vessels by affecting the smooth muscle cells in the arterial walls.

Beta-blockers

The exact mechanisms of action of beta-blockers as anti-hypertensive drugs is still largely unknown. The proposed mechanism is that beta-blockers inhibit the effects of the sympathetic nervous system on beta-adrenergic receptor of the heart [START_REF] Parati | The human sympathetic nervous system: its relevance in hypertension and heart failure[END_REF]. They reduce the work of the heart and requirement blood and oxygen. As a result, the heart doesn't have to work as hard, which decreases blood pressure. Also, they help to control heart rate and are used in the treatment of abnormal heart rhythms that may be too fast or irregular. Beta-blockers are also widely used to treat hypertension, although they are no longer a first choice for initial treatment of most patients according to current guidelines [START_REF] Hunt | ACC/AHA 2005 guideline update for the diagnosis and management of chronic heart failure in the adult a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society[END_REF].

Diuretics

Diuretics acts on kidneys to remove more sodium and water from the body, which helps to relax the blood vessel walls, thereby lowering blood pressure. Moreover, they are combined with other blood pressure medicines as they can enhance the effect of the other antihypertensive drugs and prevent the fluid retention [START_REF] Weber | Clinical practice guidelines for the management of hypertension in the community[END_REF]. Thiazide diuretics are recommended as the first line of treatment for hypertension and are usually prescribed as one of at least two medicines to control hypertension.

Vasodilators

Vasodilators are medications that cause dilatation of blood vessels predominantly by the release of NO. They act directly on the VSMC in the walls of arteries resulting in vasorelaxation and preventing vasoconstriction. As a result, blood flows more easily through arteries.

ACE inhibitors

ACE inhibitors block the conversion of angiotensin I to angiotensin II, a potent vasoconstrictor and mitogenic. Therefore, they lower arteriolar resistance and increase venous capacity;

decrease cardiac output, cardiac index, stroke work, and decrease resistance in blood vessels in the kidneys; and lead to increased natriuresis.

Angiotensin II receptor antagonists (AT1R blockers)

The angiotensin II receptor antagonists (AT1R blockers, ARBs, sartans) are a group of antihypertensive drugs that act by blocking the effects of Ang II-mediated via AT1R activation in the body, thereby lowering blood pressure. Their structure is similar to Ang II and they bind to

Ang II receptors as inhibitors. AT1 blockers are widely used drugs for mild to moderate hypertension, chronic heart failure, secondary stroke prevention and diabetic nephropathy.

AT1 blockers and ACE inhibitors directly inhibit RAS and are the most effective for the treatment of hypertension [START_REF] Taal | Renoprotective benefits of RAS inhibition: from ACEI to angiotensin II antagonists[END_REF].

Endothelial Dysfunction and Diabetes

Diabetes Mellitus (DM) is a major health problem worldwide, associated with morbidity and mortality. DM is characterized by persistent elevation of the blood glucose level [START_REF] Monesi | Prevalence, incidence and mortality of diagnosed diabetes: evidence from an Italian population-based study[END_REF]. There are two types of diabetes: the type 1 diabetes (T1DM), which occurs due to the absence of the formation of insulin, and type 2 diabetes (T2DM), which is characterized by insulin insensitivity as a result of insulin resistance usually associated with metabolic syndrome and obesity (Figure 16) [START_REF] Sharma | Targeting endothelial dysfunction in vascular complications associated with diabetes[END_REF]. Several clinical studies with both types 1 and type 2 diabetic patients have shown the presence of an endothelial dysfunction [START_REF] Mcveigh | Impaired endothelium-dependent and independent vasodilation in patients with type 2 (non-insulin-dependent) diabetes mellitus[END_REF][START_REF] Nathan | Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus[END_REF]. In addition, studies report that endothelial dysfunction appears early in the development of DM, which may suggest a role of impaired endotheliumdependent vasodilatation in the initiation and development of both macro-vascular and microvascular complications of diabetes. Individuals with type I and type II diabetes have evidence of both microvascular and macrovascular endothelial dysfunction [START_REF] Caballero | Microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes[END_REF].

Endothelial dysfunction can even be evident in healthy individuals with a family history of diabetes, suggesting a genetic link [START_REF] Alvarado Vásquez | Reduced NO synthesis and eNOS mRNA expression in endothelial cells from newborns with a strong family history of type 2 diabetes[END_REF]). Patients with diabetes often have reduced NO bioavailability which results from increased oxidative stress, and oxidation of LDL due to hyperglycaemia [START_REF] Endemann | Endothelial dysfunction[END_REF]. Patients with type 1 diabetes have shown improved endothelial function when taking ACE inhibitors, through a reduction in oxidative stress, and an increase in NO bioavailability [START_REF] Heitzer | Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease[END_REF].

Lipids

Lipids are defined as small hydrophobic or amphipathic (or amphiphilic) molecules that may originate entirely or, in part, through condensations of thioesters and/or isoprene units [START_REF] Fahy | A comprehensive classification system for lipids[END_REF] Lipids are a chemically diverse group of substances that are poorly soluble or insoluble in water, but soluble in a polar organic solvents such as chloroform, hydrocarbons, alcohols or ethers [START_REF] Rane | What determines drug solubility in lipid vehicles: is it predictable?[END_REF]. They are mostly composed of carbon, hydrogen, oxygen and also sometimes nitrogen and phosphorous. Triglyceride, phospholipids sterols and waxes are the main types of lipids [START_REF] Mcdonald | Animal nutrition[END_REF]. The triglycerides exists in both foods and in the body, and usually serve as energy sources and can be stored in the adipose tissue for later use within the body [START_REF] Turchini | Fish oil replacement with different vegetable oils in Murray Cod: evidence of an "omega-3 sparing effect" by other dietary fatty acids[END_REF] The lipid classification system enables categorization of lipids and their properties in a way that is compatible with other macromolecular databases. Using this approach, lipids from biological tissues are divided into eight categories: fatty acids (Table 1), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits); sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Each category contains distinct classes and subclasses of molecules [START_REF] Fahy | A comprehensive classification system for lipids[END_REF].

Saturated fatty acids (SFA)

SFA only have carbon-carbon single bonds in their chain and these carbons are fully occupied with hydrogen atoms, thus forming straight chains giving strength to the structure [START_REF] Brownstein | High-Resolution Nuclear Magnetic Resonance And Molucular Structure[END_REF].

They are a useful source of energy and also have some important physical properties including poorly soluble in water in their undissociated (acidic) form, whereas they are relatively hydrophilic as potassium or sodium salts. [START_REF] Fuhrhop | Molecular and supramolecular chemistry of natural products and their model compounds[END_REF]. The most prevailing SFA are palmitic acid (C16), and stearic acid (C-18), found most commonly in animal products (Table 2). Vegetable derivatives of SFA such as palm oil, palm kernel oil, and coconut oil are produced from vegetables [START_REF] Healy | Evaluation of autologous shed blood for autotransfusion after orthopaedic surgery[END_REF]. High consumption of SFA is associated with high LDL levels which

Monounsaturated fatty acids (MUFA)

Upon losing one or more pairs of hydrogen atoms from the carbon chain, the fatty acids are called unsaturated fatty acids. The fatty acids containing one double bond are MUFA (Figure 17). Some common MUFA are palmitoleic acid (16:1 n-7), cis-vaccenic acid (18:1 n-7) and oleic acid 19

Polyunsaturated fatty acids (PUFA)

PUFA contain more than one double bonds in there chains (Figure 18). According to the international nomenclature, the positioning of the first double bond is given by (n-x) notation, counting the number of carbon atoms from the methyl end [START_REF] Keller | Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers[END_REF]. For

Trans fatty acids (TFA)

Trans fatty acids are unsaturated fatty acids of plant origin that have a trans arrangement of the carbon atoms adjacent to its double bonds resulting from the hydrogenation process, which gives a more rigid molecule close to a saturated fatty acids. The two hydrogen atoms of the carbons adjacent to the double bond point to opposite directions, which is different from the double bond of cis-configuration in the fatty acids of mammals. Furthermore, the location of the double bond is not fix and it may appear anywhere along the molecule, so that many positional isomers may exist.

In trans fatty acids, angle of the double bond is smaller than the cis-isomeric configuration and hence acyl chain is more linear, resulting in a more rigid molecule with different physical properties such as greater thermodynamic stability and a higher melting point. The trans configuration is designated by a t-; the number preceding the tindicates the position of the trans bond acids counted from the carboxyl end of the molecule, and cdesignates the cis isomers. Consumption of such acids is thought to increase the risk of atherosclerosis.

n-3 polyunsaturated fatty acids (n-3 PUFAs)

Long-chain polyunsaturated fatty acids with the first double bond at the third position from the methyl terminal that are found in plants and some types of fish. n-3 PUFAs are found in short and long-chain varieties. The short chain form is alpha-linolenic acid, 18:3 n-3 (ALA) which contains 18 carbons having 3 double bonds and is considered as essential fatty acids because it can't be synthesized within the body [START_REF] Sen | Holistic supplement omega fatty acids: a challenge for smart heart by saying no to atheroscelerosis and yes to homeostasis[END_REF]. The long chain n-3 PUFA includes EPA, DPA (docosapentaenoic acid) and DHA, and as compared to ALA, these fatty acids are elongated and highly unsaturated; EPA has 20 carbons with 5 double bonds while DPA and DHA has 22 carbons with 5 and 6 double bonds, respectively (Figure 19).

Main vegetal sources of n-3 PUFA include flax seed, camelina seed, perilla and chiaseed oils which contain the 18-carbon ALA as the major n-3 PUFA [START_REF] Turchini | Fish oil replacement with different vegetable oils in Murray Cod: evidence of an "omega-3 sparing effect" by other dietary fatty acids[END_REF]). The carbon-20

and carbon-22 n-3 PUFA such as EPA and DHA are abundantly found in seafood such as fish and shellfish. These fatty acids are also considered as essential fatty acids. Fish such as tuna, sardines, salmon, mackerel and herring contains higher concentration of these fatty acids. Shellfish like abalone, oyster, mussel and scallop are also good sources of these long chain n-3 PUFA [START_REF] Su | A gene atlas of the mouse and human protein-encoding transcriptomes[END_REF]).The National Heart Foundation of Australia recommends the consumption of 500 mg per day of combined DHA and EPA, which is associated with a reduction in the risk of coronary heart disease [START_REF] Colquhoun | the Nutrition and Metabolism Committee of the Heart Foundation. Review of evidence fish, fish oils, n-3 polyunsaturated fatty acids and cardiovascular health[END_REF] .

n-6 polyunsaturated fatty acids

Long-chain polyunsaturated fatty acids with the first double bond at the sixth position from the methyl terminal that are found in plants and in animal muscles and organ meat. n-6 PUFA has its short-chain representative, linoleic acid, which is an essential fatty acid and the most prevalent one in western diets. Linoleic acid, is most abundantly found in nature, having high proportion in most of the vegetable oils such as sunflower, safflower, corn, soybean and canola oils [START_REF] Mateos | The effects of feed supplemented with omega-3 polyunsaturated fatty acids on cultured abalone[END_REF].

Evening primrose and borage oil are also enriched in linoleic acid. Animal products also serve as a major source of n-6 PUFA in the form of arachidonic acid predominantly found in both muscle and organ meats [START_REF] Sinclair | Corpus, concordance, collocation[END_REF]).

The importance of the ratio of omega-6/omega-3 essential fatty acids

Western diets are deficient in omega-3 fatty acids, and have excessive amounts of omega-6 fatty acids. Excessive amounts of omega-6 polyunsaturated fatty acids (PUFA) and a very high omega-6/omega-3 ratio promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 PUFA (a low omega-6/omega-3 ratio) exert suppressive effects [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF]) (Figure 20).

Mammalian cells cannot convert omega-6 to omega-3 fatty acids because they lack the converting enzyme, Δ3 desaturase. Linoleic acid, α-Linoleic acid and their long-chain derivatives are important components of animal and plant cell membranes (Barceló-Coblijn and Murphy 2009).

These two classes of essential fatty acids, are metabolically and functionally distinct, and often have important opposing physiological functions. An optimal balance of EFA is important for good health and normal development [START_REF] Simopoulos | Evolutionary aspects of diet, the Omega-6/Omega-3 ratio, and gene expression[END_REF]. When humans ingest fish or fish oil, the EPA and DHA from the diet partially substitute the omega-6 fatty acids, especially AA, in the membranes of probably all cells, but especially in the membranes of platelets, erythrocytes, neutrophils, monocytes, and liver cells [START_REF] Simopoulos | Omega-6/Omega-3 essential fatty acids: biological effects[END_REF].

AA and EPA are the parent compounds for eicosanoid production. Due to the increased amounts of omega-6 fatty acids in the Western diet, the eicosanoid metabolic products from AA, specifically prostaglandins, thromboxanes, leukotrienes, hydroxy fatty acids, and lipoxins, are formed in larger quantities than those formed from omega-3 fatty acids, specifically EPA [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF]). The eicosanoids from AA are biologically active in very small quantities, and, they promote to the formation of thrombus and atheromas, allergic and inflammatory disorder, particularly in susceptible people (Simopoulos 2011). Thus, a diet rich in omega-6 fatty acids shifts the physiological state to one that is prothrombotic and proaggregatory, with increases in blood viscosity, vasospasm, and vasocontriction and decreases in bleeding time [START_REF] Hussein | Cell membrane fatty acids and health[END_REF].

Omega-3 fatty acids inhibit the production of platelet-derived growth factor (PDGF) in bovine endothelial cells [START_REF] De Caterina | The omega-3 fatty acid docosahexaenoate reduces cytokine-induced expression of proatherogenic and proinflammatory proteins in human endothelial cells[END_REF]). Thus, the reduction in its production by endothelial cells, monocytes/macrophages, and platelets could inhibit both the migration and proliferation of smooth muscle cells, monocytes/macrophages, and fibroblasts in the arterial wall.

Omega-3 fatty acids also increase endothelium-derived relaxing factor (EDRF) which facilitates relaxation in large arteries and vessels [START_REF] Nicolosi | n-3 Fatty acids and atherosclerosis[END_REF]. Supplementing the diet with omega-3 fatty acids (3.2 g EPA and 2.2 g DHA) in normal subjects increased the EPA content in neutrophils and monocytes more than sevenfold without changing the quantities of AA and DHA [START_REF] Nicolosi | n-3 Fatty acids and atherosclerosis[END_REF]. The antiinflammatory effects of fish oils are partly mediated by the inhibition of the 5-lipoxygenase pathway in neutrophils and monocytes [START_REF] Lee | Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function[END_REF].

Moreover, several studies show that omega-3 fatty acids influence interleukin metabolism by decreasing IL-1 and IL-6, suggesting an important role in the prevention of atherosclerosis (Jung, Torrejon et al. 2008)

Metabolim of PUFA

Free arachidonic acid (AA) is oxidized through three major metabolic routes: (i) the cyclooxygenase (COX) pathway producing prostaglandins and thromboxanes, (ii) the lipoxygenase (LOX) pathway leading to leukotrienes, hydroxyeicosatetraeneoic acids (HETEs) and lipoxins, whereas (iii) the cytochrome P450 (CYP) pathway implies oxidation by monooxygenases to produce hydroxylated and epoxidised fatty acids [START_REF] Hong | Modulation of arachidonic acid metabolism by curcumin and related β-diketone derivatives: effects on cytosolic phospholipase A2, cyclooxygenases and 5-lipoxygenase[END_REF]). αlinolenic acid (ALA) can be metabolized to some extent by mechanisms including desaturation and elongation to yield EPA, DHA, while linoleic acid (LA) is the metabolic precursor of arachidonic acid (AA) [START_REF] Holub | Clinical nutrition: 4. Omega-3 fatty acids in cardiovascular care[END_REF]). In the omega-6 fatty acids pathway, linoleic acid can be first converted into gamma-linolenic acid (GLA, 18:3, omega-6 fatty acid) by the enzyme Δ6-desaturase before elongation leading to (DGLA) dihomo-GLA (DHGLA, 20:3, omega-6 fatty acids) [START_REF] Das | Beneficial actions of polyunsaturated fatty acids in cardiovascular diseases: But, how and why?[END_REF]. The dihomo-GLA can be further converted into arachidonic acid (AA, 20:4, omega-6 fatty acids) by the Δ5-desaturase. The omega-3 fatty acids pathway uses the same series of enzymes for converting α-linolenic acid (ALA) into EPA and then into docosapentaenoic acid (DPA) by elongase (Figure 21). The conversion of ALA to EPA and DPA occurs primarily in the liver in the endoplasmic reticulum, whereas the final conversion of DPA to DHA requires a translocation to the peroxisome for a β-oxidation reaction [START_REF] Arterburn | Distribution, interconversion, and dose response of n-3 fatty acids in humans[END_REF]). -67 -

Health benefits of n-3 polyunsaturated fatty acids 2.6.1 n-3 polyunsaturated fatty acids and diabetes

Intake of n-3 fatty acids, either as fish oil or ethyl ester formulations is related with a variety of biochemical changes that might be beneficial in diabetes: reduced triglyceridemia, mainly through enhanced triglyceride lipolysis and enhanced fatty acid oxidation [START_REF] Sirtori | N-3 fatty acids and diabetes[END_REF].

Increasing the consumption of n-3 long chain PUFAs improves several cardiovascular risk factors in persons with diabetes and may reduce the risk of conversion from impaired glucose tolerance to type 2 diabetes [START_REF] Carpentier | n-3 Fatty acids and the metabolic syndrome[END_REF]. Many epidemiologic studies reported that type 2 diabetes was less prelevant among Japanese as compared to their mainland counterparts. Lower prevalence was attributed mainly to diets rich in n-3 long chain PUFAs [START_REF] Montmayeur | Pathophysiology and evolutionary aspects of dietary fats and long-chain polyunsaturated fatty acids across the life cycle[END_REF].

n-3 long chain PUFAs have beneficial effects in lowering triglyceride levels and reducing remnant lipoprotein (RLP) levels in subjects with type 2 diabetes or hypertriglyceridemia and may increase high-density lipoprotein (HDL) cholesterol levels [START_REF] Okumura | Eicosapentaenoic acid improves endothelial function in hypertriglyceridemic subjects despite increased lipid oxidizability[END_REF]. RLPs are higly atherogenic lipoproteins produced in the hydrolysis of chylomicrons and very low density lipoprotein (VLDL) [START_REF] Shin | Remnant Lipoprotein Particles Induce Apoptosis in Endothelial Cells by NAD (P) H Oxidase-Mediated Production of Superoxide and Cytokines via Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Activation Prevention by Cilostazol[END_REF]). Modest amounts of purified EPA 0.9 to 1.8 g/day reduced the RLP levels significantly by 77% in patients with type 2 diabetes treated for 3 months [START_REF] Nettleton | n-3 long-chain polyunsaturated fatty acids in type 2 diabetes: a review[END_REF].

It is of interest that in in vitro studies EPA has been shown to increase glucose-induced insulin secretion from beta-TC3 insulinoma cells [START_REF] Dubnov | Omega-6 fatty acids and coronary artery disease: the pros and cons[END_REF]. Other potentially beneficial aspects of n-3 treatment in diabetes may be related to the role of these dietary components in providing a source of vasoactive compounds, potentially leading to reduced blood pressure and improved peripheral perfusion (Hornstra 2012).

n-3 polyunsaturated fatty acids and cardiovascular diseases

Long chain PUFAs including EPA and DHA are the key nutrients in fish responsible for the potential cardioprotective effects of fish consumption (Kris-Etherton, [START_REF] Kris-Etherton | Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease[END_REF]. A beneficial effect of fish consumption on CVDs has been suggested to be related to overall favorable

Lipid Profile

Hypertriglyceridemia is one of the components of metabolic syndrome. Several studies reported that long chain n3 PUFAs reduce the blood triglyceride levels (Kris-Etherton, [START_REF] Kris-Etherton | Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease[END_REF].

Recently, 47 trials showed that fish oil supplementation with 1 g/day was effective in reducing triglyceride levels by 0.34 mmol/L over an average treatment period of 24 weeks in participants with hypertriglyceridemia [START_REF] He | Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease-eat fish or take fish oil supplement?[END_REF].

PUFA have multiple CVD-related physiological effects such as lowering of plasma triglycerides including reduced fatty acid availability for triglyceride synthesis [START_REF] Reddy | Diet, nutrition and the prevention of hypertension and cardiovascular diseases[END_REF].

Moreover, they reduced delivery of nonesterified fatty acids to the liver reduced, hepatic enzyme activity for triglyceride synthesis; and increased hepatic synthesis of phospholipids rather than triglycerides [START_REF] Nakamura | Essential fatty acid synthesis and its regulation in mammals[END_REF].

Inflammation and endothelial function

Several epidemiological studies reported that n-3 PUFA fatty acids have beneficial effects on inflammation and endothelial function [START_REF] Mori | Omega-3 fatty acids and inflammation[END_REF]. Omega-3 fatty acids are antiinflammatory. Indeed inverse association has been found between Omega-3 or fish consumption and circulating levels of C-reactive protein, interlukin-6, endothelial-leukocytes adhesion molecule-1, soluble intercellular adhesion molecule-1, tumor necrosis factor, INF-α soluble receptor 1, and matrix metalloproteinases-3 [START_REF] He | Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease-eat fish or take fish oil supplement?[END_REF]).

Omega 3 fatty acids have recognized anti-inflammatory actions that may contribute to their beneficial cardiac effects [START_REF] Russo | Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to clinical implications in cardiovascular prevention[END_REF]. Omega 6 fatty acids can be converted into arachidonic acid and then metabolised into the omega 6 eicosanoids. Consumption of omega 3 fatty acids increases eicosapentanoic acid in the cell membrane. This competes with arachidonic acid for enzymatic conversion into its own metabolites, the omega-3-derived eicosanoids [START_REF] Wall | Fatty acids from fish: the anti-inflammatory potential of longchain omega-3 fatty acids[END_REF].

Independent of the effects on the metabolism of eicosanoids, fish oils suppress pro-inflammatory cytokines and reduce expression of cell adhesion molecules [START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF] which are critical in recruiting circulating leucocytes to the vascular endothelium, an important event in the pathogenesis of atherosclerosis and inflammation. Omega-3 fatty acids also have direct effects on endothelial vasomotor function. Higher plasma concentrations are associated with improved dilatation of the brachial artery in young adults with cardiovascular risk factors, which implies a protective effect on endothelial function [START_REF] Din | Omega 3 fatty acids and cardiovascular disease--fishing for a natural treatment[END_REF]). In hyperlipidaemic men, omega-3 fatty acid supplementation improved systemic arterial compliance, and supplementation with docosahexanoic acid increased vasodilator responses in the human forearm arteries. These effects may be mediated through actions on intracellular signalling pathways, leading to reduced activation of transcription factors such as NF-ĸB [START_REF] Egert | Impact of n-3 fatty acids on endothelial function: results from human interventions studies[END_REF]. However, the precise effects of omega 3 fatty acids on these fundamental cellular processes and their potential impact on coronary heart disease are yet to be delineated completely.

Atherosclerosis

Although high dose supplementation of omega-3 fatty acids exerts a hypotriglyceridemic effects, these fatty acids also increase and enhance oxidation of LDL cholesterol [START_REF] Larsson | Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms[END_REF]). However, data from both animal models and humans are inconsistent. It is unclear whether omega-3 fatty acids have a direct effect on the pathogenesis of atherosclerosis. Consumption of 6g/d EPA and DHA for 2 years had no major favorable effects on the thickness of atherosclerotic coronary arteries [START_REF] Woodman | The Independent Effects of Purified EPA and DHA Supplementation on Cardiovascular Risk in Treated-hypertensive Type 2 Diabetic Individuals[END_REF]. Several other studies showed that dietary intake of omega-3 fatty acids or nonfried fish is associated with a lower prevalence of subclinical atherosclerosis classified by significant changes in common carotid intima-media thickness, in percent stenosis, while no modification in coronary artery calcium score, and ankle-brachial index were observed (Von [START_REF] Schacky | The effect of dietary Ω-3 fatty acids on coronary atherosclerosis: a randomized, double-blind, placebo-controlled trial[END_REF]).

Platelets aggregation

Omega-3 FAs compete with omega-6 FAs for prostaglandin and leukotrines synthesis at the cyclooxygenase and lipoxygenase level. omega-3 fatty acids modulate prostaglandin metabolism by increasing prostaglandin E3, an active vasodilator and inhibitor of platelets aggregation, thromboxane A3, leukotrines B5, and by decreasing production of thromboxane A2, a potent platelet aggregation and vasoconstrictor, and leukotrines B4 formation (an inducer of inflammation and a powerful inducer of leukocyte chemotaxis and adherence) [START_REF] Jha | Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity[END_REF]). In addition, omega-3 fatty acids may react with reactive oxygen species because of their double bonds and lead to decreased production of hydrogen peroxide, which is a critical activator of the nuclear NF-ĸB.

Other studies also indicate that high intake of omega-3 fatty acids would produce a lower platelet count, less platelet aggregation, a longer bleeding time and lower concentrations of thromboxane metabolites.

Clinical implications of omega-3 fatty acids

Omega 3 fatty acids from fish or fish oil supplements should be considered in the secondary prevention regimen of patients after myocardial infarction [START_REF] Holub | Omega-3 fatty acids from fish oils and cardiovascular disease[END_REF]. Patients should consume about 1 g/day of eicosapentanoic acid and docosahexanoic acid, preferably by increasing their intake of oily fish to at least two servings per week [START_REF] Harris | The Omega-3 Index: a new risk factor for death from coronary heart disease?[END_REF]). Fish oil capsules may be considered for those unable to tolerate fish or change their diet effectively.

Approved pharmaceutical grade capsules should be prescribed rather than encouraging over the counter supplements [START_REF] Din | Omega 3 fatty acids and cardiovascular disease--fishing for a natural treatment[END_REF]). Recent guidelines from the American Heart Association have gone further, supporting the use of fish oil supplements for patients with "documented" coronary heart disease. However, they believe that more evidence is required before considering fish oil supplements for patients with coronary heart disease outside the specific indication of myocardial infarction. Others have argued that fish oil supplements should not be recommended routinely for patients after myocardial infarction until more definitive evidence is available [START_REF] Rahman | Fish Oil and Cardiovascular Health[END_REF]. No trial has assessed the effects of fish oils on risk of coronary heart disease in primary prevention, and therefore explicit recommendations for this group cannot be made currently. Such a trial may prove impractical in terms of the numbers required. However, on the basis of evidence from epidemiological and observational studies the consumption of (preferably oily) fish at least twice weekly should be encouraged as part of a balanced diet [START_REF] Din | Omega 3 fatty acids and cardiovascular disease--fishing for a natural treatment[END_REF]). Any recommendations regarding fish and fish oils should be balanced against safety issues. Side effects such as fishy aftertaste are uncommon, and gastrointestinal upset is infrequent at moderate intakes. Some reports show that fish oil may worsen glycemic control in diabetes, but two meta-analyses found no adverse effect. Furthermore, a recent prospective cohort study found that a higher consumption of omega 3 fatty acids was associated with a lower incidence of coronary heart disease and mortality in diabetic women [START_REF] Din | Omega 3 fatty acids and cardiovascular disease--fishing for a natural treatment[END_REF][START_REF] Borghi | Omega-3 polyunsaturated fatty acids: Their potential role in blood pressure prevention and management[END_REF]. Concerns have been raised regarding adverse effects on low density lipoprotein (LDL) cholesterol and oxidative stress, but increases in LDL cholesterol are modest and studies into oxidative stress have been contradictory. Overall these effects are unlikely to be dominant given the apparent cardiac benefits of omega 3 fatty acids. More specific concerns regarding dietary fish relate to environmental contaminants, and a recent study showed that mercury in fish may attenuate their cardioprotective effects [START_REF] Cicero | Omega-3 polyunsaturated fatty acids: their potential role in blood pressure prevention and management[END_REF].

AIM OF THE STUDY

The endothelial cells are the major regulator of vascular homeostasis and play a key role by 7. To find the source of ROS.

-75 -

RESULTS

ARTICLE I

-76 - While current antihypertensive treatment are able to potently reduce the blood pressure, they seems to have a limited ability to protect and/or improve the endothelial dysfunction, a pivotal event in the protection of the cardiovascular system. Several epidemiological and both primary and secondary prevention studies have indicated that dietary intake of omega-3 polyunsaturated fatty acids (PUFAs), including the two major compounds eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduces the risk of cardiovascular diseases. Moreover, studies have reported that purified formulations of EPA and DHA are able to induce potent and sustained endothelium-dependent relaxations of isolated artery rings via an increased formation of NO and EDH. A previous study of the research team has shown that the endothelium-dependent vasorelaxant effect of omega-3 PUFAs is dependent on both the purity and ratio of EPA:DHA, with EPA:DHA ratio of 6:1 and 9:1 being superior formulations (Zgheel et al., 2014).

The aim of the present study was to determine whether chronic oral intake of the optimized

Results

The major results of our study show that infusion of Ang II caused a significant increase in systolic blood pressure, as compare to control rats. EPA:DHA 6:1 treatment significantly prevented the increase in systolic blood pressure by about 57%, while having no effect on the systolic blood pressure of normotensive rats. The chronic intake of the optimized EPA:

Conclusions and perspective

In conclusion, the present work has assessed the potency of an optimized EPA: There are numerous reports that support an important role of omega-3 fatty acids in preventing endothelial dysfunction, hypertension and cardiovascular diseases. The impregnation of omega-3 fatty acids in vascular tissues is also important, and needs to be clarified. However, the active molecules involved in the protective effects of omega-3 intake on endothelial function remains to be identified. Recent studies have reported that metabolites of omega-3 fatty acids, such as the resolvins, are key elements in the anti-inflammatory effect of omega-3. They also have been reported to exert beneficial vascular effect such as reducing the hyperreactivity induced by endothelin-1 and pro-inflammatory cytokines in pulmonary arteries. Since Ang II-induced T cell activation and the subsequent vascular inflammatory response have been identified as key mechanisms in Ang II-induced hypertension and endothelial dysfunction, a reduced proinflammatory responses due to production of anti-inflammatory omega-3 metabolites is likely to contribute to the EPA:DHA 6:1 antihypertensive and vasoprotective effect. Thus, further work is needed for the identification of active metabolites of omega-3 fatty acids in the vascular wall including resolvins, protectins, thromboxane A3, leukotriene 5 and lipoxins.

The present study has assessed the potency of the EPA:DHA 6:1 formulation to prevent the Ang II-induced hypertension and endothelial dysfunction. However, the curative potential of the optimized EPA:DHA 6:1 formulation on an established hypertension and associated endothelial dysfunction needs to be determined. Indeed, while the current antihypertensive treatments effectively reduce blood pressure, they seems to have limited ancillary effect on the endothelial dysfunction and hence to protect the cardiovascular system. Thus, the potency of the EPA:DHA 6:1 formulation to improve the endothelial dysfunction associated with an established hypertension could be an interesting novel therapeutic approach to reduce the cardiovascular mortality risk of hypertension.

Finally, on the basis of the beneficial vasoprotective effect of the EPA:DHA 6:1 formulation demonstrated in the present study and the reported beneficial effect of omega-3 intake in humans, a clinical study can be designed to determine the potential of EPA:DHA 6:1 to reduce cardiovascular risk factors in hypertensive patients.

Arterial hypertension

Arterial hypertension is a major problem of public health, being associated with end organ damage and cardiovascular morbidity and mortality. Arterial hypertension is the most important risk factor for a number of cardiovascular diseases including CAD, CHF, peripheral vascular disease, stroke and chronic kidney disease [START_REF] Sarnak | Kidney disease as a risk factor for development of cardiovascular disease a statement from the American Heart Association Councils on kidney in cardiovascular disease, high blood pressure research, clinical cardiology, and epidemiology and prevention[END_REF]. Prevalence of arterial hypertension in United States is very high and continues to increase with a control rate of just 30 % in treated patients [START_REF] Ong | Prevalence, awareness, treatment, and control of hypertension among United States adults 1999-2004[END_REF]. At the age of 75 years or older, about 70 % of men and women develop arterial hypertension (Chobanian, Bakris et al. 2003). Although effective antihypertensive drugs such as AT1R antagonists (ARAs), angiotensin-converting enzyme inhibitors (ACEI), betablockers, and calcium channel blockers, diuretics, and vasodilators exist, the control rate of hypertension remains low possibly due to poor observance and side effects. Treatments blocking pathological effects of the RAS at different levels have been shown to limit target-organ damage in hypertension and to decrease cardiovascular morbidity and mortality. AT1R and ACE inhibitors based treatments contributed to 60 % and 65 % of overall hypertensive therapy.

Omega-3 polyunsaturated fatty acids

Interest in natural products research is strong and can be attributed to several factors, including unmet therapeutic needs, the remarkable diversity of both chemical structures and biological activities of naturally occurring secondary metabolites. In this context, the adverse effects associated with synthetic molecules, decreased patients acceptability, contributed to shift the interest into natural products.

Numerous experimental and clinical studies have documented that omega-3 fatty acids can benefit the cardiovascular system, and particularly in patients diagnosed with CAD [START_REF] Harris | Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids[END_REF]). The American Heart Association recommends the intake of 1 g/day of the two omega- Omega-3 fatty acid lower plasma triglycerides mainly through reduced fatty acid availability for triglyceride synthesis due to decreased de novo lipogenesis, increased fatty acid beta-oxidation [START_REF] Kusunoki | Modulation of fatty acid metabolism as a potential approach to the treatment of obesity and the metabolic syndrome[END_REF], reduced delivery of nonesterified fatty acids to the liver, reduced hepatic enzyme activity for triglyceride synthesis, and increased hepatic synthesis of phospholipids rather than triglycerides [START_REF] Nakamura | Essential fatty acid synthesis and its regulation in mammals[END_REF]. Omega-3 fatty acids have anti-inflammatory properties and an inverse association has been found between regular intake of fish oil or fish and C-reactive protein, interleukin-6, E-selectin, soluble intercellular adhesion molecule-1, tumor necrosis factor, IFN-α soluble receptor 1, and matrix metalloproteinase-3 [START_REF] He | Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease-eat fish or take fish oil supplement?[END_REF]. Fish oils inhibit pro-inflammatory cytokines production and reduce expression of cell adhesion molecules [START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF]). These factors are critical in recruiting circulating leucocytes to the vascular endothelium, an important early event in the pathogenesis of atherosclerosis and inflammation.

Omega-3 fatty acids compete with omega-6 fatty acids for prostaglandin and leukotrienes synthesis at the cyclooxygenase and lipoxygenase level. Omega-3 fatty acids modulate prostaglandin metabolism by increasing synthesis of prostaglandin E3, an active vasodilator and inhibitor of platelets aggregation, thromboxane A3, leukotrienes B5, and by decreasing production of thromboxane A2, a potent inducer of platelet aggregation and vasoconstrictor, and leukotriene B4 formation (an inducer of inflammation and a powerful inducer of leukocyte chemotaxis and adherence). Other studies also indicate that high intake of fish oil would produce a lower platelet count, less platelet aggregation, a longer bleeding time and lower concentrations of thromboxane metabolites. The omega-3 fatty acids-induced hypotriglyceridemia effect requires doses of DHA and EPA of 3 to 4 g/day. In American population such doses reduce triglyceride levels by 30 % to 40 % [START_REF] Harris | Safety and efficacy of Omacor in severe hypertriglyceridemia[END_REF][START_REF] Lavie | Omega-3 polyunsaturated fatty acids and cardiovascular diseases[END_REF]). However, data from both animal models and humans on cardiovascular protection are inconsistent. Consumption of 6 g/d EPA and DHA for 2 years by 59 patients had no major favorable effects on the diameter of atherosclerotic coronary arteries [START_REF] Woodman | The Independent Effects of Purified EPA and DHA Supplementation on Cardiovascular Risk in Treated-hypertensive Type 2 Diabetic Individuals[END_REF]. Several studies showed that dietary intake of fish oil or nonfried fish is associated with a lower prevalence of subclinical atherosclerosis classified by significant changes in common carotid intima-media thickness and in percent stenosis while no modification in coronary artery calcium score, and ankle-brachial index were observed (Von [START_REF] Schacky | The effect of dietary Ω-3 fatty acids on coronary atherosclerosis: a randomized, double-blind, placebo-controlled trial[END_REF]. DHA is the precursor to a newly described metabolite called 10,17S docosatriene, which is part of a family of compounds called resolvins. These have firstly been described as being released in the brain in response to an ischemia and respond to the proinflammatory actions of infiltrating leukocytes by blocking interleukin-1-beta-induced NF-ĸB activation and cyclooxygenase-2 expression [START_REF] Layé | Polyunsaturated fatty acids, neuroinflammation and well being[END_REF].

The previous studies of our research team have shown that EPA and DHA, the major omega-3 fatty acids, induced concentration-dependent relaxations in coronary artery rings (Zgheel, Alhosin et al. 2014). The relaxation in response to EPA was slightly but significantly greater than those to DHA at 0.4 % (v/v) (77.8±10.3 and 64.7±12.8 %, respectively). Then, the ability of optimized omega-3 ratios to induce endothelium-dependent relaxation was determined and the results showed that EPA:DHA at ratio of either 6:1 or 9:1 induced relaxations significantly more potent than ratio of 3:1, 1:1, 1:3, 1:6, and 1:9. Similarly, the role of the purity of the EPA:DHA ratio was determined.

The endothelium-dependent relaxation in porcine coronary artery rings in response to a product with a high purity of omega-3 EPA:DHA (694:121 mg/g) was significantly greater than those in response to a product with a lower purity of omega-3 EPA:DHA (352:65 mg/g). Thus, the biological activity of omega-3 products is critically dependent on the consumption, ratio and purity.

Beneficial effects of omega-3 fatty acid on endothelial function

Dietary omega-3 fatty acids have a variety of anti-inflammatory and immune-modulating effects that may be of relevance to atherosclerosis and its clinical manifestations of myocardial infarction, sudden death, and stroke [START_REF] Mori | Omega-3 fatty acids and inflammation[END_REF]. The omega-3 fatty acids that appear to be most potent in this respect are the long-chain polyunsaturated derived from marine oils, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [START_REF] Riediger | A systemic review of the roles of n-3 fatty acids in health and disease[END_REF]).

Several studies have indicated an inverse correlation between the risk of cardiovascular diseases

and the increased consumption of omega-3 fatty acids, such as eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3), that are found mainly in fatty fish (e.g., salmon, trout, herring, sardines, and mackerel) [START_REF] Lavie | Omega-3 polyunsaturated fatty acids and cardiovascular diseases[END_REF]). The beneficial cardiovascular effects of dietary supplementation with omega-3 fatty acids include decreased arrhythmias, decreased triglycerides plasma concentrations, decreased blood pressure, and decreased platelet aggregation, all leading to reduced risk of cardiovascular mortality in patients with cardiovascular diseases [START_REF] Harris | Omega-3 fatty acids and coronary heart disease risk: clinical and mechanistic perspectives[END_REF]). In addition, the protective effect of omega-3 fatty acids could also be explained by their ability to improve endothelium-dependent relaxation of the arteries by stimulating the formation of the endothelium vasoprotective factors NO and EDH, as well as reduction in endothelium-dependent contractile responses [START_REF] Ribeiro | Cardiovascular effects induced by northeastern Brazilian red wine: Role of nitric oxide and redox sensitive pathways[END_REF]).

Since EPA:DHA 6:1 and 9:1 are more efficient than 1:1 (which is the only available prescribed drug) for endothelium-dependent relaxations in porcine coronary artery rings (Zgheel, Alhosin et al. 2014), the EPA:DHA 6:1 formulation was selected for the present study and given at the dose of 500 mg/kg/d to rats, which is equivalent to 5.67 g/d for a 70 kg Human (Reagan-Shaw, [START_REF] Reagan-Shaw | Dose translation from animal to human studies revisited[END_REF]). This dose is in line with reported clinical trials and meta-analysis, where doses range Ang II can be infused to mice or rats subcutaneously by Alzet osmotic mini pumps over a period of 4 weeks [START_REF] Zimmerman | Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervous system[END_REF]). Ang II doses of 0.7-1 mg/kg/day in mice and of 0.3-10 mg/kg/day for rats can induce hypertension rapidly in 7-10 days or slowly in 4-8 weeks [START_REF] Edgley | Slow Pressor'Hypertension From Low Dose Chronic Angiotensin II Infusion[END_REF]. Rats develop cardiac and renal fibrosis as well as aortic and cardiac hypertrophy after two weeks of infusion [START_REF] Huentelman | Protection from angiotensin II induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats[END_REF])..

Ang II was infused at 0.4 mg/kg/day to male Wistar rats subcutaneously by Alzet osmotic mini pumps over a period of 4 weeks. Ang II is a multifunctional peptide hormone that regulates blood pressure, plasma volume, cardiac, renal and neuronal functions, and also controls thirst responses.

This peptide is of central importance in hypertension and myocardial remodeling and is the principle effector molecule of the renin-angiotensin system (RAS) playing an important role in the regulation of arterial blood pressure [START_REF] Zhuo | New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II[END_REF]). In the present study Ang II infusion induced a rapid increase in systolic blood pressure of rats within a couple of days that remained elevated throughout the study. The chronic intake of the EPA:DHA 6:1 formulation was able to partially but significantly prevent the Ang II-induced increased systolic blood pressure. This result is in line with previously published studies reporting that omega-3 intake 3.3 to 7 g/d is associated with decreased blood pressure by 2.9 and 1.6 mm Hg among hypertensive patients [START_REF] He | Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease-eat fish or take fish oil supplement?[END_REF].

Indeed, the recent meta-analysis by Miller et al. indicates that EPA+DHA provision is associated with reduced systolic blood pressure, whereas diastolic blood pressure is reduced for EPA+DHA provision exceeding 2 g/d [START_REF] Miller | Long-chain omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and blood pressure: a meta-analysis of randomized controlled trials[END_REF].

In our experimental model, we observed that the continuous infusion of 0. In the present study, infusion of Ang II in rats induced an increase in vascular oxidative stress, mainly through the AT1R-mediated overexpression and activation of NADPH oxidase, in the vascular wall, which, in turn, affects the endothelial function by reducing both NO and EDH mediated relaxations and an increased in the formation of EDCFs (Figure 23). Indeed, the increased formation of NADPH oxidase-derived superoxide anions most likely reduces the bioavailability of NO by chemical reaction leading to peroxynitrite formation, which, in turn, promotes the uncoupling of eNOS that increases further the oxidative stress [START_REF] Förstermann | Nitric oxide and oxidative stress in vascular disease[END_REF][START_REF] Rochette | Diabetes, oxidative stress and therapeutic strategies[END_REF]. Increased oxidative stress also impairs functioning of the Ca 2+ -activated K + channels, SKCa and IKCa, resulting in an impaired electrical conduction and electrical signaling, leading to reduced EDH-mediated relaxation [START_REF] Behringer | Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation[END_REF][START_REF] Ellinsworth | Endothelium Derived Hyperpolarization and Coronary Vasodilation: Diverse and Integrated Roles of Epoxyeicosatrienoic Acids, Hydrogen Peroxide, and Gap Junctions[END_REF].

This result highlights the key role of the oxidative stress in the Ang II-induced endothelial dysfunction by reducing both EDH-and NO-mediated relaxations subsequent to the upregulation of NADPH oxidase subunits (p47 phox and p22 phox ). The chronic oral intake of the optimized EPA:DHA 6:1 formulation significantly decreased the level of vascular oxidative stress, at least in part, by decreasing the expression of both the NADPH oxidase subunits p22 phox and and p47 phox and AT1R. Our results are well in line with clinical studies showing a decreased level of oxidative vascular stress following omega-3 treatment in atherosclerotic patients [START_REF] Eftekhari | Effect of conjugated linoleic acid and omega-3 fatty acid supplementation on inflammatory and oxidative stress markers in atherosclerotic patients[END_REF]. In the cardiovascular system, Ang II activates NADPH oxidase through AT1R activation which increases vascular oxidative stress that is strongly associated with the progression of cardiovascular disease [START_REF] Manrique | The renin angiotensin aldosterone system in hypertension: roles of insulin resistance and oxidative stress[END_REF]). Several signaling pathways in response to Ang II are mediated by ROS [START_REF] Mehta | Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system[END_REF]. Different enzymes have been involved in the increased arterial oxidative stress involving AT1R, NADPH oxidases, xanthine oxidases, COX-1 and COX-2, cytochrome P450 monooxygenases, enzymes of the mitochondrial respiratory chain, and eNOS uncoupling. ROS (O2 •-) can react with NO to form peroxynitrite [START_REF] Koppenol | Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide[END_REF], leading to the oxidation of the eNOS cofactor tetrahydrobiopterin (BH4) and the subsequent uncoupling of eNOS thereby further promoting oxidative stress (Cai and Harrison 2000).

The chronic intake of the optimized EPA:DHA 6:1 formulation is associated with significantly increased plasma level of omega-3 fatty acids, mainly as EPA, DHA and the intermediate elongated metabolite of EPA, the docosapentaenoic acid (DPA), resulting in a decreased omega-6/omega-3 ratio. Excessive amounts of omega-6 polyunsaturated fatty acids (PUFA) and a very high omega-6/omega-3 ratio promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 fatty acids (a low omega-6/omega-3 ratio) exert protective effects.

The reduction of this ratio has been associated with a shift towards beneficial health effects of omega-3, including reduced cardiovascular and cancer risk [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF]. Human beings have evolved on a diet with a ratio of omega-6 to omega-3 essential fatty acids of approximately 1, whereas in Western diets this ratio is 10/1-22.5/1 (Simopoulos 2011). Western diets are excessive in omega-6 fatty acids and deficient in omega-3 fatty acids as compared with the diet on which human beings evolved [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF]).

In the present study, chronic oral intake of the optimized EPA:DHA 6:1 formulation significantly decreases Ang II-induced over-expression of AT1R, COX-1 and COX-2. Omega-3 fatty acids have recognized anti-inflammatory actions in humans that may contribute to their beneficial cardiac effects [START_REF] Wall | Fatty acids from fish: the anti-inflammatory potential of longchain omega-3 fatty acids[END_REF]. Omega-6 fatty acids can be converted into arachidonic acid and then metabolized into the omega-6 eicosanoids. Consumption of omega-3 fatty acids increases EPA in the cell membrane. This PUFA competes with arachidonic acid for enzymatic conversion into its own metabolites, the omega-3 derived eicosanoids [START_REF] Wall | Fatty acids from fish: the anti-inflammatory potential of longchain omega-3 fatty acids[END_REF].

Increased concentration and activity of Ang II is strongly associated with inflammation. Ang II has a key proinflammatory effect on the cardiovascular system by stimulating vascular damage, inducing adhesion molecule-1 expression, recruiting inflammatory cells, increasing cytokine expression and tissue repairing [START_REF] Brasier | Vascular inflammation and the renin-angiotensin system[END_REF]. The physiological effects of Ang II are mediated by Ang II receptor subtype 1 (AT1R), which is widely distributed in many organs. In a rat experimental model Ang II infusion caused hypertension accompanied by marked monocyte infiltration as well as VCAM-1 and MCP-1 expression in the microvessels wall or perivascular tissue [START_REF] Cheng | Angiotensin II and vascular inflammation[END_REF]. In endothelial, VSMC and mononuclear cells, Ang II is associated with an increased expression of MCP-1, the main chemokine for monocyte/macrophages and of interleukin-8 (IL-8), which are potent chemoattractants and activators of neutrophils [START_REF] Yadav | MCP-1: chemoattractant with a role beyond immunity: a review[END_REF]. Ang II also increased IL-6 production in macrophages and vascular cells, and upregulated TNF-α and IL-6 gene expression [START_REF] Libby | Inflammation and atherosclerosis[END_REF]. ACE inhibitors and AT1 antagonists reduced the expression of inflammatory markers, adhesion molecules, and also cytokines [START_REF] Mezzano | Angiotensin II and renal fibrosis[END_REF]. Ang II activates vascular and inflammatory cells to secrete proinflammatory mediators that assist to recruit new mononuclear cells, and, hence, results in additional inflammatory response contributing to the progression of vascular damage.

Independent of their effects on the metabolism of eicosanoids fish oils suppress pro-inflammatory cytokines and reduce expression of cell adhesion molecules in humans [START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF].

Conclusions and perspective

In conclusion, the present work has assessed the potency of optimized EPA: There are numerous reports that support an important role of omega-3 fatty acids in preventing endothelial dysfunction, hypertension and cardiovascular diseases. Further work is needed for the determination of active metabolites of omega-3 fatty acids other than resolvins, protectins, thromboxane A3, leukotriene 5 and lipoxins.

Further studies are needed to determine the curative potential of the optimized EPA:DHA 6:1 formulation in treatment of endothelial dysfunction and hypertension in Ang II-induced hypertensive models of rats (Figure 24). The impregnation of omega-3 fatty acids in vascular tissues is also important, which needs to be clarified. Furthermore, additional investigations are required to determine the role of the omega-3 purity and ratio to improve hypertension-related dysfunction of the vascular system including endothelial dysfunction and vascular remodeling.

On the basis of vasoprotective results established by the current study a clinical study can be designed to determine the potential of EPA:DHA 6:1 to reduce cardiovascular risk factors in hypertensive patients (Figure 25).

Les acides gras polyinsaturés omega-3

La recherche sur les produits naturels présente un fort intérêt du fait de plusieurs critères dont la non-couverture des besoins thérapeutiques, et la diversité remarquable des métabolites secondaires naturels au niveau à la fois des structures chimiques et des activités biologiques. Dans ce contexte, les effets indésirables associés aux molécules de synthèse et la diminution de l'acceptabilité des patients contribuent à augmenter l'intérêt pour les produits naturels.

De nombreuses études expérimentales et cliniques ont montré que les acides gras omega-3 peuvent avoir une répercussion bénéfique sur le système cardiovasculaire, et plus particulièrement chez les patients souffrant de maladie coronarienne [START_REF] Harris | Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids[END_REF]. Ainsi, l'American

Heart Association recommande la prise quotidienne de 1 g des deux acides gras omega-3 EPA et DHA dans le cadre de la prévention primaire et secondaire des maladies cardiovasculaires, après un infarctus du myocarde, et pour la prévention des morts subites cardiovasculaires [START_REF] Von Schacky | Cardiovascular benefits of omega-3 fatty acids[END_REF].

Les acides gras omega-3 réduisent le taux plasmatique en triglycérides principalement en réduisant la disponibilité des acides gras pour la synthèse de triglycérides par diminution de la lipogenèse de -131 -novo, augmentation de la bêta-oxydation des acides gras [START_REF] Kusunoki | Modulation of fatty acid metabolism as a potential approach to the treatment of obesity and the metabolic syndrome[END_REF], Ainsi, les effets bénéfiques des omega-3 dépendent fortement de la dose consommée, de la composition et de la pureté des omega-3.

réduction

Effets bénéfiques des acides gras omega-3 sur la fonction endothéliale

Les acides gras omega-3 de l'alimentation présentent divers effets anti-inflammatoire et de modulation du système immunitaire qui pourrait jouer un rôle dans le ralentissement du développement de l'athérosclérose et de l'apparition de ses signes cliniques comme l'infarctus du myocarde, la mort subite et l'accident vasculaire cérébral [START_REF] Mori | Omega-3 fatty acids and inflammation[END_REF]. Les acides gras omega-3 qui semblent les plus efficaces dans ce cadre sont les polyinsaturés à longues chaines des huiles d'origine marine, c'est-à-dire l'acide ecosapentaénoïque (EPA) et l'acide docosahexaénoïque (DHA) [START_REF] Riediger | A systemic review of the roles of n-3 fatty acids in health and disease[END_REF]. Plusieurs études ont montré une corrélation inverse entre le risque de maladies cardiovasculaires et une augmentation de la consommation d'acides gras omega-3, comme l'EPA et le DHA, qui sont principalement retrouvés dans les poissons gras (saumon, truite, hareng, sardines, maquereau, etc.) [START_REF] Lavie | Omega-3 polyunsaturated fatty acids and cardiovascular diseases[END_REF]. Les effets bénéfiques d'une augmentation de la consommation alimentaire en acides gras omega-3 sur le système cardiovasculaire incluent une baisse des arythmies, de la triglycéridémie, de la pression artérielle et de l'agrégation plaquettaire, l'ensemble de ces effets résulte en une baisse de risque de mortalité cardiovasculaire chez les patients atteints de maladies cardiovasculaires [START_REF] Harris | Omega-3 fatty acids and coronary heart disease risk: clinical and mechanistic perspectives[END_REF]. De plus, les effets protecteurs des omega-3 peuvent être attribués à leur capacité à améliorer les relaxation dépendante de l'endothélium dans les artères en stimulant la formation endothéliale des facteurs vasoprotecteurs NO et EDH, ainsi qu'en réduisant les réponses contractiles dépendantes de l'endothélium [START_REF] Ribeiro | Cardiovascular effects induced by northeastern Brazilian red wine: Role of nitric oxide and redox sensitive pathways[END_REF].

Du fait que les formulations EPA:DHA 6:1 et 9:1 sont plus efficaces que le ratio 1:1 (qui correspond à la seule spécialité avec AMM sur le marché) pour l'induction des relaxations dépendantes de l'endothélium dans des anneaux d'artère coronaire de porc (Zgheel, Alhosin et al. 2014), la formulation EPA:DHA 6:1 a été retenue pour l'étude présente et donnée à des rats à la dose de 500 mg/kg/j, ce qui équivaut à 5,67 g/j for pour un homme de 70 kg [START_REF] Reagan-Shaw | Dose translation from animal to human studies revisited[END_REF]. Cette dose est cohérente avec les études cliniques et les méta-analyses, où les doses rapportées vont de 0,18 à 10 g/j sous forme de divers produits à base omega-3 (Delgado-Lista, [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Enns | The impact of omega-3 polyunsaturated fatty acid supplementation on the incidence of cardiovascular events and complications in peripheral arterial disease: a systematic review and meta-analysis[END_REF].

L'angiotensine II (Ang II) peut être infusé à des rats ou des souris grâce à des mini pompes osmotiques sur une durée allant jusqu'à 4 semaines [START_REF] Zimmerman | Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervous system[END_REF]).

L'infusion d'Ang II à la dose de 0,7-1 mg/kg/j chez la souris ou de 0,3-10 mg/kg/j chez le rat va induire une hypertension de facon rapide en 7 à 10 jours ou de façon lente en 4 à 8 semaines [START_REF] Edgley | Slow Pressor'Hypertension From Low Dose Chronic Angiotensin II Infusion[END_REF]. Les rats recevant l'Ang II en infusion vont développer en 2 semaines des fibroses rénales et cardiaques ainsi qu'une hypertrophie aortique et cardiaque [START_REF] Huentelman | Protection from angiotensin II induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats[END_REF].

Dans notre étude, l'Ang II a été infusée pendant 4 semaines à la dose de 0,4 mg/kg/j à des rats vasodilatations indépendantes de l'endothélium (Wang, Liang et al. 2012). La dysfonction endothéliale a été associée à une atteinte des relaxations dépendantes de l'endothélium impliquant une réduction de la biodisponibilité du NO dans la plupart des maladies cardiovasculaires dont l'hypertension, l'athérosclérose, la néphropathie chronique ou le diabète [START_REF] Griendling | Oxidative stress and cardiovascular injury: Part II: animal and human studies[END_REF][START_REF] Rush | Vascular nitric oxide and oxidative stress: determinants of endothelial adaptations to cardiovascular disease and to physical activity[END_REF][START_REF] Félétou | Endothelial dysfunction: a multifaceted disorder (the Wiggers Award Lecture)[END_REF]. Le mécanisme sousjacent à la dysfonction endothéliale a été lié à une augmentation du stress oxydant vasculaire luimême associé à une diminution de la biodisponibilité du NO, une altération de la production des prostanoïdes tels que la prostacycline, le thrombaxane A2 ou les isoprostanes, une diminution de la composante EDH de relaxation ainsi qu'à une augmentation de formation d'endotheline-1, l'ensemble de ces phénomènes pouvant contribuer à la dysfonction endothéliale de manière isolée ou ensemble.

Dans la présente étude, l'infusion d'Ang II est associée à une augmentation du stress oxydant vasculaire chez les rats, principalement par la surexpression et l'activation de la NADPH oxydase induite par AT1R dans la paroi vasculaire, ce qui va à son tour affecter la fonction endothéliale en réduisant les deux composantes de la relaxation NO et EDH, et en augmentant la formation d'EDCFs (Figure 23). En effet, l'augmentation de la formation par la NADPH oxydase d'anions superoxyde diminue probablement la biodisponibilité du NO par la réaction chimique produisant des peroxynitrites, qui peuvent à leur tour favoriser le découplage de la eNOS ce qui augmente encore le stress oxydant vasculaire [START_REF] Förstermann | Nitric oxide and oxidative stress in vascular disease[END_REF][START_REF] Rochette | Diabetes, oxidative stress and therapeutic strategies[END_REF].

L'augmentation du stress oxydant vasculaire va aussi altérer le fonctionnement des canaux potassiques dépendants du calcium SKCa and IKCa, résultant dans une altération de la conductivité électrique et des voies de signalisation électriques, conduisant à la réduction de la composante EDH de la relaxation [START_REF] Behringer | Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation[END_REF][START_REF] Ellinsworth | Endothelium Derived Hyperpolarization and Coronary Vasodilation: Diverse and Integrated Roles of Epoxyeicosatrienoic Acids, Hydrogen Peroxide, and Gap Junctions[END_REF].

Ces résultats soulignent le rôle majeur du stress oxydant dans la dysfonction endothéliale induite par l'Ang II via la diminution des composantes NO et EDH de la relaxation qui suivent la surexpression des sous-unités de la NADPH oxydase (p47 phox and p22 phox ). La consommation chronique de la formulation EPA:DHA 6:1 a significativement diminué le niveau du stress oxydant vasculaire, du moins en partie, en prévenant la surexpression de la NADPH oxydase et de AT1R.

Ces résultats sont en accord avec ceux d'étude cliniques qui montrent une diminution du stress oxydant vasculaire chez des patients athérosclérotiques recevant des omega-3 [START_REF] Eftekhari | Effect of conjugated linoleic acid and omega-3 fatty acid supplementation on inflammatory and oxidative stress markers in atherosclerotic patients[END_REF].

Dans le système cardiovasculaire, l'Ang II active la NADPH oxydase via l'activation de AT1R, ce qui augmente le stress oxydant vasculaire qui est fortement associé au développement des maladies cardiovasculaires [START_REF] Manrique | The renin angiotensin aldosterone system in hypertension: roles of insulin resistance and oxidative stress[END_REF]). Plusieurs voies de signalisation en réponse à l'Ang II impliquent ainsi des espèces réactives de l'oxygène (ROS) [START_REF] Mehta | Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system[END_REF].

Diverses enzymes ont été impliquées dans l'augmentation du stress oxydant vasculaire tels que AT1R, la NADPH oxydase, la xanthine oxydase, les COX-1 et COX-2, le cytochrome P450, les enzymes de la chaine respiratoire mitochondriale, et la eNOS découplée. Les ROS (O2 •-) peuvent réagir chimiquement avec le NO pour former des peroxynitrites [START_REF] Koppenol | Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide[END_REF], ce qui conduit à l'oxydation du cofacteur de la eNOS, la tétrahydrobioptérine (BH4), et subséquemment au découplage de la eNOS augmentant à son tour le stress oxydant (Cai and Harrison 2000).

La consommation chronique de la formulation EPA:DHA 6:1 est associée à une augmentation significative des proportions plasmatiques en acides gras omega-3, principalement sous forme d'EPA, de DHA et de l'intermédiaire métabolique allongé de l'EPA, l'acide docosapenténoïque (DPA), conduisant à la diminution significative du ratio omega-6/omega-3. Des quantités excessives d'acides gras polyinsaturés omega-6 ainsi qu'un ratio omega-6/omega-3 élevé promeuvent la genèse de nombreuses pathologies, dont les maladies cardiovasculaires, les cancers, les maladies inflammatoires et auto-immunes, alors qu'à l'inverse des niveaux importants en omega-3 (et un faible ratio omega-6/omega-3) ont des effets protecteurs. La réduction de ce rapport omega-6/omega-3 a été associée à un basculement vers les effets bénéfiques pour la santé des omega-3 [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF]. Les êtres humains ont évolués avec un régime alimentaire ayant un rapport entre acides gras omega-6 etomega-3 approchant de 1/1, alors que l'alimentation occidentale a un rapport de l'ordre de 10/1-22.5/1 (Simopoulos 2011). De ce fait, l'alimentation occidentale est trop riche en omega-6 et présente un déficit en omega-3 [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF].

Dans la présente étude, la consommation chronique de la formulation EPA:DHA 6:1 a significativement diminué la surexpression induite par l'Ang II de AT1R, de COX-1 et de COX-2.

Les acides gras omega-3 ont des effets antiinflammatoires reconnus chez l'homme qui pourrait contribuer à leur effets bénéfiques vis-à-vis du système cardiovasculaire [START_REF] Wall | Fatty acids from fish: the anti-inflammatory potential of longchain omega-3 fatty acids[END_REF].

Les acides gras omega-6 sont convertis en acide arachidonique qui est lui-même métabolisé en eicosanoïdes de la série omega-6. La consommation des omega-3 augmente le taux d'EPA dans les membranes cellulaires, où il va entrer en compétition avec l'acide arachidonique pour la conversion en ses propres métabolites, les eicosanoïdes de la série omega-3 [START_REF] Wall | Fatty acids from fish: the anti-inflammatory potential of longchain omega-3 fatty acids[END_REF].

L'augmentation de la concentration et de l'activité de l'Ang II est fortement associée à l'inflammation. L'Ang II exerce un effet pro-inflammatoire sur le système cardiovasculaire qui va stimuler les dommages vasculaires, induire l'expression de molécules d'adhésion, recruter des cellules inflammatoires, et augmenter l'expression de cytokines [START_REF] Brasier | Vascular inflammation and the renin-angiotensin system[END_REF].

Dans un modèle expérimental de rat, l'infusion d'Ang II induit une hypertension associée à une forte infiltration monocytaire et une expression de VCAM-1 et MCP-1 pro-inflammatoire dans la paroi des microvaisseaux ou dans l'espace périvasculaire [START_REF] Cheng | Angiotensin II and vascular inflammation[END_REF]. Dans les cellules endothéliales, les cellules musculaires lisses vasculaires et les mononucléaires, l'Ang II induit une augmentation de l'expression de MCP-1, la principal chimiokine recrutant les monocytes/macrophages, et d'interleukine-8, un puissant chimioattractant et activateur des neutrophiles [START_REF] Yadav | MCP-1: chemoattractant with a role beyond immunity: a review[END_REF]). L'Ang II augmente aussi la production d'interleukine-6 dans les macrophages et les cellules vasculaires, et régule positivement l'expression des gènes du TNF-α et de l'interleukine-6 [START_REF] Libby | Inflammation and atherosclerosis[END_REF]. Les inhibiteurs de l'enzyme de conversion (IEC) et les antagonistes du receptuer AT1R (ARAs) réduisent l'expression des marqueurs inflammatoires, des molécules d'adhésion et des cytokines [START_REF] Mezzano | Angiotensin II and renal fibrosis[END_REF]).

L'Ang II active les cellules vasculaires et inflammatoires pour induire la sécrétion des médiateurs pro-inflammatoires qui vont recruter de nouveau mononucléaire, ce qui va encore augmenter la réponse inflammatoire contribuant à la progression de la lésion vasculaire. Les huiles de poisson peuvent limiter les cytokines pro-inflammatoires et réduire l'expression des molécules d'adhésion cellulaires chez l'homme de façon indépendante de leurs effets sur le métabolisme des eicosanoïdes [START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF].

Conclusions et perspective

En De même, des études seront nécessaires pour déterminer le potentiel curative de la formulation EPA:DHA 6:1 vis-à-vis de la dysfonction endothéliale et de l'hypertension dans des modèles d'hypertension induite par l'Ang II chez le rat (Figure 24).

Enfin, sur la base des effets vasoprotecteurs mis en évidence par la présente étude, il serait intéressant de proposer une étude clinique visant à démontrer le potentiel de la formulation EPA:DHA 6:1 à réduire la dysfonction endothéliale et les facteurs de risques cardiovasculaires chez les patients hypertendus (Figure 25).

Introduction

Pulmonary arterial hypertension (PAH) is defined by a mean pulmonary arterial pressure (mPAP) ≥ 25 mmHg at rest [START_REF] Galiè | Guidelines for the diagnosis and treatment of pulmonary hypertension[END_REF]. It is a chronic and progressive lung disease characterized by pronounced small pulmonary artery remodeling leading to chronic elevation of pulmonary vascular resistance and subsequence right ventricular failure [START_REF] Galiè | Guidelines for the diagnosis and treatment of pulmonary hypertension[END_REF](2)(3). It is also characterized by an endothelial dysfunction involving decreased NO and thrombosis [START_REF] Humbert | Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension[END_REF]. Moreover, oxidative stress and inflammatory responses have been shown to play a critical role in both human and experimental PAH (5)(6)(7). Indeed, pro-inflammatory cytokines including interleukin (IL)-1β

and IL-6 and intense lung perivascular infiltrates of macrophages and lymphocytes are observed in human idiopathic PAH [START_REF] Humbert | Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension[END_REF][START_REF] Montani | Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension[END_REF] and monocrotaline (MCT)-induced PAH (5,[START_REF] Chaumais | N-acetylcysteine improves established monocrotaline-induced pulmonary hypertension in rats[END_REF].

High levels of oxidative stress are observed in pulmonary vascular lesions of patients with severe PAH as a consequence of tissue hypoxia [START_REF] Hoshikawa | Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia[END_REF], ischemia [START_REF] Al-Mehdi | Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K +[END_REF] and possible also involving of inflammatory response [START_REF] Demarco | Contribution of oxidative stress to pulmonary arterial hypertension[END_REF][START_REF] Bowers | Oxidative stress in severe pulmonary hypertension[END_REF].

Several studies have reported that long chain polyunsaturated omega-3 fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acids (DHA, 22:6n-3) have a beneficial effect on the cardiovascular system [START_REF] Shimokawa | Dietary omega 3 polyunsaturated fatty acids augment endothelium-dependent relaxation to bradykinin in coronary microvessels of the pig[END_REF][START_REF] Auger | Potential of food and natural products to promote endothelial and vascular health[END_REF][START_REF] Alter | OMEGA, a randomized, placebo-controlled trial to test the effect of highly purified omega-3 fatty acids on top of modern guideline-adjusted therapy after myocardial infarction[END_REF]. Their potential protective effects include the antioxidant, anti-inflammatory, anti-proliferative and the endothelial formation of NO, a vasoprotective factor [START_REF] Cottin | The differential effects of EPA and DHA on cardiovascular risk factors[END_REF][START_REF] Abeywardena | Longchain n-3 polyunsaturated fatty acids and blood vessel function[END_REF][START_REF] Thioub | Effects of n-3 fatty acids and acute exercise on endothelium-dependent vasorelaxation in healthy rat aorta[END_REF][START_REF] Wang | Effect of omega-3 fatty acids supplementation on endothelial function: a meta-analysis of randomized controlled trials[END_REF][START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA: DHA 6: 1[END_REF]. Indeed EPA:DHA 6:1 a superior oméga-3 formulation caused pronounced endothelium-dependent relaxations of porcine coronary artery rings, and increased the endothelial formation of NO subsequent to the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of eNOS at Ser 1177 [START_REF] Shimokawa | Dietary omega 3 polyunsaturated fatty acids augment endothelium-dependent relaxation to bradykinin in coronary microvessels of the pig[END_REF][START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA: DHA 6: 1[END_REF].

Therefore, the aim of the present study was to determine the ability of EPA:DHA 6:1 to prevent the development of PAH using MCT-induced PAH in rats. To test this hypothesis, we investigated the chronic efficacy of oral EPA:DHA 6:1 treatment in MCT-treated rats, and, if so to characterized the underling mechanism. The MCT-induced PAH is a well-established experimental model of PAH causing similar morphologic damage as that observed in humans with idiopathic PAH [START_REF] Lalich | Pulmonary arteritis produced in rat by feeding Crotalaria spectabilis[END_REF][START_REF] Meyrick | Crotalaria-induced pulmonary hypertension. Uptake of 3H-thymidine by the cells of the pulmonary circulation and alveolar walls[END_REF]. The MCT-induced PAH is associated with endothelial cell injury followed by an inflammatory response and vascular oxidative stress [START_REF] Schultze | Chronic pulmonary hypertension--the monocrotaline model and involvement of the hemostatic system[END_REF][START_REF] Wilson | Progressive inflammatory and structural changes in the pulmonary vasculature of monocrotaline-treated rats[END_REF].

Materials and methods

Animals

Experiments were conducted according to the European Union regulations (Directive 86/609 EEC) for animal experiments, and complied with our institution's guidelines for animal care and handling. This study was approved by the Strasbourg Regional Committee of Ethics in Animal Experimentation and with the French law for the protection of animals.

The monocrotaline (MCT) of pulmonary arterial hypertension

Adult male Wistar rats (200 to 220 g) were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and maintained in a temperature-controlled room with a 12:12 light-dark cycle.

Rats were randomly divided into 4 groups: control group (n = 7), EPA:DHA 6:1 group (n = 7), MCT group (n = 13) and MCT + EPA:DHA 6:1 group (n = 13). Rats received daily by oral intake gavage 500 mg/kg/day of either EPA:DHA 6:1 (Pivotal Therapeutics, Inc Woodbridge, ON, Canada) or corn oil as control. One week after the beginning of gavage, rats received a single subcutaneous injection of either an isotonic saline solution (control groups)

or MCT (60 mg.kg -1 ). The pyrrolizidine toxic alkaloid MCT (Sigma Aldrich, Saint-Quentin Fallavier, France) was dissolved in 1 N HCl and neutralized to pH 7.4 with 1 N NaOH.

Thereafter, the oral intake of either corn-oil or EPA:DHA 6:1 was continued for 3 weeks.

Body weight was measured weekly to adjust the dose accordingly.

Echocardiographic and hemodynamic studies

Three weeks after MCT-injection, transthoracic two-dimensional, M-mode, and Doppler pulse wave images were obtained in rats anesthetized with sodium pentobarbital (50 mg/kg ip) to evaluate the progression of PAH. Both long-and short-axis views at the papillary muscle level and apical-4 chamber views were done with a Sonos 5500 (Philips, USA) equipped with 12-MHz sectorial transducer.

M-mode measurements

Pulmonary artery diameter is obtained in the parasternal long-axis. The left ventricular (LV)

and right ventricular (RV) end-diastolic and end-systolic diameters were measured in the parasternal short axis view. The fractional area change (FAC) for the RV was measured at the apical 4-chamber view and calculated as [(end-diastolic -end-systolic area)/end-diastolic area] ( 27). Cardiac output and stroke volume were obtained from the B-mode long axis according to Simpson's method [START_REF] Lang | Recommendations for chamber quantification[END_REF].

Doppler imaging

Pulse-wave Doppler of pulmonary outflow was recorded in the parasternal long-axis view at pulmonary valve leaflets. In addition, to characterize the pulmonary outflow Doppler envelope, the pulmonary artery acceleration time (PAAT) and the velocity time integral (VTI)

were measured. The VTI was obtained by tracing the outer edge of the pulmonary outflow Doppler profile. PAAT was measured from the time of onset of systolic flow to peak pulmonary outflow velocity. The tricuspid valve was used to determine the tricuspid regurgitation velocity (TR) with color flow and pulsed-wave Doppler in the apical 4-chamber view so that the tricuspid and mitral valves could be clearly visualized. If TR was observed, the transducer was aligned to achieve the maximal peak velocity. The RV systolic pressure was calculated using the peak TR velocity (Vmax) in the modified Bernoulli (RVSP = 4 × Vmax 2 ) [START_REF] Lindqvist | Echocardiography in the assessment of right heart function[END_REF][START_REF] Daskalov | Guidelines for the Echocardiography Assessment of the Right Heart in adults: a report from the American Society of Echocardiography endorsed by the European Association of Echocardiography, a registered branch of the European Society of Cardiology, and the Canadian Society of Echocardiography[END_REF]. The pulmonary vascular resistance was calculated as [PVR = Vmax (m/s)/VTI (cm)] of flow wave of pulmonary artery. Right atrial area from the 4-chamber apical view and the inferior vena cava diameter and collapsibility were measured. All measurements and calculated indexes are presented as the average of three cardiac cycles.

Hemodynamic Measurements and Tissue Preparation

Following echocardiography, the mean pulmonary arterial pressure (mPAP) was monitored with a heparinized saline catheter inserted into the RV through the jugular vein and placed in the lumen of the pulmonary artery. The systemic arterial blood pressure was monitored with a pressure catheter inserted into the femoral artery, and steady-state hemodynamic was recorded using a blood pressure transducer (EMKA Technologie, Paris, France).

Subsequent to hemodynamic measurements, rats were euthanized and the hearts and lungs were isolated, the left lungs lobe and some group of hearts were in fixated in 4% paraformaldehyde for 48 h for morphology study. The RV and right lungs were embedded in Tissue-Tek ® O.C.T. and snap-frozen on liquid nitrogen and quantitative as immunofluorescence and Western blot analysis.

Assessment of right ventricular hypertrophy and remodeling

Subsequent to hemodynamic measurements, rats were euthanized and the hearts were isolated to separate the right ventricle (RV) wall from the left ventricle wall and the septum (LV + S) and then weighed. The RV hypertrophy was assessed by the weight ratio of RV to LV plus the septum [RV/LV + S] as a Fulton's index (Mam et al. 2010). Cardiomyocytes crosssectional area and interstitial collagen content were determined in the RV and LV to determine cardiac ventricle tissue remodeling. Four μm sections of heart were stained with hematoxylin and eosin (H-E) or Gomori's trichrome to assess fibrosis. 50 cardiomyocytes in each ventricle per rat were determined on transversely cut myocardial.

Assessment of pulmonary arteriolar wall thickness

The left lung lobe from each rat was isolated, harvested and perfused via the trachea with a 4% paraformaldehyde solution, and then immersed in the fixative solution for 48 h. Following dehydration, lungs were embedded in paraffin blocks and cut into 4 μm thick sections. Lungs sections were stained with H-E for morphology analysis (Olympus camera and microscope).

The images of terminal arterioles were captured with magnification 20X and measured using ImageJ Software. Pulmonary vascular remodeling was evaluated by determining the percentage wall thickness (%WT) in H-E stained sections and wall areas were measured in smooth muscle α-actin-stained sections (% WA). A minimum of 16 arterioles of comparable size (< 50 μm) per lung sections were examined for each group. The percent WT of pulmonary arterioles was calculated as follows: % WT = [(2 x medial thickness/external diameter) x 100] and the percent wall area, %WA = [(vessel wall area /vessel lumen ratio) x 100].

Immunohistochemistry

The antibodies against smooth muscle α-actin (mouse monoclonal, 1:20000; Santa Cruz) to assess the degree of muscularization of small peripheral pulmonary arterioles, CD68 (mouse monoclonal [ED1], 1:1000; Abcam) for macrophages and CD3 (rabbit monoclonal [SP7],

1:100; Abcam) for lymphocytes staining were used for this study. Microwave antigen retrieval (10 mM citrate buffer, pH 6.0) of 4 μm paraffin sections was followed by incubation in blocking buffer. Endogeneous peroxidases were blocked (3% H2O2 for 10 min) before incubation with a primary antibody overnight at 4°C. Thereafter, sections were incubated with a secondary biotinylated antibody for 2h, and then streptavidin-biotin-peroxidase complex linked to HRP (Vectastain Elite ABC kit, Vector Laboratories, AbCys, Paris, France) for 30 min. VIP peroxidase substrate kit (Vector Laboratories) was used as chromogen. Sections were counterstained with methyl green, air-dried and cover slipped with Eukitt (Labonord, Templemars, France). Ten photographs were taken from each lung sample, and the fraction area occupied by the macrophages and the number of lymphocytes was evaluated from each photograph using the ImageJ software (National Institutes of Health, http://rsweb.nih.gov/ij/).

Immunofluorescence studies

Right lung lobes were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek, Villeneuve d'Ascq, France), frozen in liquid nitrogen bath and cryosectioned at 14 μm. Lung sections were first fixed with 4% paraformaldehyde, washed and treated with 5% bovine serum albumin in PBS containing 0.1% Triton X-100 for 1 h at room temperature to block non-specific binding. Lung sections were then incubated overnight at 4°C with an antibody directed against either eNOS (mousse monoclonal, 1/1000, Santa Cruz), angiotensin II type 1 receptor (rabbit polyclonal AT-1, 1/500; Santa Cruz), endothelin-1 type A and B receptors (rabbit polyclonal ETA, ETB, 1/1000; Abcam), cyclooxygenase (rabbit monoclonal COX-1 and COX-2, 1/1000, Abcam) or the NADPH oxidase subunits (p22 phox and p47 phox , 1/500, Santa Cruz). Sections were then washed with PBS, incubated with the secondary antibody (1/400, immunoglobulin G coupled to Alexa 488-or 633) for 2h at room temperature in the dark before being washed with PBS and mounted in Dako fluorescence mounting medium (Dako France SAS, Les Ulis, France) and cover-slipped. All samples for immunofluorescence studies were observed using a confocal laser-scanning microscope (Leica SP2 UV DM IRBE;

Leica, Heidelberg, Germany) with a 20X magnification lens. Quantification of fluorescence levels was performed using the ImageJ software.

Determination of vascular oxidative stress

The redox-sensitive fluorescent dye dihydroethidium (DHE, 2.5 μM) was applied onto 25 μm unfixed lung cryosections for 30 min at 37°C in a light protected humidified chamber to determine the in situ formation of ROS. To characterize the source of ROS, sections were incubated with either N-acetylcysteine (NAC, antioxidant 1 mM,), VAS-2870 (VAS, NADPH oxidase inhibitor, 10 μM), N-nitro-L-arginine (L-NA, NO synthase inhibitor, 300 μM), indomethacin (Indo, cyclooxygenase inhibitor, 10 μM), or MRK (inhibitors of the mitochondrial respiration chain ( myxothiazol, 0.5 μM + rotenone, 1 μM + potassium cyanide (KCN), 1 μM) for 30 min at 37°C before DHE staining. Sections were then washed three times, mounted in DAKO and cover-slipped. The level of fluorescence in each section was examined under a confocal laser-scanning microscope (Leica SP2 UV DM IRBE; Leica, Heidelberg, Germany) with a 20X magnification lens. Quantification of fluorescence levels was performed using the ImageJ software.

Evaluation of O2 -and NO levels

The levels of NO and O2 -in lungs was determined by electron paramagnetic resonance (EPR)

in frozen tissues at liquid nitrogen temperature.

Nitric oxide (NO) determination

Lung samples were incubated for 30 min in Krebs-Hepes buffer containing bovine serum albumin (20.5 g/L), CaCl2 (3 mM), and L-arginine (0.8 mM). NaDETC (3.6 mg) (DETC: diethyldithiocarbamate) and FeSO4, 7H2O (2.55 mg) were separately dissolved under nitrogen gas bubbling in 10-mL volumes of ice-cold Krebs-Hepes buffer. These compounds were rapidly mixed to obtain a pale yellow-brown opalescent colloid Fe(DETC)2 solution (0.4 mM), which was used immediately. The colloid Fe(DETC)2 solution was added to the incubation solution of lungs for 45 min at 37°C. The level of NO was determined using a table-top x-band spectrometer Miniscope (Magnettech, MS200, Berlin, Germany). The quantification of the signal is based on the mean of the height (amplitude) of the three signals.

Values are expressed in signal amplitude (amplitude, arbitrary units).

Superoxide anion (O2 -)

Lung samples were allowed to equilibrate in deferoxamine-chelated Krebs-Hepes solution containing 1 hydroxy-3 methoxycarbonyl 2,2,5,5-tetramethylpyrrolidin (CMH, Noxygen, Germany,500 μM), deferoxamine (25 μM), and DETC (5 μM) under constant temperature (37°C) for 1 h. The reaction was stopped by freezing the samples in liquid nitrogen before EPR spectroscopy analysis. Values were expressed as arbitrary units per milligram weight of dried tissue (A/Wd).

Vascular reactivity studies

Vascular reactivity studies are performed in main pulmonary artery and secondary mesenteric artery rings. Briefly, the arteries were excised, carefully cleaned of connective tissue in Krebs bicarbonate solution and cut into rings (3 mm length). Rings were suspended in organ chambers 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,pH 7.4 at37°C) for the determination of changes in isometric tension. After an equilibration period, rings were subjected to functional test before construction of either a concentrationcontraction curve to phenylephrine, or a concentration-relaxation curve to acetylcholine on rings precontracted with phenylephrine (10 μM). The concentration-response curves were constructed both in absence or presence of an eNOS inhibitor (L-NA, 300 μM) to assess the role of the basal endothelial NO formation.

Statistical analysis

All values are expressed as the mean ± SEM of n different rats. Statistical analysis was performed using either a one-way or a two-way analysis of variance test (ANOVA), followed by Bonferroni's post-hoc test as appropriate using GraphPad Prism software (version 5.04 for Windows, GraphPad software, Inc., San Diego, CA, USA). A P value < 0.05 was considered to be statistically significant.

RESULTS

EPA:DHA 6:1 prevents the MCT-induced increase in mPAP and RVSP Three weeks after MCT injection, hemodynamic parameters were monitored by echocardiography and right ventricular catheterization. MCT-treated rats consistently developed pulmonary hypertension associated with a significant increase in mPAP from 16 ± 0.3 to 35.2 ± 0.7 mmHg and RVSP from 17.4 ± 0.5 to 40.5 ± 1.9 mmHg (Fig. 1A,B). In addition, CO was significantly decreased in the MCT group compared with the control group (124 ± 7.4 to 67.7 ± 3.7 ml/min) (Figure 1C). Oral intake of EPA:DHA 6:1 significantly reduced mPAP to 29.6 ± 0.8 mmHg and RVSP to 31.9 ± 0.8 mmHg and also improved CO to 90.2 ± 4.4 mL/min (Fig. 1A,C).

The heart rate was slightly but significantly lowered from 392.7 ± 8.1 bpm to 333.2 ± 4.9

bpm by the MCT treatment and the mean systemic arterial pressure is not significantly different in the four groups (Fig. 1D,E). Right ventricular hypertrophy was assessed by the weight ratio of RV/(LV+S). The MCT group developed severe RV hypertrophy manifested by an RV/(LV+S) ratio of 0.54 ± 0.03 compared with 0.22 ± 0.01 in the control group (Fig. 2E). Similarly, the ratio of RV weight to body weight was significantly higher in the MCT-treated group compared to the control group. The marked MCT-induced RV hypertrophy was reduced significantly to 0.33 ± 0.01 by the EPA:DHA 6:1 treatment indicating that chronic oral intake of EPA:DHA 6:1 was able to prevent PAH-induced RV hypertrophy.

EPA

EPA:DHA 6:1 prevents the MCT-induced RV cardiomyocytes hypertrophy and macrophages infiltration

In the RV, representative H-E staining showed that the MCT treatment induced a significant cardiomyocytes hypertrophy as compared to the control group (Figure 3A). The RV cardiomyocytes hypertrophy was associated with enhanced perivascular infiltration of macrophages detected by anti-CD68 staining (Figure 3B). The EPA:DHA 6:1 treatment prevented the MCT-induced RV cardiomyocytes hypertrophy by restored RV area and reduced infiltration of macrophages (Fig. 3A,B). In contrast, in the left ventricle, cardiomyocytes size was similar in all group studied, and only a low level of infiltration of macrophages was observed.

EPA:DHA 6:1 prevents MCT-induced pulmonary arterial remodeling

Pulmonary vascular remodeling was assessed by pulse-wave Doppler of pulmonary outflow.

MCT significantly increased the pulmonary artery diameter and pulmonary vascular resistance (control vs. MCT: 0.37 ± 0.03 vs. 0.84 ± 0.05 unite wood), indicating hypertrophy and stiffness of the pulmonary artery, and reduced the velocity-time integral (VTI) and pulmonary artery acceleration time (43.05 ± 2.39 vs. 26.06 ± 0.91 ms), indicating an increased pulmonary arterial pressure. EPA:DHA 6:1 treatment significantly prevented the MCT-induced changes on pulmonary artery diameter, PVR, PAAT, and pulsatility (Fig. 4).

Indeed, to examine the level of pulmonary vascular remodeling, morphometric analysis was performed on lung tissue sections stained with H-E or α-actin. Quantitative morphometric analyses showed pulmonary arterioles remodeling in MCT group (Figure 5). The medial thickness of pulmonary arterial was markedly increased in arterioles with external diameter < 50 μm in the MCT group (control vs. MCT: 25.9 ± 0.6 vs. 73.7 ± 0.5). The medial wall hypertrophy was accompanied with muscularization of small pulmonary arteries evidenced by enhanced α-actin staining (Fig. 5). The EPA:DHA 6:1 treatment significantly attenuated the MCT-induced increase in wall thickness and pulmonary arterioles muscularization. In addition, an increased collagen deposition in MCT group as assessed by Gomori's Trichrome, which was prevented by the EPA:DHA 6:1 treatment (Fig. 5).

To determine the perivascular inflammatory cell infiltration, immunohistochemical staining was performed in lung sections with CD68 for macrophages and CD3 for T lymphocytes. The MCT-treatment was associated with a significantly macrophages and lymphocytes infiltration surrounding pulmonary remodeled arterioles. The EPA:DHA 6:1 treatment significantly prevented the MCT induced infiltration of macrophages and lymphocytes (Fig. 5B). To obtain further evidence for a role of eNOS, NADPH oxidase and COXs. Their expression level was determined by immunofluorescence staining in the pulmonary arterioles. A significantly increased immunofluorescence signal of the NAPDH oxidase subunits p22 phox , p47 phox , and of COX-1 and COX-2 was observed in pulmonary arterioles of the MCT group compared to the control group (Fig. 7). The EPA:DHA 6:1 treatment significantly reduced the MCT-induced stimulatory effect for p22 phox , p47 phox , COX-2 and COX-1 (Figure 7A). In addition, an upregulation of eNOS was observed in the MCT group which was prevented by the EPA:DHA 6:1 treatment (Fig. 7).

EPA

EPA :DHA 6 :1 prevents the expression of endotheline-1 receptors (ETA, ETB) and angiotensin II receptor on pulmonary arterioles wall.

It has been suggested that PAH is associated with the increased expression of endothelin-1 [START_REF] Prie | EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension[END_REF] and angiotensin II [START_REF] Demarco | Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat[END_REF]

DISCUSSION

The present study demonstrated that the optimized EPA:DHA 6:1 formulation prevents the development of PAH in MCT-treated rat. Since, rats were treated with EPA:DHA 6:1 one week before the induction of PAH, the findings indicate the capacity of the omega-3 products to prevent the disease and not to cure it. The MCT-induced PAH rat model has been widely

used as an experimental model of PAH [START_REF] Homma | Genistein, a phytoestrogen, attenuates monocrotaline-induced pulmonary hypertension[END_REF] and it is tough to the most similar animal model to the human form of the disease [START_REF] Robbins | Advancing therapy for pulmonary arterial hypertension: can animal models help? American journal of respiratory and critical care medicine[END_REF]. It is characterized by pulmonary endothelial cell damage, associated with an inflammatory response and vascular oxidative stress. It also leads to small pulmonary arterial and RV remodeling and dysfunction [START_REF] Stenmark | Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure[END_REF][START_REF] Huang | Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced pulmonary artery hypertension[END_REF]. Previous studies focused on the effects of omega-3 on the systemic circulation but provided little information on its pulmonary effects.

The presents findings indicate that daily oral treatment with EPA:DHA 6:1 significantly prevented an elevation of mean pulmonary arterial pressure, RV systolic pressure and improved cardiac output in rats model of MCT-induced PAH. EPA:DHA 6:1 treatment also prevented pulmonary arterioles remodeling and dysfunction, RV hypertrophy and dilation, attenuated oxidative stress and inhibited inflammation. Our studies show that EPA:DHA 6:1 exerts anti-inflammatory, antioxidant and antiproliferative effects in the pulmonary arteries, which may contribute to prevent pulmonary hypertension. Recent study showed that DHA therapy reduced mPAP in a rat model of hypoxia-induced PAH and this effect was linked with inhibition of pulmonary vascular remodeling [START_REF] Chen | Effects of docosahexaenoic acid on hypoxiainduced pulmonary arterial hypertension[END_REF].

Echocardiography is widely used in the evaluation of PAH in the rat MCT model [START_REF] Howard | Echocardiographic assessment of pulmonary hypertension: standard operating procedure[END_REF]. The echocardiography analysis of control and MCT groups indicates that oral treatment of EPA:DHA 6:1 prevented in the pulmonary arteries of the MCT-treated rats the elevation of vascular resistance and the reduction of pulmonary acceleration time compared to control rats, suggesting that this formulation has the beneficial effect of reducing the stiffness of the pulmonary artery. In addition, oral treatment with EPA:DHA 6:1 significantly prevented the MCT-induced RV dilatation as indicated by increased RV area and decreased RV fractional area change (RVFAC), suggesting a beneficial effect of EPA:DHA 6:1 to reduce RVSP. The FAC is an ideal echocardiography parameter reflecting RV dysfunction and disease severity in PAH [START_REF] Kimura | Evaluation of right ventricle by speckle tracking and conventional echocardiography in rats with right ventricular heart failure[END_REF]. Oral treatment with EPA:DHA 6:1 prevent RV hypertrophy characterized by increased RV/(LV + S) ratio, cardiomyocytes hypertrophy and extracellular matrix changes with fibrosis.

Previous studies have been revealed that inflammation plays a key role in human PAH as well as in experimental models including MCT-induced PAH (7,[START_REF] Dorfmuller | Inflammation in pulmonary arterial hypertension[END_REF] . In response to injury and stress, a pronoucial lung vascular inflammatory response is observed including macrophages, monocytes, lymphocytes and mast cells [START_REF] Voelkel | Primary pulmonary hypertension between inflammation and cancer[END_REF][START_REF] Huertas | Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: a complex interplay[END_REF] and they have been involved in the initiation of pulmonary vascular remodeling by matrix remodeling, collagen deposition, and vascular cell proliferation and migration in PAH [START_REF] Hassoun | Inflammation, growth factors, and pulmonary vascular remodeling[END_REF][START_REF] Pullamsetti | Inflammation, immunological reaction and role of infection in pulmonary hypertension[END_REF]. These process lead to increased pulmonary resistance and right heart failure. Consistent with these previous findings, a substantial increased number of CD68 (ED-1) and CD3 + positive cells were observed in the lungs of MCT-treated rats. In addition an increased pulmonary arterial medial thickness and RV cardiomyocytes hypertrophy were observed and associated with an increased the number of macrophages in perivascular intra-alveolar spaces, and of lymphocytes around the pulmonary arteries in the MCT-treated rats relative to the control group. Importantly, oral intake of EPA:DHA 6:1 significantly prevented the stimulatory effect of MCT on macrophages and T lymphocytes infiltration in the lung and right ventricle. Thus, these findings suggest that the EPA:DHA 6:1 formulation prevented MCT-induced PHA by reducing the inflammatory responses.

In addition to the inflammatory response, oxidative stress has been implicated in the pathogenic mechanism underling the vascular remodeling and heart failure observed in pulmonary hypertension [START_REF] Bowers | Oxidative stress in severe pulmonary hypertension[END_REF][START_REF] Demarco | Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat[END_REF][START_REF] Barman | NADPH oxidase 4 is expressed in pulmonary artery adventitia and contributes to hypertensive vascular remodeling[END_REF]. Dysregulation of the pro-oxidant/antioxidant balance contributes to impair vascular tone and the pathological activation of anti-apoptotic and mitogenic pathways, leading to cell proliferation and obliteration of the vasculature [START_REF] Tabima | Reactive oxygen and nitrogen species in pulmonary hypertension[END_REF].

There is solid evidence in MCT-treated rats, that oxidative injury to the pulmonary vascular Thus, it could be hypothesized that EPA:DHA 6:1-induced elevation of cGMP. It is possible that up-regulation of eNOS contributes to the therapeutic action of omega-3. Furthermore, EPA:DHA 6:1 has been shown to stimulate the endothelial release of NO and to improve endothelial function subsequent to phosphorylation of PI3K/Akt/eNOS signaling pathway [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA: DHA 6: 1[END_REF].

The endothelin (ET) system is activated in human pulmonary hypertension (PH) of various pathogeneses [START_REF] Jasmin | Effectiveness of a nonselective ETA/B and a selective ETA antagonist in rats with monocrotaline-induced pulmonary hypertension[END_REF][START_REF] Vincent | Relation of elevated plasma endothelin in congenital heart disease to increased pulmonary blood flow[END_REF]. ET-1 could contribute to the development of human PH through its strong vasoconstrictive and promitogenic properties [START_REF] Giaid | Expression of endothelin-1 in the lungs of patients with pulmonary hypertension[END_REF]. Previous studies have shown enhanced ET-1-induced pulmonary vasoconstriction and vascular resistance in the MCT-PAH [START_REF] Nikitopoulou | Vascular endothelial cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension[END_REF] and increased vasomotor tone in hypoxic PAH (5,[START_REF] Demarco | Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat[END_REF]. The present findings support a role for the ET-1 in the MCT-induced endothelial dysfunction. Indeed, an increased expression of ETA and ETB receptors was observed throughout the arterial wall of the MCT treatment group. The ETA receptors are located on smooth muscle cells, where they mediate vasoconstrictive and proliferative effects [START_REF] Montani | Endothelin-1/endothelin-3 ratio: a potential prognostic factor of pulmonary arterial hypertension[END_REF]. The ETB receptor is the only subtype found predominantly on the vascular endothelium, where it promotes vasodilation through the release of nitric oxide and prostacyclin [START_REF] Prie | EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension[END_REF]. There is also evidence that the ETB receptor indirectly modulates ET-1 synthesis through negative feedback under the action of nitric oxide [START_REF] Jasmin | Effectiveness of a nonselective ETA/B and a selective ETA antagonist in rats with monocrotaline-induced pulmonary hypertension[END_REF]. They are also consistent with the fact that chronic bosentan, a non-selective ET-1 receptor antagonist reduced pulmonary hypertension in MCT-treated rats and prevented the MCT-induced endothelial dysfunction and oxidative stress [START_REF] Prie | EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension[END_REF][START_REF] Hill | Nonspecific endothelinreceptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats[END_REF]. Our data show that EPA:DHA 6:1 prevented the MCT-induced upregulation of the expression ETA and ETB receptors in MCT-induced PAH rats.

However, the mechanism explaining best the beneficial effect of omega-3 treatment to prevent pulmonary hypertension most likely includes its antioxidant, anti-inflammatory and endothelial protective properties. Two previous studies have demonstrated potential benefits of oral administration of MAG-DPA to reduce inflammation (decreased NF-kB and p38

MAPK activation) and proliferation (reduction in MMP-2, MMP-9 and VEGF expression levels in lung tissue homogenates) of pulmonary artery smooth muscle cells in MCT-treated rats [START_REF] Morin | Docosapentaenoic acid monoacylglyceride reduces inflammation and vascular remodeling in experimental pulmonary hypertension[END_REF] and DHA to inhibit the development of hypoxic pulmonary hypertension in vitro and in vivo studies [START_REF] Yan | Docosahexaenoic acid inhibits development of hypoxic pulmonary hypertension: in vitro and in vivo studies[END_REF]. The effects of EPA and DHA have not be investigated in PAH; however, these two fatty acids affect cell function differently [START_REF] Gorjao | Comparative effects of DHA and EPA on cell function[END_REF][START_REF] Dhein | Antiarrhythmic and electrophysiological effects of long-chain omega-3 polyunsaturated fatty acids[END_REF].

Moreover, the beneficial role of omega-3 in cardiovascular health is supported by its metabolism. Indeed, omega-3, unlike omega-6, decreases the production of pro-inflammatory cytokines and leukocyte reactive oxygen species, increase the synthesis of anti-inflammatory cytokines, lead to the release of resolvins and modulate the activation of genes involved in the inflammatory process [START_REF] Calder | Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology?[END_REF][START_REF] Mozaffarian | Omega-3 fatty acids and cardiovascular disease: effects on risk factors, molecular pathways, and clinical events[END_REF]. Furthermore, when administrated per os, omega-3 represents a stable compound that could serve as a precursor to generate proresolving (resolvins and protectins) molecules, which are known for their anti-inflammatory properties [START_REF] Aoki | Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma[END_REF][START_REF] Serhan | Resolvins and protectins in inflammation resolution[END_REF].

Study limitation

This In presence of L-NA (300 μM)

In presence of L-NA (300 μM)

Introduction

Hypertension is a major public health problem (WHO, 2013). In chronic situation, hypertension increases the risk factor for cardiovascular diseases including atherosclerosis, coronary disease, congestive heart failure and stroke [START_REF] Kannel | Risk stratification in hypertensive: New insights from the Framinghan study[END_REF]Tsotetsi et al., 2001;[START_REF] D'agostino | General cardiovascular risk profile for use in primary care: the Framingham heart study[END_REF]. For a better understanding of hypertensive disease physiopathology, several animal models such as spontaneously hypertensive rats (SHR), angiotensin II (Ang II)-induced hypertension, Dahl salt-sensitive hypertensive rats, DOCA-salt rats and other models are usedas a mimetic of human essential hypertension [START_REF] Galisteo | Effects of quercetin treatment on vascular function in deoxycorticosterone acetate-salt hypertensive rats.Comparative study with verapamil[END_REF]Wilcox and Pearlman, 2008). A DOCA-salt model isa characteristic of human volume-overload induced hypertension with sodium retention [START_REF] Galisteo | Effects of quercetin treatment on vascular function in deoxycorticosterone acetate-salt hypertensive rats.Comparative study with verapamil[END_REF]Iyer et al., 2010 ), obtainedthrough several weeks administration of a synthetic mineralocorticoid deoxycorticosterone acetate (DOCA) to rat allowed free access to water containing NaCl.This model isalso associate to a low rennin and potassium-depleted [START_REF] Galisteo | Effects of quercetin treatment on vascular function in deoxycorticosterone acetate-salt hypertensive rats.Comparative study with verapamil[END_REF]Liu et al., 2014).

Almost deleterious effects found in human hypertension are observed in DOCA-salt hypertensive rat as a model ofendothelial dysfunction, cardiac hypertrophyand renal damage, and inflammation [START_REF] Fenning | L-Arginine attenuates cardiovascular impairement in DOCA-salt hypertensive rats[END_REF].These phenomena are related to an excessive production of reactive oxygen species (ROS) by NADPH oxidase [START_REF] O'brien | L-Carnitine attenuates cardiac remodelling rather than vascular remodelling in deoxycorticosterone acetate-salt hypertensive rats[END_REF]. Left ventricular hypertrophy is found in most animal models of hypertension (Perez-Vizcaino et al., 2009). Numerous studies have indicated that endothelial dysfunction in pathological modelswas linked to animpairment of endothelium-derived relaxing factors mediatedrelaxations, such as NO and EDH component [START_REF] Li | Upregulation of heme oxygenase-1 potentiates EDH-type relaxations in the mesenteric artery of the spontaneously hypertensive rat[END_REF][START_REF] Rashid | Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system[END_REF].

Hypertension management required the use of classic antihypertensive drug agents such as diuretics, beta-blockers, angiotensin converting enzyme inhibitors or angiotensin II type 1 receptor (AT1R) blockers, and calcium channel blockers. Since efficacy of antihypertensive inhibition of iNOS, COX-2, and cytokines via the NF-kB pathway [START_REF] Kiemer | Phyllanthus amarus induced a potent anti-inflammatory effect resulting by inhibition of iNOS, COX-2, and cytokines via the NF-kB pathway[END_REF]. The antihypertensive activity has been shown to be related to stimulation of muscarinic receptor and involving endothelium-derived nitric oxide (NO) and also to blocking of calcium channel (Amaechina et al., 2007;[START_REF] Amonkan | Comparative effects of two fractions of Phyllanthus amarus (Euphorbiaceae) on the blood pressure in rabbit[END_REF]. Little data are available about cardioprotective and antihypertensive effects of P. amarus in chronic hypertension. Therefore, the aim of the present study was to determine if an aqueous extract of P. amarus (AEPA)

obtained by decoction is able to prevent hypertension, cardiac hypertrophy and endothelial dysfunction in DOCA-salt hypertensive rats, and, if so, to clarify the underlying mechanism.

Materials and Methods

Plant material and extraction procedure

The whole plant of Phyllanthus amarus (Euphorbiaceae) was collected in the district of Cocody (Abidjan, Côte d'Ivoire) in April 2014. The plant is registered under No. 3, 141 and 248 at the Centre National de Floristique (CNF). Extraction procedure was similar to a method previously described [START_REF] Amonkan | Comparative effects of two fractions of Phyllanthus amarus (Euphorbiaceae) on the blood pressure in rabbit[END_REF]. Briefly, the whole plant was harvested, washed, and extracted in boiling distilled water for 30 min at ratio of 500 g of plant for 1 liter.

The decoction was filtered and then lyophilized to obtain a powder of aqueous extract of Phyllanthus amarus (11.72 g from 1 kg of plant).

Animals and experiments groups

Experiments were performed in accordance to the Guidelines for experiments involving animals (McGrath et al., 2010).Male Wistar rats (Janvier, Le Genest-Saint-Isle, France)

weighing between 180 and 200 g were maintained under standard laboratory conditions (21-22 °C) with dark and light cycle (12/12 h) and had free access to a standard dry pellet diet from Scientific Animal Food and Engineering (SAFE, France) and water ad libitum. Animals were randomly assigned 7-8 rats per group, into the control group, the AEPA group (100 mg/kg/day, by gavage), the DOCA-salt group (50 mg/kg, s.c, per week), and the DOCA-salt + AEPA groups (100 or 300 mg/kg/day, by gavage). DOCA-salt-treated rats were allowed free access to water containing 1% NaCl.

Blood pressure measurements

Prior to start the blood pressure monitoring, rats were subjected to a period (one week) of adaptation to the system. Systolic blood pressure (SBP) was determined by tail-cuff sphingomanometry twice weekly during 5 weeks using blood pressure analysis system (Bioseb, Vitrolles, France). Blood pressure was monitoredat 9 a.m.,and at least 12 determinations were made for each session.

Echocardiographic studies

After the 5 weeks treatment, the rats were anaesthetized by intraperitoneal injections of pentobarbital (50 mg/kg). Cardiac structure and function were determined by echocardiography transthoracic using the Phillips Sonos 5500 machine equipped with a probe

Assessment of cardiac and kidney weight indices

At the end of study, rats were prior weighed, anaesthetized by intraperitoneal injections of pentobarbital (50 mg/kg) and sacrificed. Blood was taken directly by cardiacpuncture. The heart and the left kidney, werecarefully taken, cleaned, and then weighed. The heart was after separation into left and right ventriclerespectively,also weighed. Thereafter, cardiac and renal weight ratios were determined.

Biochemical analysis

After taken blood pressure, it was put into heparinized tubes and, thereafter the plasma was obtained by centrifugation 1500 g for 15 min. Plasma aliquots were stored at -80°C for subsequent determination of the electrolyte content, urea and uric acid.

Vascular studies

Vascular studies were performedusing main mesenteric artery rings according to a similar method described previously in our team [START_REF] Lee | An ethanolic extract of Linderaobtusiloba Stems, YJP-14, improves endothelial dysfunction, metabolic parameters and physical performance in diabetic db/db mice[END_REF].Briefly,rings were contracted with 1 μM of phenylephrine (PE) before the application of increasing concentrations of acetylcholine (ACh)ranging from0.1 nM -10 μM to construct concentration-response curves.

In some experiments, rings were exposed to an inhibitor for 30 min before contraction with GraphPad software, Inc, San Diego, CA, USA). The difference was considered to be significant when the P <0.05.

Results

Intake of AEPA improves the DOCA-salt-induced hypertension

After 5 weeks, the SBP (mmHg) increased significantly in DOCA-salthypertensive rats, compared to the control group (P < 0.05; Fig. 1). Daily oral administration with AEPA (100 or 300 mg/kg/day) significantly prevented the increasing of SBP observed in DOCA-saltrats by 24 and 21 mmHg, respectively at the end of treatment. The SBP of animal receiving only AEPA (100 mg/kg/day) remained unaffected compared to the control groups (P >0.05; Fig. 1).

Effect of AEPA treatment in cardiac structure and function

Transthoracic echocardiography revealed that after 5 weeks, DOCA-salthypertensive rats developed an important modification of cardiac structure, marked by left ventricular hypertrophy, compared to the control group. We observed asignificant increase of PWT (1.58± 0.02 mmversus 2.24±0.05 mm in the control and DOCA-salthypertensive group respectively; Fig. 2A) and SWT (1.54±0.03 mm versus 1.77±0.05 mm in the control and DOCA-salt hypertensive group respectively; Fig. 2B). The LVM was also significantly greater in DOCA-salt hypertensive group than control (1103.61±40.13mg versus 755.29±24.66 mg; Fig. 2C). No significant modification of LVEDD was observed (P >0.05; Fig. 2D). The AEPA treatment (100 or 300 mg/kg/day) reduced significantly the increase of PWT by 0.36 mm and 0.30 mm respectively,SWT by 0.18 mm and 0.17 mm, LVM by 268.55mg and 251.61mg respectively. Parameters for AEPA (100 mg/kg/day) remained unaffected compared to the control groups (P >0.05).

Concerning cardiac function, our results show that, in DOCA-salt rats, IVRT was significantly increased compared to the control (35.5± 2.83ms versus 45.71±1.96 ms, respectively; Fig. 3A). AEPA treatment (100 or 300 mg/kg/day) improves these parametersat a value close to normal, 36.67±3.47 and 36.88± 2.37 ms respectively (Fig. 3A). No significant difference was observed between rats treated only with AEPA (100 mg/kg/day) and control group. The ejection fraction was affected slightly but not significantly in DOCA-salt rats compared to the control (P >0.05; Fig. 3B).

Analyse of morphological parameters

Post mortem morphometric analysis of organ shows that except right ventricularDOCA-salt rats increase significantly the cardiac (3.4 ± 0.23 versus 2.73 ± 0.08), left ventricular (1.34 ± 0.11 versus 1.04 ± 0.05) and , left kidney (3.71 ± 0.18 versus 3.18 ± 0.12) weight indices, as compared respectively to the control group (Table1). The cardiac and kidneyweight indices were reduced in the DOCA-salt + AEPA groups (100 or 300 mg/kg/day), compared to the DOCA-salt rats (P ˂ 0.05; Table 1). These parameters remained similar between AEPA (100 mg/kg/day) group and control rats (P > 0.05; Table 1).

Effect of DOCA-salt on plasma electrolyte levels DOCA-salt treatment increases the plasma uric acid level (11.25 ± 0.48 in DOCA-salt rats versus 7.00 ± 1.00 in control; Table 2) and decrease the plasma potassium level (4.18 ± 0.08 versus 5.54 ± 0.2 in the control; Table 2). EAPA treatments (100 or 300 mg/kg/d) normalize the plasma levels of potassium (5.26 ± 0.3 and 5.14 ± 0.3 respectively; Table 2) and uric acid level (6.67 ± 0.52 and 8.0 ± 0.50 respectively; Table 2).

At the end of experiment, the plasma sodium level in DOCA-salt rat increase slightly, but not significantly compare to the control group (147.50 ± 0.65 in DOCA-rats versus 143. 20 ± 1.11 in control; Table 2). The plasma chlorine and urea levels remained also unaffected (Table 2).

AEPA improves the DOCA-salt-induced endothelial dysfunction in the mesenteric artery

In mesenteric artery rings with endothelium, DOCA-salt treatment increase significantly phenylephrine-induced vasoconstriction compared to the control rats (P ˂ 0.05; Fig. 4A). In the DOCA-salt + AEPA groups (100 or 300 mg/kg/day), the phenylephrine inducedcontractile response was shifted to the right (Fig. 4A). The significant effect was obtained with AEPA at dose of 300 mg/kg/day (P ˂ 0.05; Fig. 4A).

DOCA-salt group, present an endothelial dysfunction, marked by a significant reduction ofacetylcholine induced endothelium-dependent vasorelaxation, compared to the control group (P ˂ 0.05; Fig. 4B).Treatment with AEPA (100 or 300 mg/kg/day) improve these endothelial dysfunction (P ˂ 0.05; Fig. 4B).

AEPA improves the DOCA-salt-blunted NO-and EDH-mediated relaxations in the mesenteric artery

In mesenteric artery rings with endothelium, both the NO-mediated (assessed in the presence of indomethacinand Tram-34 plus Apamin) and the endothelium-dependent hyperpolarization (EDH)-mediated (assessed in the presence of indomethacinand N ω -nitro-L-arginine) relaxation to acetylcholine were significantly reduced in the DOCA-salt group compared to the control group (P˂ 0.05; Fig. 5A and Fig. 5B). Treatment with AEPAimproves the DOCAsalt-blunted NO-and EDH-mediated relaxations in the mesenteric artery (Fig. 5A and Fig. 5B).

AEPA prevents the DOCA-salt-induced increased vascular oxidative stress and up-regulation of NADPH oxidase

Immunofluorescence studies show that DOCA-salt treatment induced an important increased of vascular oxidative stress, attest by the significant increase of DHE fluorescence signal amounted to 124.17 ± 4.89 %, compared to control (100.00 ± 3.97 % ; Fig. 6A). This oxidative stress was accompanied by a significant increase of NADPH oxidase subunits p22phox (198.82 ± 17.17% in DOCA-salt rat versus 100.00 ± 17.34 %; Fig. 6B). AEPA treatmentprevents the DOCA-salt-induced increased vascular oxidative stress and upregulation of NADPH oxidase (P˂ 0.05; Fig. 6A and Fig. 6B).

AEPA prevents the DOCA-salt-induced increased expression of eNOS and decreased expression of Cx37

Immunofluorescence staining indicated a significant increased expressionofeNOS (188. 22 ± 12.91 %; Fig. 7A) and a decreased expression of Cx37 (32.39 ± 3.23 %;Fig. 7B) in DOCAsalt rats compared to control rats. (P ˂ 0.05). AEPA treatment normalized these deleterious effect by preventing the DOCA-salt-induced increased expression of eNOS and decreased expression of Cx37 (P ˂ 0.05; Fig. 7A and Fig. 7B).

DOCA-salt-induced increased expression of COX-2, but not expression of COX-1

The expression of COX-2 signal was significantly increased in DOCA-salt rats compared to the control (153.72 ± 12.44 %, versus 100.00 ± 9.07%; Fig. 8A). After treatment, a preventing effect was observed by a significant reduction of COX-2 levels expression in DOCA + AEPA (100 or 300 mg/kg/day) group (P ˂ 0.05; Fig. 8A). In contrast to COX-2 signal, the expression COX-1 levels expression remained unaffected (P ˂ 0.05; Fig. 8B).

Discussion

The present findings show that after 5 weeks ofinjection of deoxycorticosterone acetate (DOCA) followed by1% NaCl diet, ratsbecome hypertensive. This hypertension is associated to left cardiac hypertrophy and endothelial dysfunction.

Concerning cardiac structure, our result is in agreement with previous study indicated that DOCA-salt induces cardiac hypertrophy, especially in left ventricular in rat [START_REF] Fenning | L-Arginine attenuates cardiovascular impairement in DOCA-salt hypertensive rats[END_REF]. Transthoracic echocardiography result show an increase of PWT and SWT associated with an increase of LVM without any significant dilation of the left ventricular chamber, indicating that hypertrophy observed in rat was concentric type [START_REF] Chan | Improved cardiovascular function with aminoguanidine in DOCA-salt hypertensive rats[END_REF].

Analyses of cardiac function show an impairment of diastolic function in DOCA-salt rats, attest by an increase of IVRT. Ejection fraction (LVEF) was slightly reduced, in DOCA-salt group. However, this parameter remained in the normal range i.e. upper than 50 % [START_REF] Kawaguchi | Combined ventricular systolic and arterial stiffening in patients with heart failure and preserved ejection fraction. Implications for systolic and diastolic reserve limitations[END_REF]. This result is in agreement with previous study indicating that in hypertension, at early stages, cardiac diastolic function was prior affected before systolic function [START_REF] Ayme-Dietrich | Contribution of serotonin to cardiac remodeling with hypertensive diastolic ventricular dysfunction in rats[END_REF].

Moreover, the beneficial cardioprotective effect of AEPA in DOCA-salt rats was related to its ability to exhibit an antihypertrophic effectby reducing near to the normal PWT,SWT and LVM, together with an improvement of cardiac function. Similar effect was reported with oral extract of Moringaoleiferaseeds which decreases the LVM and improves diastolic function in SHR [START_REF] Randriamboavonjy | Cardiac protective effects of Moringaoleiferaseeds in spontaneous hypertensive rats[END_REF].

The experimental models of salt-insensitive hypertension induce also a renal damage [START_REF] Manning | Oxidative stress and antioxidant treatment in hypertension and the associated renal damage[END_REF].In this study, we found an increase of kidneyweight indices, an increase of uric acid and a depletion of potassium plasma levels in untreated pathological rats.

Chronic hypokalemia has been involved incardiac hypertrophy, thus, potassiumsupplementations seem to be a strategy to prevent the development of cardiac hypertrophy (Wang et al., 2005;[START_REF] Liu | Sesame oil therapeutically ameliorates cardiac hypertrophy by regulating hypokalemia in hypertensive rats[END_REF]. Our result shows that in hypertensive rats,

EAPA treatment was able to restore near to the normal, the potassium plasma level. The same result was obtained with sesame oil and quercetin in DOCA-salt hypertensive rats [START_REF] Galisteo | Effects of quercetin treatment on vascular function in deoxycorticosterone acetate-salt hypertensive rats.Comparative study with verapamil[END_REF][START_REF] Liu | Sesame oil therapeutically ameliorates cardiac hypertrophy by regulating hypokalemia in hypertensive rats[END_REF].

Endothelial dysfunction is a common vascular deleterious effect found in hypertension (Perez-Vizcaino et al., 2009). In the present study, we demonstrated that AEPA treatment prevent endothelial dysfunction by reducing the maximal contractile response to phenylephrine and an improvement of vascular effect of acetylcholine in DOCA-salt hypertensive rat. Moreover, endothelial dysfunction was corroborating by an impairment of both NO-and EDH-mediated relaxations in the mesenteric artery in DOCA hypertensive rats.

Previous reports indicatethat in animal artery, endothelium-dependent relaxations to acetylcholine most likely involve a NO component (Furchgott and Zawadzki 1980) and an EDH component [START_REF] Pannirselvam | Pharmacological characteristics of endothelium-derived hyperpolarizing factor-mediated relaxation of small mesenteric arteries from db/db mice[END_REF]. Thus EAPA treatment prevent endothelial dysfunction by improvement of the bluntedof NO-and EDH-mediated relaxations in the mesenteric artery. Other natural product derived from plant extract such as red wine polyphenols (RWPs) or the sesame lignan, sesamin have been reported to restore endothelial function in DOCA-salt hypertension mainly by preventing vascular oxidative stress (Jiménezet al., 2007;[START_REF] Nakano | Dietary sesamin suppresses aortic NADPH oxidase in DOCA-salthypertensive rats[END_REF]. We therefore examine the possibility that AEPA exert a beneficial effect on cardiovascular system by such mechanism, since the DOCA-salt hypertensive rat is described as a model of cardiovascular oxidative and inflammatory stress (Iyer et al., 2010). Interestingly, in the present study,in mesenteric artery of DOCAsalthypertensive rat, we observed an increaseof reactive oxygen species production and an overexpression of NADPH oxidase subunits p22phox. It is well established that NADPH oxidase activation induces a release of ROS production, contributing to vascular endothelial dysfunction [START_REF] Nediani | NADPH oxidase dependent redox signaling in human heart failure: relationship between the left and right ventricle[END_REF].

eNOS expression was up-regulated in the mesenteric artery of DOCA-salt hypertensive. We can explain this phenomenon by the fact that, in presence of vascular oxidative stress, tetrahydrobiopterin (BH4) can promote oxidation, anda compensatory mechanism take place and lead consequently to eNOs uncoupled [START_REF] Landmesser | Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension[END_REF].Thus, the effect of AEPA was similar in part toresveratrol which attenuates hypertension spontaneously inhypertensive rats by preventing endothelial nitric oxide synthase uncoupling [START_REF] Bhatt | Resveratrol prevents endothelial nitric oxide synthase uncoupling and attenuates development of hypertension in spontaneously hypertensive rats[END_REF].Connexin (Cx) protein subunits present into the gap junctions play a crucial role in EDH component of endothelium-dependent relaxation [START_REF] De Wit | Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses[END_REF]. In accordance with our results, previous results haveindicated that vascular oxidative stress also reducesconnexin subunits expression such as Cx37, and thus leading to an impairment of EDH-mediated relaxation [START_REF] Rashid | Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system[END_REF].

DOCA-salt hypertension promotes an overexpression of COX-2 in artery [START_REF] Callera | Endothelin-1induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidase-independent mechanisms[END_REF].Such effect was most likely restored and the value observed was close to the normal level after AEPA treatment. These resultsare in agreement with previousin vivo and invitro studies, which have been showed that Phyllanthus species such as P. amarus or P.acidusare able to reduce inflammatory stress by inhibiting COX-2 overexpression [START_REF] Kiemer | Phyllanthus amarus induced a potent anti-inflammatory effect resulting by inhibition of iNOS, COX-2, and cytokines via the NF-kB pathway[END_REF][START_REF] Hossen | In vitro and in vivo anti-inflammatory activity of Phyllanthusacidusmethanolic extract[END_REF].

In most human and experimental salt sensitivehypertension, antioxidant level is downregulated, thus antioxidant therapy can be helpful [START_REF] Manning | Oxidative stress and antioxidant treatment in hypertension and the associated renal damage[END_REF]. Taken together, the antioxidant properties ofP. amaruspreviously described [START_REF] Roengrit | Antioxidant and anti-nociceptive effects of Phyllanthus amarus on improving exerciserecovery in sedentary men: a randomized crossover (double-blind) design[END_REF], might contribute to explain theability of AEPAto counteract the deleterious effectsdescribed in DOCA-salt rats.

Altogether, our study indicates that AEPA has antihypertensive properties, and prevents endothelial dysfunction and cardiac hypertrophy in DOCA-salt hypertensive rats, in part, by preventing vascular oxidative stress. The antihypertensive effect of AEPA was associated with an improvement of the blunted NO-and EDH-mediated relaxation, and a reduced vascular oxidative stress level and improvement of target proteins (eNOS, Cx37, COX-2 and p22phox) and cardiac hypertrophy in DOCA-salt rats.

Tsotetsi, O.J., Woodiwiss, A.J. ,Netjhard, M., Qubu, D., Brooksbank, R., Norton, G.R., 2001.

Attenuation of cardiac failture, dilatation, damage, and detrimental interstitial remodeling without regression of hypertrophy in hypertensive rats. Hypertension 38, 846-851. Wang, Q., Domenighetti, A.A. Thierry Pedrazzini, T., Burnier, M., 2005

Résumé en anglais

EPA:DHA 6:1 has been shown to be a superior omega-3 formulation inducing a sustained endothelial NO synthase-derived formation of nitric oxide. This study examined whether chronic intake of EPA:DHA 6:1 prevents hypertension and endothelial dysfunction induced by angiotensin II (Ang II) in rats. Ang II-induced hypertension was associated with endothelial dysfunction characterized by blunted components of relaxation and increased endothelium-dependent contractile responses. Ang II increased the vascular oxidative stress, and the expression of NADPH oxidase subunits, COXs, eNOS, and AT1R whereas SKCa and connexin 37 were down-regulated. Intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction, and improved expression of target proteins. In conclusion, chronic intake of EPA:DHA 6:1 prevented the Ang IIinduced hypertension and endothelial dysfunction in rats, most likely by preventing NADPH oxidaseand cyclooxygenase-derived oxidative stress.

Figure

  Figure 1. Endothelial dysfunction; A hallmark of major cardiovascular and other diseases .................. --Figure 2. Structure of the blood vessel (adapted from Boston University School of Public Health). ..... --Figure 3. Endothelium-dependent release of vasocontracting and vasorelaxing factors. ..................... --Figure 4. NO generation from L-Arginine and its functional properties (Ghalayini 2004) ..................... --Figure 5. Nitric oxide synthesis pathway in the endothelial cell and its actions in the vascular smooth muscle cell. .............................................................................................................................................. --Figure 6. The pleiotropic effects of NO. .................................................................................................. --Figure 7. The activation and inhibition sites of eNOS. ............................................................................ --Figure 8. Regulation of eNOS activity. .................................................................................................... --Figure 9. Hypothesis describing the endothelium-derived hyperpolarizing pathway. ........................... --Figure 10. Production and action of prostaglandins (Araujo, Soeiro et al. 2005). ................................. --Figure 11. Summary of acute and chronic stimulation of angiotensin II receptors (Dasgupta and Zhang 2011). ...................................................................................................................................................... --Figure 12. The biological effect of endothelin-1 on arteries in physiological and pathophysiological conditions................................................................................................................................................ --Figure 13. Prostanoids biosynthesis and response pathways (Zhang, Gong et al. 2010). ...................... --Figure 14. The effects of vascular endothelial factors on the function of vascular smooth cells in healthy and pathological conditions. ................................................................................................................... --Figure 15. Mechanisms implicated in essential hypertension-associated endothelial dysfunction ...... --Figure 16. Progression of endothelial dysfunction in relation to the progression of insulin resistance (Cosentino and Lüscher 1997). ............................................................................................................... --Figure 17. Monounsaturated fatty acids MUFAs .................................................................................... --Figure 18. Major Polyunsaturated fatty acids (PUFA). (Nair, Leitch et al. 1997). ................................... --Figure 19. Structures of dietary ω 3 and ω 6 polyunsaturated fatty acids. A: C18 ω 3 and ω 6 PUFA. B: C20-22 ω 3 and ω 6 PUFA (Jump, Depner et al. 2012). .......................................................................... --Figure 20. Importance of omega-6/omega-3 ratio. ................................................................................ --Figure 21. Synthesis pathway of omega-6 and omega-3 fatty acids in mammals. ................................ --Figure 22. Physiological effects of n-3 PUFA that might influence CVD Risk (Mozaffarian and Wu 2011). .. -68 -Figure 23. Omega-3 EPA:DHA 6:1 prevents Ang II-induced endothelial dysfunction and hypertension in rats. ....................................................................................................................................................... --Figure 24. Perspectives in animal models. ............................................................................................ --Figure 25. Clinical perspective. ............................................................................................................. --

1 .

 1 Figure 1. Endothelial dysfunction; A hallmark of major cardiovascular and other diseases .................. --Figure 2. Structure of the blood vessel (adapted from Boston University School of Public Health). ..... --Figure 3. Endothelium-dependent release of vasocontracting and vasorelaxing factors. ..................... --Figure 4. NO generation from L-Arginine and its functional properties (Ghalayini 2004) ..................... --Figure 5. Nitric oxide synthesis pathway in the endothelial cell and its actions in the vascular smooth muscle cell. .............................................................................................................................................. --Figure 6. The pleiotropic effects of NO. .................................................................................................. --Figure 7. The activation and inhibition sites of eNOS. ............................................................................ --Figure 8. Regulation of eNOS activity. .................................................................................................... --Figure 9. Hypothesis describing the endothelium-derived hyperpolarizing pathway. ........................... --Figure 10. Production and action of prostaglandins (Araujo, Soeiro et al. 2005). ................................. --Figure 11. Summary of acute and chronic stimulation of angiotensin II receptors (Dasgupta and Zhang 2011). ...................................................................................................................................................... --Figure 12. The biological effect of endothelin-1 on arteries in physiological and pathophysiological conditions................................................................................................................................................ --Figure 13. Prostanoids biosynthesis and response pathways (Zhang, Gong et al. 2010). ...................... --Figure 14. The effects of vascular endothelial factors on the function of vascular smooth cells in healthy and pathological conditions. ................................................................................................................... --Figure 15. Mechanisms implicated in essential hypertension-associated endothelial dysfunction ...... --Figure 16. Progression of endothelial dysfunction in relation to the progression of insulin resistance (Cosentino and Lüscher 1997). ............................................................................................................... --Figure 17. Monounsaturated fatty acids MUFAs .................................................................................... --Figure 18. Major Polyunsaturated fatty acids (PUFA). (Nair, Leitch et al. 1997). ................................... --Figure 19. Structures of dietary ω 3 and ω 6 polyunsaturated fatty acids. A: C18 ω 3 and ω 6 PUFA. B: C20-22 ω 3 and ω 6 PUFA (Jump, Depner et al. 2012). .......................................................................... --Figure 20. Importance of omega-6/omega-3 ratio. ................................................................................ --Figure 21. Synthesis pathway of omega-6 and omega-3 fatty acids in mammals. ................................ --Figure 22. Physiological effects of n-3 PUFA that might influence CVD Risk (Mozaffarian and Wu 2011). .. -68 -Figure 23. Omega-3 EPA:DHA 6:1 prevents Ang II-induced endothelial dysfunction and hypertension in rats. ....................................................................................................................................................... --Figure 24. Perspectives in animal models. ............................................................................................ --Figure 25. Clinical perspective. ............................................................................................................. --

  ...................................................... --Table

  et de la prolifération et de la migration des cellules musculaires lisses vasculaires. De plus, plusieurs études ont montré que la dysfonction endothéliale et le développement des maladies cardiovasculaires sont associés à une augmentation du stress oxydant vasculaire et à une surexpression du système angiotensine local. L'angiotensine II (Ang II) participe à la physiopathologie de l'athérosclérose et des maladies vasculaires non seulement de par son rôle dans l'hypertension, mais aussi de par son effet direct sur la prolifération et la migration des cellules vasculaires. L'Ang II contribue aussi au développement des maladies cardiovasculaires de par l'augmentation du stress oxydant vasculaire induit par la surexpression de la NAPDH oxydase, la principale source des espèces réactives de l'oxygène dans la paroi vasculaire. L'alimentation peut jouer un rôle dans le développement des maladies cardiovasculaires. Ainsi, alors que le régime occidental riche en graisses saturées a été associé à une augmentation du risque de maladies cardiovasculaires, d'autres types d'alimentation tels que le régime Méditerranéen riche en graisse non-saturées ont montrées une réduction de l'incidence de maladies cardiovasculaires allant jusqu'à 30 % de réduction. De nombreuses études épidémiologiques ou d'intervention ont montré que la consommation alimentaire de poisson, d'huile de poisson ou d'acides gras polyinsaturés omega-3 (n-3 PUFAs) exerçait des effets bénéfiques vis-à-vis de la maladie coronarienne, des accidents vasculaires cérébraux et de l'hypertension. De plus, la consommation des acides gras n-3 PUFAs majeurs, à savoir l'acide eicosapentaénoïque (EPA) et l'acide docosahexaénoïque (DHA), est associée à une réduction de la morbi-mortalité cardiovasculaire. Les mécanismes par lesquels les n-3 PUFAs inhibent le développement de l'athérosclérose restent à éclaircir, mais pourraient être dû, au moins partiellement, à une modulation des métabolites lipidiques, une diminution de la production de cytokines proinflammatoires et une réduction des processus inflammatoires. Il a été montré que dans les cellules endothéliales, l'EPA stimule l'activation de la NO synthase endothéliale (eNOS) en induisant sa dissociation d'avec la protéine inhibitrice cavèoline, alors que le DHA stimule la formation endothéliale de NO en augmentant les interactions entre la eNOS et la protéine chaperonne HSP-90 qui active la voie PKB/Akt conduisant à la phosphorylation activatrice de la eNOS. Dans les situations physiopathologiques comme l'hypertension ou l'insuffisance rénale, les n-3 PUFAs peuvent réduire l'augmentation des niveaux circulants de diméthylarginine (ADMA, un inhibiteur endogène de la eNOS), ce qui induit une augmentation de l'activité de la eNOS. De plus, notre équipe de recherche a récemment démontré que la stimulation de la fonction endothéliale par les n-3 PUFAs dépend à la fois du ratio et du degré de pureté de la formulation en EPA et DHA. En effet, la formulation optimisée EPA:DHA 6:1 est un puissant activateur de la formation endothéliale de NO, et de façon moindre de l'augmentation de la réponse d'hyperpolarisation dépendante de l'endothélium (EDH). L'induction par les n-3 PUFAs de la formation endothéliale de NO due à l'activation de la eNOS via les voies de signalisation redoxsensibles Src/PI3-kinase/Akt et MAPKs, est dépendante du ratio et de la quantité de EPA et DHA dans la formulation. Un grand nombre d'études clinique montre que la consommation des n-3

  études de réactivité vasculaire dans les branches secondaires de l'artère mésentérique montrent que l'Ang II induit une dysfonction endothéliale caractérisée à la fois par une diminution des relaxations en réponse à l'acétylcholine affectant les composantes NO et EDH de la relaxation, et par une augmentation de la formation des facteurs constricteurs dérivés de l'endothélium (EDCFs) en réponse à l'acétylcholine. La consommation chronique de EPA:DHA 6:1 normalise les réponses NO, EDH et EDCFs dans les branches secondaires de l'artère mésentérique. Afin de mieux caractériser les mécanismes moléculaires impliqués dans l'effet protecteur de EPA:DHA 6:1, des analyses quantitatives des niveaux d'expression de protéines ont été effectués dans les branches secondaires de l'artère mésentérique par immunofluorescence sur coupes congelées. Dans un premier temps, nous avons étudié l'expression de la eNOS et de l'arginase 1 (composante NO de la relaxation), de SKCa et Cx37 (composante EDH de la relaxation), et de cyclooxygénases (COXs, impliquées dans les réponse EDCFs). Par rapport aux animaux contrôles, l'Ang II induit une augmentation significative de l'expression de la eNOS, d'arginase 1, et de COX-1 et COX-2, la forme inductible des COXs, et une diminution significative de l'expression de SKCa et Cx37. La prise chronique de EPA:DHA 6:1 prévient significativement les effets de l'Ang II sur l'expression des protéines cibles. Comme la dysfonction endothéliale est associée à un stress oxydant vasculaire, nous avons évalué le niveau de stress oxydant dans les branches secondaire d'artère mésentérique à l'aide de la sonde fluorescente redox-sensible dihydroethidium (DHE). L'Ang II induit une augmentation significative de la fluorescence dans l'ensemble de la paroi vasculaire en comparaison des rats contrôles, qui est significativement prévenue par la prise chronique de EPA:DHA 6:1. Du fait que l'augmentation de stress oxydant dans l'hypertension induite par l'Ang II est due, du moins partiellement, à une surexpression de la NAPDH oxydase liée à l'activation du récepteur de l'angiotensine II de type 1 (AT1R), les niveaux d'expression d'AT1R et des sous-unités p22 phox et p47 phox de la NADPH oxydase ont été déterminés. En comparaison des branches secondaires d'artère mésentérique de rat contrôles, les rats recevant l'Ang II montrent une augmentation significative des niveaux d'expression d'AT1R, p22 phox et p47 phox . Le traitement avec EPA:DHA 6:1 prévient significativement cette augmentation de stress oxydant vasculaire et de l'expression d'AT1R et des deux sous-unités de la NADPH oxydase. Afin de confirmer ces résultats obtenus par immunofluorescence et histochimie fluorescente sur les branches secondaires d'artère mésentérique, l'analyse par Western blot de l'expression des protéines eNOS, COX-2, et la sous-unité p22 phox de la NADPH oxydase a été réalisée dans l'artère mésentérique principale. L'Ang II induit une augmentation significative de l'expression de eNOS, COX-2, et p22 phox , et cette surexpression est significativement prévenue par la prise de EPA:DHA 6:1. L'ensemble des résultats obtenus lors de la présente étude indique que la prise chronique de la formulation optimisée EPA:DHA 6:1 prévient le développement de l'hypertension et de la dysfonction endothéliale induites par l'infusion d'Ang II chez le rat. La dysfonction endothéliale induite par l'Ang II est associée à une régulation positive du système angiotensine local et une augmentation du stress oxydant vasculaire. Les effets bénéfique de la consommation chronique de EPA:DHA 6:1 impliquent une amélioration des composantes de relaxations NO et EDH, et à une diminution des réponses contractiles dépendantes de l'endothélium, probablement via la prévention du stress oxydant vasculaire induit par la régulation positive du système angiotensine local.

Figure 1 .

 1 Figure 1. Endothelial dysfunction: A hallmark of major cardiovascular and other related diseases.

Figure 2 .

 2 Figure 2. Structure of the blood vessel (adapted from Boston University School of Public Health).

Figure 3 .

 3 Figure 3. Endothelium-dependent release of vasocontracting and vasorelaxing factors. NO, nitric oxide; PGI2, prostacyclin; EDHF, endothelium-derived hyperpolarizing factor; A-II, angiotensin II; ET, endothelin; TxA2/PGH2, thromboxaneA2/prostaglandin H2O2, hydrogen peroxide (Abeywardena and Head 2001).

Figure 4 .

 4 Figure 4. NO generation from L-Arginine and its functional properties (Ghalayini 2004)

Figure 5 .

 5 Figure 5. Nitric oxide synthesis pathway in the endothelial cell and its actions in the vascular smooth muscle cell. ACh, acetylcholine; BK, bradykinin; ADP, adenosine diphosphate; 5-HT, serotonin; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; Src, Sarcoma-family kinases; PI3/kinase, phosphoinositide 3kinase; Akt, Protein kinase B; Ca 2+ / CaM, calcium calmodulin; L-Arg, L-arginine; eNOS, endothelial Nitric Oxide Synthase; NO, nitric oxide; sGC, soluble guanylyl cyclase; GTP, Guanosine 5'-Triphosphate; cGMP, cyclic Guanosine 3'-5' monophosphate

Figure 6 .

 6 Figure 6. The pleiotropic effects of NO. VCAM-1; Vascular cell adhesion molecule-1, MCP-1; Monocyte chemoattractant protein-1, LDL; Low density lipoproteins.

Figure 7 .

 7 Figure 7. The activation and inhibition sites of eNOS. Green arrows for activation, red arrows for inhibition, black arrow for no direct effect on enzyme activity. The numbers refer to the human sequence (Fleming 2010).

Figure 8 .

 8 Figure 8. Regulation of eNOS activity.(1) At rest, the eNOS is coupled to cav-1 (caveolin-1, a structural protein of caveolae) that decreases its activity. (2) eNOS is constitutively phosphorylated at Thr 495 preventing its activation by the Ca 2+ /CaM. (3) eNOS may be inhibited in response to oxidative stress by tyrosine phosphorylation by PYK2 (proline-rich tyrosine kinase). (4) eNOS can be activated by both Ca 2+ /CaM (calcium / calmodulin) and phosphorylation of Ser1177. Hsp90 (heat shock protein) facilitates the recruitment of Akt responsible for the phosphorylation of eNOS[START_REF] Fleming | Molecular mechanisms underlying the activation of eNOS[END_REF].

Figure 9 .

 9 Figure 9. Hypothesis describing the endothelium-derived hyperpolarizing pathway. AA, arachidonic acid; ACh, acetylcholine, [Ca 2+ ]i, intracellular calcium concentration; CYP, cytochrome P450 epoxygenase; EC, endothelial cell; EETs, epoxyeicosatrienoic acids; ER, endoplasmic reticulum; GPCR, G proteincoupled receptor; BKCa, large conductance Ca 2+ -activated K+ channel; SKCa, small-conductance Ca 2+ -activated K + channel subtype 3; IKCa, intermediate-conductance Ca 2+ -activated K+ channel; Kir, inwardly rectifying K + channel; meGJ, myo-endothelial gap-junction; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; VDCC, voltagedependent Ca 2+ channel; VSMC, vascular smooth muscle cell[START_REF] Grgic | Endothelial Ca+-activated K+ channels in normal and impaired EDHF-dilator responses--relevance to cardiovascular pathologies and drug discovery[END_REF]).

Figure 10 .

 10 Figure 10. Production and action of prostaglandins (Araujo, Soeiro et al. 2005).

Figure 11 .

 11 Figure 11. Summary of acute and chronic stimulation of angiotensin II receptors (Dasgupta and Zhang 2011).

Figure 12 .

 12 Figure 12. The biological effect of endothelin-1 on arteries in physiological and pathophysiological conditions.In healthy arteries the production of ET-1 is small and the bioavailability of NO is preserved. In endothelial dysfunction there is increased expression of ET-1 in smooth muscle cells and macrophages (MØ). Both the ETA and the ETB receptor on smooth muscle cells may mediate formation of superoxide (O2 -). Collectively the balance of effects is shifted towards more vasoconstriction, inflammation and oxidative stress in endothelial dysfunction[START_REF] Böhm | The importance of endothelin-1 for vascular dysfunction in cardiovascular disease[END_REF].

Figure 13 .

 13 Figure 13. Prostanoids biosynthesis and response pathways (Zhang, Gong et al. 2010).

Figure 14 .

 14 Figure 14. The effects of vascular endothelial factors on the function of vascular smooth cells in healthy and pathological conditions.In the healthy endothelium, the eNOS is responsible for most of the vascular NO production. However, eNOS becomes a potential ROS generator when in the pathological uncoupled state, due to oxidative stress. ACE, angiotensin-converting enzyme; Ach, acetylcholine; AT-I, angiotensin I; AT-II, angiotensin II; AT1, angiotensin 1 receptor; BH4, tetrahydrobiopterin; BK, bradykinin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ECE, endothelin converting enzyme; eNOS, endothelial nitric oxide synthase; EDHF, endothelium derived hyperpolarizing factor; ETA and ETB, endothelin A and B receptors; ET-1, endothelin-1; L-Arg, L-arginine; L-Cit, L-citruline; M, muscarinic receptor; O2 -, superoxide anion; ONOO -, peroxynitrite; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOX, nicotinamide adenine dinucleotide phosphate oxidase; PGH2, prostaglandin H2; PGI2, prostaglandin I2; ROS, reactive oxygen species; S1B, serotonin receptor; TP, thromboxane prostanoid receptor; TXA2, thromboxane; 5-HT, serotonin; Θ, inhibition; , stimulation.[START_REF] Park | Endothelial dysfunction: clinical implications in cardiovascular disease and therapeutic approaches[END_REF].

Figure 15 .

 15 Figure 15. Mechanisms implicated in essential hypertension-associated endothelial dysfunction. (a): In healthy artery endothelium produces vasoprotectors (PGI2, NO and EDH) which induces relaxation in vascular smooth muscle cells. (b) In pathological artery, the release and activity of vasoprotectors is decreased while the local angiotensin system is activated resulting in the increased production of vasoconstrictors (ROS, EDCFs and ET1) which induces contraction in the vascular smooth muscle cells leading to endothelial dysfunction and hypertension.

Figure 16 .

 16 Figure 16. Progression of endothelial dysfunction in relation to the progression of insulin resistance (Cosentino and Lüscher 1997).

  is an independent risk factors of cardiovascular diseases (CVD)[START_REF] Siri-Tarino | Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease[END_REF][START_REF] Colquhoun | the Nutrition and Metabolism Committee of the Heart Foundation. Review of evidence fish, fish oils, n-3 polyunsaturated fatty acids and cardiovascular health[END_REF]).

( 18 :

 18 1 n-9). Palmitoleic acid has 16 carbon atoms with the first double bond occurring 7 carbon atoms away from the methyl group and 9 carbons from the carboxyl end, which can be lengthened to the 18-carbon cis-vaccenic acid. Oleic acid has 18 carbon atoms with the first double bond occurring after the ninth carbon atom from the methyl end of fatty acid chain. Vegetable oils such as olive oil and canola oil are good sources of MUFA. MUFA consumption is strongly associated with a decrease in LDL cholesterol[START_REF] Corrao | Exploring the dose response relationship between alcohol consumption and the risk of several alcohol related conditions: a meta analysis[END_REF]).

Figure 17

 17 Figure 17. Monounsaturated fatty acids MUFAs

  example, in omega-3 (n-3) and omega-6 (n-6) fatty acids, the first double bond starts at 3 and 6 carbons from the methyl end, respectively. Thus α-Linoleic acid symbol 18:3 n-3 identifies a fatty acid having 18 carbon atoms and 3 double bonds, the first double bond occurring after the third carbon atom from the methyl end of the fatty acid chain, known as the n end (Mateos 2012).

Figure 18 .

 18 Figure 18. Major Polyunsaturated fatty acids (PUFA). (Nair, Leitch et al. 1997).

Figure 19 .

 19 Figure 19. Structures of dietary ω 3 and ω 6 polyunsaturated fatty acids. A: C18 ω 3 and ω 6 PUFA. B: C20-22 ω 3 and ω 6 PUFA (Jump, Depner et al. 2012).

Figure 20 .

 20 Figure 20. Importance of omega-6/omega-3 ratio.A very high omega-6/omega-3 ratio promotes the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 PUFA (a low omega-6/omega-3 ratio) exert suppressive effects. PGH2, Prostaglandin H2; TXA2, Thromboxane A2; LT E4, Leukotriene E4; LT B4, Leukotriene B4; PDGF, Platelet derived growth factor; IL-1, Interleukin-1; IL-2, Interleukin-2.

Figure 21 .

 21 Figure 21. Synthesis pathway of omega-6 and omega-3 fatty acids in mammals.

3 .

 3 synthesis and secretion of various potent vasodilatating and vasoconstricting factors. In addition, they also inhibit platelet aggregation, decrease the endothelial expression of adhesion molecules and smooth muscle cell proliferation, thus reducing the risk of cardiovascular diseases. Endothelial dysfunction is an early hallmark for the development and progression of most of the cardiovascular diseases, like hypertension, atherosclerosis, myocardial infarction, cerebrovascular diseases (stroke), peripheral artery diseases, rheumatic heart diseases, congenital heart diseases and heart failure, which are the leading cause of mortality and morbidity worldwide. Endothelial dysfunction is associated with an impairment of endothelium-dependent relaxations involving a reduced NO bioavailability, EDH component of relaxation and an increased production of endothelium derived contractile factors (EDCF). Endothelial dysfunction could lead to hypertension.Several naturally occurring constituents like red wine polyphenols, caffeine, omega-3 fatty acids, carotenoids, vitamins E and C have received significant consideration because of their potential antioxidant activity. Consuming a diet rich in these natural antioxidants has been associated with prevention and treatment of endothelial dysfunction and associated cardiovascular diseases[START_REF] Potashkin | Splice Variant Biomarkers for Parkinson's Disease[END_REF].Antihypertensive treatments affecting RAS (AT1R and ACE inhibitors) contributes to 60 % to 65 % of overall hypertensive therapy and, therefore, Ang II-induced hypertension is one of the most widely used pharmacological model of hypertension in rats. In several experimental models of endothelial dysfunction, an overexpression of the local angiotensin system associated to a vascular oxidative stress has been described. Red wine polyphenols (RWPs) are able to prevent the Ang II-induced endothelial dysfunction mostly due to their antioxidant properties(Kane, Etienne-Selloum et al. 2010). RWPs intake caused a persistent improvement of the endothelial function, particularly the EDH component of relaxation, in middle-aged rats and this effect seems to involve the normalization of the expression of IKCa, SKCa and the angiotensin system[START_REF] Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF].Various studies demonstrates that regular intake of fish products and dietary consumption of fish or fish oil rich in omega-3 PUFAs, particularly EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) has been related to a reduced risk of cardiovascular disease morbidity/mortality.In a previous study of our group different ratio of EPA and DHA (EPA:DHA 1:1, EPA:DHA 3:1, EPA:DHA 6:1, EPA:DHA 1:3, EPA:DHA 1:6) and alone EPA and DHA were studied and they found that omega-3 EPA:DHA 6:1 is the most potent formulation for the induction of both NOmediated and EDH-mediated response in porcine arteries by the activation of eNOS through Src PI3K Akt Pathway(Zgheel, Alhosin et al. 2014). Being the most superior formulation of omega-3 EPA:DHA 6:1 was selected to treat angiotensin II induced endothelial dysfunction and hypertension in rats. The major goal of this thesis was to determine whether chronic intake of EPA: DHA 6:1 affects hypertension and endothelial dysfunction induced by angiotensin II infusion in rats.More specifically the aims were 1. To study the effect of EPA:DHA 6:1 in angiotensin induced hypertension.2. To study the impregnation of EPA:DHA 6:1 in plasma and its effects on omega6/omega3 ratio. To study the effect of EPA:DHA 6:1 in the endothelium -dependent relaxation in second branch mesenteric arterial rings. 4. To characterize the mechanisms underlying EPA:DHA 6:1 induced endothelium-dependent relaxation mediated by NO and EDH, in second branch mesenteric arteries. 5. Evaluate the ability of EPA:DHA to reduce contractile responses in second branch mesenteric arterial rings. 6. To study Ang II induced oxidative stress responses (ROS) in second branch mesenteric arteries.

EPA:DHA 6 :

 6 1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase and COX-derived oxidative stressZahid RasulNiazi, Grazielle C. Silva G, Thais Porto Ribeiro, Antonio J León-González, Mohamad Kassem, Abdur Mirajkar, Azhar Alvi, Malak Abbas, Faraj Zgheel, Valérie B. Schini-Kerth, Cyril Auger Submitted to the British Journal of Pharmacology Cardiovascular diseases are the major of death worldwide and should remain the leading cause of mortality and morbidity over the next few decades. Hypertension and other risk factors of cardiovascular diseases are associated early with the development of an endothelial dysfunction. The endothelial dysfunction is generally characterized by a reduced formation of vasoprotective factors including nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), and an increased production of vasocontracting factors such as cyclooxygenase (COX)-derived metabolites of arachidonic acid involved in the endothelium-dependent contractile factors.

  DHA 6:1 formulation is associated with significantly increased plasma levels of omega-3 fatty acids, mainly as EPA, DHA and the intermediate elongated metabolite of EPA, the docosapentaenoic acid (DPA), resulting in a decreased omega-6/omega-3 ratio. The reduction of this ratio have been associated with a shift towards beneficial health effects of omega-3, including reduced cardiovascular and cancer risk.Vascular reactivity studies in the secondary branch of the mesenteric artery indicate that Ang II induced an endothelial dysfunction characterized by reduced relaxations in response to acetylcholine affecting both the NO-and EDH-mediated components, and increased formation of endothelium-derived contractile factors (EDCFs) in response to acetylcholine. The chronic intake of EPA:DHA 6:1 normalized both the NO, EDH and EDCF responses in the secondary branch of the mesenteric artery. To better characterize the molecular mechanisms involved in the protective effects of EPA:DHA 6:1 intake, we performed quantitative analysis of protein expression in the secondary branch of the mesenteric artery by immunofluorescence. The Ang II treatment increased the level of vascular oxidative stress and the expression of NADPH oxidase subunits p22 phox and p47 phox , AT1R, AT2R, eNOS, arginase-1, COX-1 and COX-2, and decreased the expression of SKCa and connexin 37, which were improved by the omega-3 treatment. To confirm the results obtained by immunofluorescence in the secondary branch of the mesenteric artery, we performed Western blot analysis of the expression levels of eNOS, COX-2, and the NADPH oxidase subunit p22 phox in the secondary branch mesenteric artery. The Ang II group presented a significantly increased expression of eNOS, COX-2, and the NADPH oxidase subunit p22 phox , that was prevented by the chronic oral intake of EPA:DHA 6:1.Altogether, the present findings indicate that chronic intake of the optimized EPA:DHA 6:1 formulation prevented the development of hypertension and endothelial dysfunction induced by the infusion of Ang II in rats. The Ang II-induced endothelial dysfunction is associated to an upregulation of the local angiotensin system and an increased vascular oxidative stress. The beneficial effect of EPA:DHA 6:1 is mediated by an improvement of both the NO-and the EDH-mediated relaxations and a reduction of endothelium-dependent contractile response, most likely by preventing the oxidative stress induced by the up-regulation of the local angiotensin system.

  DHA 6:1 formulation to protect the endothelial function in vivo. The present findings shows that Ang II infusion is associated with the development of endothelial dysfunction in the secondary branch of the mesenteric arteries, which affects markedly the EDH-mediated component of relaxation and also, to some extent, the NO-mediated component of relaxation. The Ang II-induced endothelial dysfunction involves a redox-sensitive mechanism implicating NADPH oxidase, COX-1 and COX-2. The chronic oral intake of the optimized EPA:DHA 6:1 formulation prevents Ang IIinduced endothelial dysfunction and hypertension most likely by decreasing oxidative stressmediated impairment of both NO and EDH components as well as a reduction of endotheliumdependent contractile response.

3

  fatty acids EPA and DHA for cardiovascular disease prevention, treatment after a myocardial infarction, prevention of sudden death, and secondary prevention of cardiovascular disease (Von Schacky and Harris 2007).

  from 0.18 up to 10 g/d of various omega-3 products (Delgado-Lista, Perez-Martinez et al. 2012; Enns, Yeganeh et al. 2014).

  4 mg/kg/d of Ang II in rats induced endothelial dysfunction in secondary branch of the mesenteric artery that is characterized by reduced endothelium-dependent relaxing responses, involving mainly blunted EDH-mediated responses and to a lesser extent NO-mediated relaxations, and an increased formation of EDCFs. As a result, omega-3 prevented Ang II induced endothelial dysfunction as indicated by a significant improvement of both components of the relaxation and a decreased in endothelium-dependent contractile response. The chronic oral intake of the optimized EPA:DHA 6:1 significantly reduced Ang II induced over expression of eNOS, arginase-1, COX-1 and COX-2 as well as prevented the down-regulation of SKCa and Cx37. Supplementation with omega-3 fatty acids also significantly improved the endothelial function in Humans without affecting endothelium-independent dilation(Wang, Liang et al. 2012). Endothelial dysfunction has been associated with an impairment of endothelium-dependent relaxations involving a reduced bioavailability of NO in major CV diseases such as hypertension, atherosclerosis, chronic renal failure, and diabetes[START_REF] Griendling | Oxidative stress and cardiovascular injury: Part II: animal and human studies[END_REF][START_REF] Rush | Vascular nitric oxide and oxidative stress: determinants of endothelial adaptations to cardiovascular disease and to physical activity[END_REF][START_REF] Félétou | Endothelial dysfunction: a multifaceted disorder (the Wiggers Award Lecture)[END_REF]. The mechanism underlying endothelial dysfunction has been linked to increased oxidative stress which is associated with a reduced bioavailability of (NO), an alteration of the production of prostanoids, including prostacyclin, thromboxane A2, and/or isoprostanes, an impairment of endothelium-dependent hyperpolarization, these phenomena being able to contribute to endothelial dysfunction individually or in association.

Figure 23 .

 23 Figure 23. Omega-3 EPA:DHA 6:1 prevents Ang II-induced endothelial dysfunction and hypertension in rats.

  DHA 6:1 formulation to protect the endothelial function in vivo. The present findings shows that Ang II infusion is associated with the development of endothelial dysfunction in second branch mesenteric arteries, which affects markedly EDH-mediated component and also, to some extent, the NO-mediated component of relaxation. The Ang II-induced endothelial dysfunction involves a redox-sensitive mechanism implicating NADPH oxidase, COX-1 and COX-2. The chronic oral intake of the optimized EPA:DHA 6:1 formulation prevents Ang II-induced endothelial dysfunction and hypertension most likely by decreasing oxidative stress-mediated impairment of both NO and EDH components as well as a reduction of endothelium-dependent contractile response.

Figure 24 .

 24 Figure 24. Perspectives in animal models. Determination of active metabolites of omega-3 fatty acids, curative potential of the optimized EPA:DHA 6:1 formulation, importance of the omega-3 purity and ratio and impregnation of omega-3 fatty acids in vascular tissues.

Figure 25 .

 25 Figure 25. Clinical perspective. Determination of EPA:DHA 6:1 potential to reduce cardiovascular risk factors in hypertensive patients.

9 .

 9 de l'influx hépatique d'acides gars non-estérifiés, diminution des activités enzymatiques hépatiques de synthèse des triglycérides, et augmentation de la synthèse hépatique des phospholipides au dépend des triglycérides[START_REF] Nakamura | Essential fatty acid synthesis and its regulation in mammals[END_REF]. De plus, les acides gras omega-3 ont des propriétés anti-inflammatoires, et une corrélation négative a été montrée entre la consommation régulière de poisson ou d'huile de poisson et les taux circulants de protéine Créactive, d'interleukine 6, d'E-selectin, de sICAM-1, de TNF, de la forme soluble du récepteur à l'interferon alpha, et de la métalloprotéase matricielle 3[START_REF] He | Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease-eat fish or take fish oil supplement?[END_REF]. Les huiles de poisson inhibent la production des cytokines pro-inflammatoires et réduisent l'expression de molécules d'adhésion cellulaire[START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF]. Ces facteurs sont critiques dans le recrutement des leucocytes circulants sur l'endothélium vasculaire, un événement précoce clé dans la pathogenèse de l'athérosclérose.Les acides gras omega-3 entrent en compétition avec les acides gras omega-6 au niveau des cyclooxygénases et des lipoxygénases pour la synthèse des prostaglandines et des leucotriènes.Ainsi, les acides gras omega-3 modulent le métabolisme des prostaglandines en augmentant la synthèse de prostaglandine E3, un vasodilatateur et inhibiteur de l'agrégation plaquettaire, du thromboxane A3, de leucotriène B5 et en diminuant la synthèse de thromboxane A2, un puissant vasoconstricteur et inducteur de l'agrégation plaquettaire, et de leucotriène B4 (un inducteur de l'inflammation et la chimiotaxie et de l'adhérence des leucocytes). D'autres études ont aussi montré que la consommation élevée d'huile de poisson conduisait à une diminution du taux de plaquettes, de l'agrégation plaquettaire, de la concentration des métabolites du thromboxane, ainsi qu'à une augmentation du temps de saignement. Les effets hypotriglycéridémiants des acides gras omeag-3 requièrent des doses d'EPA et DHA de 3 ou 4 g par jour. Dans les populations américaines, ces doses peuvent réduire le taux de triglycérides de l'ordre de 30 à 40 %[START_REF] Harris | Safety and efficacy of Omacor in severe hypertriglyceridemia[END_REF][START_REF] Lavie | Omega-3 polyunsaturated fatty acids and cardiovascular diseases[END_REF]). Cependant, la consommation quotidienne de 6 g d'EPA et DHA pendant 2 ans par 59 patients n'a pas montré d'effet favorable sur l'évolution du diamètre d'artères coronaires athéromateuses[START_REF] Woodman | The Independent Effects of Purified EPA and DHA Supplementation on Cardiovascular Risk in Treated-hypertensive Type 2 Diabetic Individuals[END_REF]. A l'inverse, plusieurs études ont montré que la consommation d'huile de poisson ou de poissons non frits était associée à une diminution de la prévalence de l'athérosclérose subclinique caractérisée par un changement significatif de l'épaisseur intima-media de l'artère carotide commune et du pourcentage de sténose, alors qu'aucun changement n'ont été observé pour la quantification du calcium dans l'artère coronaire ou pour l'index de pression systolique (Von[START_REF] Schacky | The effect of dietary Ω-3 fatty acids on coronary atherosclerosis: a randomized, double-blind, placebo-controlled trial[END_REF]. Le DHA est le précurseur d'un métabolite récemment décrit sous le nom de 10,17S docosatriène, qui est un membre de la famille des résolvines. Ces molécules ont d'abord été décrites comme relâchées par le cerveau en réponse à l'ischémie, et elles s'opposent à l'effet pro inflammatoire des infiltrats leucocytaires en bloquant l'activation de NF-κB et l'expression de la cyclooxygénase-2 induites par l'interleukine 1-bêta[START_REF] Layé | Polyunsaturated fatty acids, neuroinflammation and well being[END_REF]. Les études précédentes de notre équipe de recherche ont montré que l'EPA et le DHA, les acides gars omega-3 majeurs, induisent des relaxations dépendantes de la concentration dans les anneaux d'artères coronaires de porc(Zgheel, Alhosin et al. 2014). La relaxation en réponse à l'EPA est légèrement mais significativement plus importante que celle en réponse au DHA (77.8±10.3 et 64.7±12.8 % à 0.4 % v/v, respectivement).Ensuite, la capacité de ratios optimisés d'omega-3 à induire la relaxation dépendante de l'endothélium a été déterminée, et les résultats indiquent que les ratios EPA:DHA de 6:1 et 9:1 induisent des relaxations significativement plus importantes que celles en réponse aux ratios 3De plus, la relaxation dépendante de l'endothélium obtenu en réponse à un produit ayant un degré de pureté en EPA:DHA élevé (694:121 mg/g) était significativement plus importante celle obtenu en réponse à un produit ayant un degré de pureté plus faible (352:65 mg/g).

  Wistar males à l'aide de mini-pompes osmotiques Azlet. L'ang II est une hormone peptidique multifonctionnelle qui régule la pression sanguine, le volume plasmatique, les fonctions cardiaques, rénales et neuronales, et qui contrôle aussi la soif. Ce peptide joue un rôle central dans le développement de l'hypertension artérielle et dans le remodelage cardiaque, et il est l'effecteur principal du système rénine-angiotensine qui joue un rôle majeur dans la régulation de la tension artérielle[START_REF] Zhuo | New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II[END_REF]. Dans la présente étude, l'infusion d'Ang II a induit une augmentation rapide de la pression artérielle chez les rats en quelques jours et qui resté élevée tout au long de l'étude. La prise quotidienne de la formulation EPA:DHA 6:1 a permis de prévenir partiellement mais significativement l'augmentation de tension artérielle induite par l'Ang II. Ce résultats est en accord avec les études publiées préalablement qui montraient que la consommation d'acides gras omega-3 à des doses de 3,3 à 7 g/j était associée à une diminution de la tension artérielle de 2,9 et 1,6 mmHg chez les patients hypertendus[START_REF] He | Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease-eat fish or take fish oil supplement?[END_REF]. Ainsi, la méta-analysede Miller et al. rapporte que la consommation de EPA+DHA est toujours associée à une diminution de la pression sanguine systolique, alors que la pression sanguine diastolique est diminuée significativement pour des consommations d'EPA+DHA supérieures à 2 g/j[START_REF] Miller | Long-chain omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and blood pressure: a meta-analysis of randomized controlled trials[END_REF].Dans notre modèle expérimental, nous avons observé que l'infusion d'Ang II à la dose de 0,4 mg/kg/j induisait l'apparition dans les branches secondaires de l'artère mésentérique d'une dysfonction endothéliale caractérisée par une diminution des réponses vasorelaxantes dépendantes de l'endothélium, impliquant principalement une réduction de la composante EDH et dans une moindre mesure de la composante NO, et d'une augmentation de la formation des EDCFs. La consommation chronique de la formulation EPA:DHA 6:1 a eu pour effet de prévenir la dysfonction endothéliale induite par l'Ang II comme l'indique l'amélioration significative des deux composantes de la relaxation et la réduction des réponses contractiles dépendantes de l'endothélium. La consommation chronique de la formulation EPA:DHA 6:1 a significativement diminué la surexpression induite par l'Ang II de la eNOS, de l'arginase-1, des COX-1 et COX-2, et a prévenu la sous-expression de SKCa et Cx37. La supplémentation alimentaire en acides gras omega-3 améliore aussi la fonction endothéliale chez l'homme, sans pour autant affecter les

  conclusion, la présente étude a évalué le potentiel d'une formulation optimisée EPA:DHA 6:1 à protéger la fonction endothéliale in vivo. Les résultats obtenus montrent que l'infusion d'Ang II est associée avec le développement dans les branches secondaires de l'artère mésentérique d'une dysfonction endothéliale affectant fortement la composante EDH de la relaxation ainsi que plus légèrement la composante NO. La dysfonction endothéliale induite par l'Ang II passe par un mécanisme redox-sensible impliquant la NADPH oxydase, COX-1 et COX-2. La consommation chronique de la formulation EPA:DHA 6:1 a prévenu la dysfonction endothéliale et l'hypertension induite par l'Ang II fort probablement en diminuant le stress oxydant conduisant à l'atteinte des composantes NO et EDH de la relaxation et à l'augmentation des réponses contractile dépendantes de l'endothélium. De nombreuses études supportent le rôle clé des acides gras omega-3 dans la prévention de la dysfonction endothéliale, de l'hypertension et des maladies cardiovasculaires. De plus amples études sont nécessaires pour déterminer les métabolites actifs des acides gras omega-3 en sus des resolvines, protectines, thromboxane A3, leucotriène 5 and lipoxines. Il serait aussi nécessaire de déterminer le degré d'imprégnation des tissus vasculaires en omega-3.

  :DHA 6:1 prevents the MCT-induced RV remodeling and hypertrophy RV morphology and function were evaluated by echocardiography. The MCT group presented a significant dilatation of the RV indicated by the increased RV area, end-diastolic and systolic diameters compared to the control group. The dilatation of the RV was associated with a decreased of the RV fractional area chance (RVFAC %) (control vs MCT: 42.4 ± 1.6 vs 27.6 ± 3.1). Oral intake of EPA:DHA 6:1 prevented RV remodeling by improving RV morphology and function (Fig. 2A-D).

  :DHA 6:1 treatment prevents MCT-induced vascular oxidative stress involving several sources in pulmonary arteriolesReactive oxygen species (ROS) have been proposed as a pathogenic mechanism underlying the vascular remodeling observed in MCT-induced PAH(5). The pulmonary vascular level of oxidative stress was assessed using the redox-sensitive fluorescent probe DHE. The MCT treatment increased the formation of ROS as indicated by the markedly increased the DHE fluorescence signal throughout the medial wall in comparison to the control group. These effects were significantly prevented by EPA:DHA 6:1 treatment (Figure6A). In order to determine the source of ROS, lung sections were treated with different inhibitors major vascular sources of ROS. The MCT-induced formation of ROS in the pulmonary arterioles wall was significantly inhibited by N-acetylcysteine (NAC, antioxidant), VAS-2870 (an NADPH oxidase inhibitor and antioxidant), L-NA (an eNOS inhibitor), indomethacin (a cyclooxygenase inhibitor) and by a combination of inhibitors of the mitochondrial respiration chain (MRK: myxothiazol, rotenone and KCN) suggesting the involvement of NADPH oxidase, COXs, uncoupled eNOS and the mitochondrial respiration chain (Fig.6B). The levels of NO -and O2 -in lungs was determined by electron paramagnetic resonance (EPR) in frozen tissues at liquid nitrogen temperature. The MCT treatment increased NO -and O2 - production in lungs. EPA:DHA treatment significantly prevented the increased of NO -and O2 - production (Fig.6 C, D).

  may contribute to the arterioles remodeling. An increased immunofluorescence level of endothelin-1 receptors (ETA, ETB) and angiotensin II receptor (AT1) throughout the pulmonary arterioles wall was observed in the MCT group (Fig7). The EPA:DHA 6:1 treatment prevents MCT-induced changes of ETA, ETB and AT1 expression in pulmonary arterioles (Fig.7).EPA:DHA 6:1 prevents MCT-induced endothelial dysfunction in the pulmonary arteryVascular reactivity was performed in primary pulmonary artery rings to evaluate the endothelial function using organ chambers. The MCT treatment significantly reduced the contractile responses to phenylephrine, and also the acetylcholine-induced endotheliumdependent relaxation of pulmonary artery rings pre-contracted with phenylephrine. The concentration-response curves were constructed both in absence or presence of an eNOS inhibitor (L-NA) to assess the role of the basal endothelial NO formation. The MCT treatment increased basal nitric oxide (NO) production. The EPA:DHA 6:1 prevented MCT-induced endothelial dysfunction as indicated by the increased relaxation to Ach (Fig.8).

  endothelium precedes pulmonary arterial smooth muscle cells proliferation and medial hypertrophy in the distal pulmonary vascular bed and the rise in pulmonary artery pressure Small pulmonary arterioles and RV remodeling is associated with an increased inflammatory infiltrate and oxidative stress as indicated by high levels of ROS formation throughout the pulmonary arterioles wall of MCT-treated rats. The characterization of the cellular sources of ROS in the small pulmonary arterioles indicated the involvement of several sources including NADPH oxidase, uncoupled eNOS, cyclooxygenase (COXs) and the mitochondrial respiration chain. Moreover, an up-regulation of several pro-oxidant enzymes including NADPH oxidase submits p22 phox and p47 phox , COX-1, and COX-2 is observed in the small pulmonary arteries of MCT-treated rats. Oral intake treatment of EPA:DHA 6:1 substantially attenuated the level of vascular oxidative stress and remodeling by reducing in arterioles as well as the upregulation of the NADPH oxidase submits p22 phox and p47 phox , COX-1 and 2 and eNOS expression in MCT-treated rat. Both in vitro and in vivo studies have shown that ROS promote cardiomyocyte hypertrophy as well as fibrosis[START_REF] Takimoto | Role of oxidative stress in cardiac hypertrophy and remodeling[END_REF] and also right ventricular failure in the MCT-induced PAH,[START_REF] Redout | Right-ventricular failure is associated with increased mitochondrial complex II activity and production of reactive oxygen species[END_REF]. EPA:DHA 6:1 due to its antioxidant, antiinflammatory and cardioprotective properties, improved right ventricular function as indicated by an increased CO and with inhibition of cardiomyocyte hypertrophy and fibrosis. Endothelial dysfunction has been shown to plays a key role in the development of PAH and results in a decrease of vasodilatator and antiproliferative factors (prostacyclin, nitric oxide) and in an increase in vasoconstrictor and proliferative factors (endothelin [ET]-1) (49).Moreover, inflammation and oxidative stress have been shown to contribute to the MCTinduced endothelial dysfunction most likely by reducing the bioavailability of NO and oxidizing tetrahydrobiopterin, an essential cofactor for eNOS(5,[START_REF] Chaumais | N-acetylcysteine improves established monocrotaline-induced pulmonary hypertension in rats[END_REF]. Although decreased endothelium-dependent relaxation was observed in the pulmonary artery (50). The present findings indicated that MCT-induced pulmonary hypertension is associated with an endothelial dysfunction in main pulmonary artery without any effect on systemic vascular functions. The MCT treatment significantly reduced the contractile response of pulmonary artery rings to α-adrenergic agonist PHE. Blockade of NO production by L-NA enhanced PHE potency in MCT-treated rats. In addition, ACh-induced endothelium-dependent relaxation was reduced in pulmonary arteries of MCT treated-rats. Although the reduced ACh relaxation in the PH rats could be due to increased oxidative stress and decreased NO bioavailability in MCT treated-rats. The decreased bioavailability of NO reduces the antiproliferative effects of NO and thus contributes to the increased of pulmonary vascular remodeling and resistance. Previous studies have shown in isolated pulmonary arteries a reduced responsiveness to endothelium-dependent vasodilators including ACh and A23187, whereas others have suggested an increased basal NO production in the MCT-treated rats from[START_REF] Altiere | Altered pulmonary vascular smooth muscle responsiveness in monocrotaline-induced pulmonary hypertension[END_REF][START_REF] Mathew | Role of nitric oxide and endothelin-1 in monocrotaline-induced pulmonary hypertension in rats[END_REF]. We have showed that inflammation and oxidative stress of the lung in PAH stimulates the eNOS uncoupled. Oral intake of EPA:DHA 6:1 prevented the MCT-induced the development of PAH, reversed vascular remodeling, reduced vascular inflammation and improved the endothelial function, as indicated by an improvement ACh-induced relaxation.

  study indicates the ability of a formulation omega-3 to prevent MCT-induced PAH, in which inflammatory mechanisms related to pulmonary vascular endothelial dysfunction and remodeling may contribute to the development and progression of the pathology. A subsequent study will be necessary to determine the efficacy of omega-3 in the treatment of an established PAH. CONCLUSION The present findings indicate that EPA:DHA 6:1 exerts a significant anti-oxidant, antiinflammatory and endothelial protective effect in the pulmonary circulation and prevents the progression of pulmonary hypertension in MCT-treated rats by reducing pulmonary vascular remodeling, endothelial dysfunction, right ventricular hypertrophy and failure. The beneficial effects were associated with a reduced vascular inflammatory responses and oxidative stress mostly by preventing overexpression of NADPH oxidases, COXs and also of uncoupled eNOS. Further investigations are necessary to better assess the biological effects, elucidate the underlying mechanisms and to evaluate the political of this omega-3 formulation in the clinical setting.

Figure 1 .

 1 Figure 1. Oral intake of EPA:DHA 6:1 prevents MCT-induced pulmonary hypertension and right heart dysfunction. (A) Mean pulmonary artery pressure (mPAP) was monitored by catheter inserted into the jugular vein and advanced to the pulmonary artery. (B) Right ventricular systolic pressure (RVSP) was evaluated by echocardiography from tricuspid regurgitation velocity (RVSP = 4 × Vmax 2 ). (C) Cardiac output (CO) and (D) heart rate (HR) were monitored by pulsed wave Doppler. (E) Systemic arterial pressure (SAP). All measurements are presented as the average of three cardiac cycles. Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 2 .

 2 Figure 2. The EPA:DHA 6:1 treatment prevents MCT-induced right ventricular remodeling and hypertrophy. (A) Echocardiographic views of the right ventricle. (B) RV end-diastolic (RVEDD), (C) end-systolic diameter (RVESD), (D) RV fractional area changes (RVFAC). Images were obtained by two-dimensional and M-mode echocardiography from a parasternal short-axis view. (E) RV hypertrophy was assessed by Fulton's index, calculated as RV to LV weight ratio [RV/(LV+S)], (F) RV weight/body weight and (G) LV+S weight/body weight.Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 3 .

 3 Figure 3. The EPA:DHA 6:1 treatment prevents the MCT-induced RV cardiomyocytes hypertrophy and macrophages infiltration. (A) Quantitative morphometric analyses of cardiomyocytes section area were done using hematoxylin and eosin and gomori's blue trichrome staining of right and left ventricle walls. (B) Evaluation of macrophage infiltration was performed by quantitative analysis of the macrophages counted in 10 fields of immuhistochemical staining with CD68 antibody (20X, objective). Results are presented as representative micrographies (left) and corresponding cumulative data (right). Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 4 .

 4 Figure 4. The EPA:DHA 6:1 treatment prevents MCT-induced pulmonary artery remodeling. (A) Notching of the pulse wave Doppler profile in the pulmonary artery outflow, (B) pulmonary artery diameter, (C) pulmonary artery acceleration time (PAAT), (D) pulmonary vascular resistance (PVR) and (E) pulsatility. Pulse-wave Doppler of pulmonary outflow was recorded in the parasternal view at the level of the aortic valve. PAAT was measured from the time of onset of systolic flow to peak pulmonary outflow velocity. Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 5 .

 5 Figure 5. The EPA:DHA 6:1 treatment prevents pulmonary vascular remodeling, pulmonary macrophages and lymphocytes infiltration in MCT-treated rats. (A) Representative images of hematoxylin-eosin, gomori's blue trichrom and immunohistochemical (α-actin as a mesure of the dedree of muscularization) staining of lung section in rats for determination of the wall thickness and wall area of small pulmonary arteries sized < 20 μm. Images are shown on the left and quantification analysis on the right. Sixteen pulmonary arteries from 6 rats/experimental group were analyzed (n= 8). (B) Immunofluorescent (green) and immunohistochemical (purple arrows) staining with antibodies against CD68 [ED-1] for detection of macrophages and CD3 for lymphocytes T, respectively. Quantification of positive cells was analyzed of the fraction area occupied macrophages and lymphocytes T were counted in 10 different fields. Scale bar, 20 μm (20X objective). Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 6 .

 6 Figure 6. The EPA:DHA 6:1 treatment prevents MCT-induced vascular oxidative stress in pulmonary arterioles. (A) The determination of the vascular formation of ROS formation was done in unfixed cryosections of the right lung using the redox-sensitive probe dihydroethidium (DHE). (B) The characterization of the source of ROS formation was performed in presence of either N-acetylcysteine (NAC, antioxydant 1 mM), VAS-2870 ( NADPH oxidase inhibitor, 10 μM), N-nitro-L-arginine (LNA, NO synthase inhibitor, 300 μM), indomethacin (Indo, cyclooxygenase inhibitor, 10 μM), or MRK (inhibitors of the mitochondrial respiration chain, myxothiazol, 0.5 μM + rotenone, 1 μM + potassium cyanide (KCN), 1 μM) for 30 min before DHE staining. Thereafter, ethidium fluorescence was determined by confocal laser-scanning microscope (Leica SP2 UV DM IRBE). (C and D) Nitric oxide (NO.) and superoxide anion (O2.-) measured by electron paramagnetic resonance in small pulmonary arterioles. Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 7 .

 7 Figure 7. The EPA:DHA 6:1 treatment prevents MCT-induced changes of protein expression in pulmonary arterioles. Protein expression levels were determined in unfixed cryosections of right lung, the determination of the expression level of endothelial NO synthase (eNOS), NADPH oxidase subunits p22 phox and p47 phox , cyclooxygenase (COX-1 and COX-2), ET-1 receptors (ETA, and ETB) and the AT1R was done by immunofluorescence. All samples for immunofluorescence studies were observed using a confocal laser-scanning microscope (Leica SP2 UV DM IRBE).

Figure 8 .

 8 Figure 8. The EPA:DHA 6:1 treatment prevents MCT-induced endothelial dysfunction in the main pulmonary artery as assessed in organ chamber. The concentration-contraction curves in response to phenylephrine (A and B) and the concentration-relaxation curves to acetylcholine in pulmonary artery rings pre-contracted by phenylephrine (10 -6 mol/L) (C and D) were constructed in the absence (A and C) and the presence (B and D) of eNOS inhibitor (L-NA, 300 μM) to assess the role of the basal formation of endothelial NO. (n = 7 to11). Values are given as mean ± SEM. *P < 0.05 versus control, # P < 0.05 versus MCT.

Figure 1 .

 1 Figure 1.

12

  MHz, transducer. Two-dimensional short axis views of the left ventricle and M-mode tracings were recorded through anterior and posterior LV walls at the papillary muscle level. Morphological characterization of the cardiac left ventricle (LV) was assayed following the determination of these parameters: LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), posterior and septum diastolic wall thickness (PWT and SWT, respectively). Left ventricular mass (LVM) and LV ejection fraction (% LVEF) were subsequently derived from these parameters. The pulsed Doppler was used to assess the isovolumetric relaxation time (IVRT), measured as the interval between aortic closure and the start of the mitral flow.

  PE. The NO-mediated component of relaxation was determined in the presence of indomethacin (10 μM) and TRAM-34 (1 μM) plus apamin (100 nM) to inhibit the formation of prostanoids and EDH-mediated relaxation, respectively. The EDH-mediated component of the relaxation was determined in the presence of indomethacin (10 μM) and N ω -nitro-L-Data are expressed as means ± standard error of mean (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.

Acetylcholine Angiopoletin ATP BK Estrogen S1P Thapsigergin VEGF1 Constitutive O2 OX-LDL constitutive VEGF Hypoxia Adiponectin Fluid shear stress VEGF Bradykinin Fluid shear stress Insulin Angiotensin II VEGF Insulin Estrogen Fluid shear stress Bradykinin [Ca 2+ ]

	Activity
	-31 -

Table 1 . Structure and taxonomy of fatty acids (Simopoulos 1998)

 1

Table 2 . Structure of different unbranched fatty acids with a methyl end and a carboxyl (acidic) end (Hagan 2015).

 2

Table 1 .

 1 Characteristics of the different omega-3 fatty acid products

	Fatty acids Purity (in %)	Ratio EPA:DHA	Content of Omega-3 EPA-DHA as EE	Sum Omega-3 as EE (mg/g)
				EPA (mg/g)	DHA (mg/g)
	EPA:DHA	93.4	01:01	476	386	934
	EPA:DHA	91.3	06:01	694	121	913
	EPA:DHA	46.0	06:01	352	65	460
	EPA	99.5		991		995
	DHA	98.6			945	986

Table 2 .

 2 Echocardiography parameters of the inferior vena cava, the right atrium and the left ventricular.All

		Control	Omega	MCT	MCT+OM
	RA diameter (mm)	1.6 ± 0.1	1.8 ± 0.1	2.9 ± 0.2 *	2.2 ± 0.1 #
	RA area (mm 2 )	9 ± 0.2	9.1 ± 0.1	13.3 ± 0.1 *	10.2 ± 0.1 #
	IVC diameter (mm)	1.6 ± 0.2	1.8 ± 0.2	2.7 ± 0.1 *	2.4 ± 0.1
	IVC systolic area (mm 2 )	3.6 ± 0.7	3.6 ± 0.4	4.5 ± 0.3 *	3.9 ± 0.2
	LV Strock volume (ml)	0.32 ± 0.02	0.31 ± 0.01	0.20 ± 0.01 *	0.24 ± 0.01
	LV End-diastolic (mm)	7.9 ± 0.1	7.8 ± 0.1	6 ± 0.1 *	6.4 ± 0.1
	LV End-systolic (mm)	4.3 ± 0.1	4.4 ± 0.2	3.6 ± 0.1	3.7 ± 0.2
	Septal wall thikness (mm)	1.5 ± 0.1	1.6 0.1	1.8 ± 0.1	1.7 ± 0.1
	LVP wall thickness (mm)	1.7 ± 0.1	1.7 ± 0.1	1.9 ± 0.1	1.8 ± 0.1
	LV area (mm 2 )	53.3 ± 1.1	50.2 ± 3.4	35.1 ± 0.7	39.6 ± 2.0
	LV fractional shortening (%) 45.3 ± 1.5	42.9 ± 2.2	39.4 ± 1.4	42.5 ± 2.4
	LV ejection fraction (%)	70 ± 1.7	67.2 ± 2.4	62.1 ± 1.6	66.4 ± 2.9

values are mean ± SEM, n = 6-11/group. IVC, inferior vena cave, MCT, monocrotaline, LV, left ventricle; RV, right ventricle. * P < 0.05 versus control; # P < 0.05 versus MCT.

DHA 6:1 formulation prevents Angiotensin-II induced hypertension and endothelial dysfunction in rats

  .Potassium supplementation reduces cardiac and renal hypertrophy independent of blood pressure in DOCA/salt mice.Hypertension 46, 547-554. WHO., 2013. A global brief on hypertension: silent killer, global public health crisis. Wilcox, C.S., Pearlman, A., 2008. Chemistry and antihypertensive effectsof tempol and other nitroxides.Pharmacol. Rev. 60, 418-469.Zapata-Sudo, G., Silva, J.S., Pereira, SL., Souza, P.J.C., Moura RS.,Sudo R.T., 2014. Oral treatment with Euterpeoleracea Mart. (açaí) extract improves cardiac dysfunction and La présente étude évalue la capacité de EPA:DHA 6:1, une formulation d'omega-3 capable d'induire la formation continue de monoxyde d'azote par la NO synthase endothéliale, à prévenir l'hypertension et la dysfonction endothéliale induites par l'angiotensine II (Ang II) chez le rat. L'hypertension induite par l'Ang II est associée à une dysfonction endothéliale caractérisée par une altération des composantes de la relaxation et une augmentation des réponses contractiles dépendantes de l'endothélium. L'Ang II augmente le stress oxydant vasculaire et l'expression de NADPH oxydase, COXs, eNOS, et AT1R, alors que SKCa et connexin 37 sont sous-exprimés. EPA:DHA 6:1 prévient l'hypertension, la dysfonction endothéliale et la surexpression des protéines cibles. En conclusion, la consommation chronique de EPA:DHA 6:1 prévient l'hypertension et la dysfonction endothéliale induites par l'Ang II chez le rat, probablement en prévenant le stress oxydant dû à la NADPH oxydase et aux cyclooxygénases.

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	Résumé
	exercise	intolerance	in	rats	subjected	to	myocardial	infarction.	BMC
	Complement.Altern.Med. 14, 227.

Optimized EPA:

Chapter 2 Omega-3 and cardiovascular effects

British Pharmacological SocietyBritish Journal of Pharmacology

Georg Thieme Publishers KG, Rüdigerstraße 14, 70469 Stuttgart, GermanyManuscript Submitted to HMR

Acknowledgments

This study was supported by a grant of the Service de Coopération et d'ActionCulturelle of French Embassy of Côte d'Ivoire (Campus France N° 767294B and 796998H) and by a scholarship from the Ministère de l'EnseignementSupérieur et de la RechercheScientifique of Côte d'Ivoire (Décision N° 1548/MERS/DB/SD-BHCI/SD/CBK du 26/08/2015).

ER Estrogen Receptor FAME Fatty Acid Methyl-Esters FAs Fatty Acids GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase GC/MS Gas Chromatography/Mass Spectrometry GLA Gamma-Linolenic Acid GLC Gas Liquid Chromatography GPx Glutathione Peroxidase GTP Guanosine Triphosphate H2O2 Hydrogen Peroxide HDL High Density Lipoprotein HETEs Hydroxyeicosatetraeneoic Acids HO-1 Hemeoxygenase-1 HSP-90 Heat Shock Protein 90 IKCa Intermediate conductance calcium-activated potassium channel IL-6 Interleukin-6 Inos ω -Nitro-L-Arginine Methyl Ester LOX Lipoxygenases LPC Lysophosphatidylcholine LPS Lipopolysaccaride LTs Leukotrienes MAPKs Mitogen

effects on lipid profile, threshold for arrhythmias, platelets activity, inflammation, endothelial function, atherosclerosis and hypertension (Figure 22).

GENERAL DISCUSSION AND PERSPECTIVES

The omega-3 EPA:DHA 6:1 formulation prevents the monocrotaline-induced pulmonary hypertension, endothelial dysfunction and vascular remodeling in rats Amissi Said 1 , Zahid Rasul Niazi 1 , Mélanie Burban 1 , Romain Kessler 2 , Mathieu Canuet 3 , Florence

inflammation . oxidative stress

Conflict interests

The authors declare that they have no conflict of interest.

Authors' contributions

AS performed most of the experiments and analyzed data and wrote the manuscript. NZ and MB performed some experiments. AS, CA and VSK performed contributed to the discussion and wrote. RK, LM, NB and VSK designed the experiments and edited the manuscript.

Abstract

Ethnopharmacology relevance: Phyllanthusamarus(Euphorbiaceae family) has been reported in traditional medicine to possess beneficial effects in the management of hypertension. In different animal models of hypertension, an impairment of the vascular function has been linked to an endothelial dysfunction. Therefore, the aim of the present study was to determine if an aqueous extract of Phyllanthus amarus (AEPA)obtained by decoction was able to prevent hypertension and endothelial dysfunction in DOCA-salt rats, and, if so, to clarify the underlying mechanism.

Materials and methods:Male Wistar rats were randomly assigned into the control group, the AEPA group (100 mg/kg/day, by gavage), the DOCA-salt group (50 mg/kg, s.c, per week), and the DOCA-salt + AEPA group (100 or 300 mg/kg/day, by gavage). DOCA-salt-treated rats were allowed free access to water containing 1% NaCl. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography twice a week, in the morning during 5 weeks.

Vascular reactivity using main mesenteric artery rings was assessed in organ chambers. Dihydroethidine (DHE) and immunofluorescence methods were used for the determination of the vascular formation of reactive oxygen species (ROS) and the expression level of proteins, respectively.

Results: After 5 weeks, SBP (mmHg) increased significantly in DOCA-salt hypertensive rats.

It was significantly lowered by the treatment with AEPA (100 or 300 mg/kg/day) by 24 and 21mmHgrespectively. In mesenteric artery rings, the phenylephrine induced contractile response was increased significantly in the DOCA-salt group in comparison to the control group. After treatment by AEPA, the contractile response was shifted to the right. Both the NO-mediated (assessed in the presence of indomethacin and TRAM-34 plus apamin) and the endothelium-dependent hyperpolarization (EDH)-mediated (assessed in the presence of arginine (L-NA, 300 μM) to inhibit the formation of prostanoids and NO, respectively. In endothelial-intact rings, rings were contracted with an increasing concentration of PE (0.1 nM-10 μM) in order to obtain PE induced contractile responses.

Immunofluorescence studies

Main mesenteric arteries ring were removed, embedded in OCT compound (Tissue-Tek®, Sakura Finetek, Villeneuve d'Ascq, France) and snap-frozen in liquid nitrogen. Frozen arteries rings were cryosectioned at 14 μm. 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-100for 1 h at room temperature to block non-specific binding.

Next, overnight at 4 °C, mesentery artery sections were incubated with an antibody directed against either eNOS(1:50), NADPH oxidase subunits p22phox (1:50),cyclooxygenase-1(COX-1, 1:250), cyclooxygenase-2 (COX-2, 1:200), or connexin 37 (Cx, 1: 100). Sections were then washed with PBS, incubated with the secondary antibody (1/400, 633-conjugated goat anti-rabbit or anti mouse IgG ) for 2 h at room temperature in the dark before being washed with PBS and mounted in Dako fluorescence mounting medium (Dako, Carinteria, USA) and cover-slipped. For negative controls, primary antibodies were omitted. The samples were kept in the dark until, observation using a confocal laser-scanning microscope (Leica TSC SPE-Mannheim, Germany).Analyse for quantification of protein levels expression was performed using Image J software (NIH, Bethesda, Maryland, USA).

Determination of vascular reactive oxygen species formation

Statistical analysis

Conflits of Interests

The authors declare that they have no conflicts of interest