Faraj Zgheel

Mahmoud Alhosin

Cyril Auger

Valérie B Schini-Kerth

Zgheel Faraj

Sherzad K Rashid

Rasul Zahid

Thais Graziella C Silva

Porto Ribeiro

Zahid Rasul

to activate eNOS by phosphorylation at ser1177. 10èmes Journées du

The highly purified EPA:DHA 6:1 product evokes potent endothelium-dependent relaxations of porcine coronary artery involves a copper-dependent event triggering the redox-sensitive PI3kinase/Akt-dependent

LIST OF FIGURES AND TABLES

ABSTRACT

The endothelium lining the inner wall of blood vessels is a complex, highly specialized and metabolically active organ, which plays a pivotal role in the maintenance of vessel wall EPA and DHA, the major omega-3 fatty acids, induced concentration-dependant relaxations in coronary artery rings with functional endothelium, whereas these relaxations were absent in rings without endothelium. 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 endotheliumdependent 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).

The endothelium-dependent relaxation induced by the high purity omega-3 EPA:DHA 6:1 optimized formulation was slightly but significantly inhibited by N Ȧ -nitro-L-arginine (eNOS inhibitor) but not affected by the EDH inhibitors (TRAM 34 plus apamin), suggesting that EPA:DHA 6:1 exerts its vasorelaxant properties mainly through the activation of eNOS.

Moreover, the NO-mediated relaxation was significantly reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, JNK or MEK, indicating that EPA:DHA 6:1 induces NO activation through a redox-sensitive activation of PI3-kinase/Akt and MAPKs pathways.

In cultured endothelial cells, EPA: DHA 6:1 induced a time-dependent phosphorylation of p38 MAPK, ERK, JNK, Src, Akt and eNOS that were markedly reduced by MnTMPyP and PEG-catalase, confirming that EPA:DEA 6:1 induces the redox-sensitive activation of eNOS through PI3-kinase/Akt and MAPKs pathways. Indeed, EPA:DHA 6:1 induced the formation of ROS in cultured endothelial cells including both superoxide anions and hydrogen peroxide.

In conclusion, the ability of omega-3 fatty acids to stimulate the endothelial formation of NO is dependent on the ratio and amount of the EPA:DHA formulation and it involved an original pathway triggered by the intracellular oxidative stress leading to the activation of the Src/PI3kinase/Akt and MAPKs pathways to increase ultimately eNOS activity.

The second study evaluated the potency of the optimized EPA:DHA 6:1 formulation to prevent the platelet-induced vasocontraction, and determine the underlying mechanisms. In porcine coronary artery rings, human washed platelets induced dose-dependent contractions that were significantly inhibited by the 5-HT 2A receptor antagonist ketanserin, suggesting that the platelet-induced contraction is mainly due to the release of serotonin from activated platelets. Moreover, the short-term incubation of the rings with the EPA:DHA 6:1 optimized formulation, but not with the EPA:DHA 1:1 formulation, resulted in the abolition of the platelet-induced contraction. The EPA:DHA 6:1 formulation also dose-dependently prevented the 5-HT-induced contraction, whereas it was without effect on that induced by the thromboxane A 2 analogue (U46619). The fact that the prevention of the platelets-induce contraction by EPA:DHA 6:1 0.4% (v/v) was abolished both in absence of a functional endothelium and in presence of an eNOS inhibitor suggests that the EPA:DHA 6:1 exert its inhibitory effects through an increased endothelial formation of NO.

Moreover, similar results were observed in human internal mammary artery rings, where the EPA:DHA 6:1 formulation at 0.4 % (v/v) significantly prevented the 5-HT-induced contraction and induced vasorelaxation through NO-mediated mechanisms.

Altogether, these findings indicate that the optimized EPA:DHA 6:1 formulation is able prevents the 5-HT-mediated platelet-induced contraction in porcine coronary and human internal mammary artery rings through endothelium-dependent NO-mediated mechanisms.

In conclusion, the present work has assessed the potency of EPA:DHA formulations to protect the endothelial function ex vivo. The ability of EPA:DHA formulations to induce endothelium-dependent relaxations is dependent on both the ratio and the purity of the formulation, with the maximal effect obtained with a highly purified EPA:DHA 6:1 ratio. which is thought to contribute to the initiation and the development of these diseases.

The endothelium

Blood vessels, including arteries, capillaries, and veins are part of the circulatory system with the heart. The YHVVHO ¶V ZDOO H[FHSW IRU WKe smallest, consists of three individualized layers: the intima, the media and the adventitia (Figure 1). The intima (tunica interna) is the innermost layer, which is characterized by the presence of the endothelium, a monocellular layer located at the interface between the blood flow and the vessel wall, supported by connective tissue and the internal elastic lamina. The media (tunica media) is mainly build of smooth muscle cells with an elastic fiber layer, the later increasing with the size of the artery. The adventitia (tunica externa or tunica adventitia) is the outermost layer containing collagen and elastin, fibroblastes, macrophages, vasa vasorum and fibers which protect the blood vessels [START_REF] Mulvany | Structure and function of small arteries in hypertension[END_REF].

The endothelium, which covers the luminal surface of all blood vessels, plays a pivotal role in the regulation of blood flow and of blood homeostasis, in part through the synthesis and secretion of various active molecules, including vasodilatating factors and vasoconstricting factors controlling vascular tone.

Control of vascular tone by the endothelium

In 1980, Furchgott and Zawadzki reported that acetylcholine induced relaxation in an arterial ring through the release of an endothelium-derived relaxing factor (EDRF) [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF]. Thereafter, it was shown that EDRF induces the relaxation of the vascular smooth muscle cells through the stimulation of the soluble guanylyl cyclase [START_REF] Ignarro | Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid[END_REF], and that EDRF is inactivated by superoxide anions [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]. EDRF was later identified as the free radical gas nitric oxide (NO). After this discovery, it became clear that endothelial cells have a pivotal role in the regulation of vascular homeostasis, endothelial permeability, smooth muscle cell proliferation, as well as of the immune response [START_REF] Santiago-Delpin | The endothelium and early immune activation: new perspective and interactions[END_REF]. The regulation of vascular homeostasis mainly consists in the ability of the endothelium to maintain a balance between vasodilatation and vasoconstriction (Figure 2), and to modulate the proliferation and migration of smooth muscle cells, thrombosis and fibrinolysis, through the release of various factors [START_REF] Davignon | Role of endothelial dysfunction in atherosclerosis[END_REF]. Endothelial cells also respond to physical and chemical stimuli such as pressure, shear stress, and pH [START_REF] Feletou | Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)[END_REF] and to numerous physiological stimuli such as circulating hormones, by releasing various factors, known as endothelium-derived vasoactive factors, which include both endothelium-derived relaxing factors and endothelium-derived contraction factors [START_REF] Moncada | Nitric oxide and the vascular endothelium[END_REF]. The main endothelium-derived vasorelaxing factors include nitric oxide (NO), endothelium-dependent hyperpolarization (EDH) and prostacyclin (PGI 2 ). The endothelium-derived contracting factors include reactive oxygen species (ROS), thromboxane A 2 (TxA 2 ) angiotensin II, prostaglandin H 2 (PGH 2 ), and endothelins [START_REF] Feletou | Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)[END_REF][START_REF] Mombouli | Endothelial dysfunction: from physiology to therapy[END_REF].

The endothelium-derived vasorelaxing factors 1.2.1.1 Nitric oxide (NO)

The endothelium-derived nitric oxide, or NO, is produced by the endothelial NO synthase (eNOS) from L-arginine, and plays a critical role in normal vascular biology and pathophysiology. 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). The two isoforms nNOS and eNOS are being constitutively expressed whereas iNOS is an inducible isoform [START_REF] Mombouli | Endothelial dysfunction: from physiology to therapy[END_REF][START_REF] Stuehr | Mammalian nitric oxide synthases[END_REF]. NO diffuses towards the underlying smooth muscle cells to active soluble guanylyl cyclase by converting GTP into F\FOLF JXDQRVLQH ¶- ¶PRQRSKRVSKDWH (cGMP) leading to smooth muscle cells relaxation (Figure 3). In addition, NO exerts several vasoprotective and anti-atherogenic effects including inhibition of platelets aggregation, monocyte adhesion, vascular smooth muscle cell migration and proliferation, oxidation of LDL, expression of pro-inflammatory and proatherothrombotic mediators such as monocyte chemoattractant protein-1 (MCP-1), adhesion molecules and tissue factor [START_REF] Dimmeler | Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like 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][START_REF] Tsao | Fluid flow inhibits endothelial adhesiveness. Nitric oxide and transcriptional regulation of VCAM-1[END_REF].

The eNOS enzyme can be activated either by receptor-dependent or receptor-independent mechanisms, leading to an increase of the intracellular concentration of cytosolic free calcium ([Ca 2+ ] i ) and the subsequent formation of the Ca 2+ /calmodulin (CaM) complex that binds to eNOS [START_REF] Fleming | Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity[END_REF]. Indeed, both the NO formation and the subsequent vasorelaxation are abolished by the removal of calcium from the extracellular space as well as by calmodulin antagonists [START_REF] Fleming | Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity[END_REF]. eNOS can also be regulated in endothelial cells at a post-translational level primarily through protein/protein interactions and multisite phosphorylations. The main sites of phosphorylation on eNOS are at the residue Ser1177 and Ser 615 (activating sites), and the residue Thr497 and Ser114 [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] Bohm | Combined endothelin receptor blockade evokes enhanced vasodilatation in patients with atherosclerosis[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)-32-like proteases[END_REF]). Moreover, eNOS has been shown to be regulated by the interaction with positive and negative protein modulators such as caveolin-1 (Cav-1) and heat shock protein 90 [START_REF] Garcia-Cardena | Dynamic activation of endothelial nitric oxide synthase by Hsp90[END_REF][START_REF] Ju | Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity[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]). In the basal state, the majority of eNOS appears to be bound to caveolin-1 with its enzymatic activity being repressed in the caveolae [START_REF] Michel | Reciprocal regulation of endothelial nitricoxide synthase by Ca2+-calmodulin and caveolin[END_REF].

This tonic inhibition of eNOS can be released by displacing caveolin-1 with Ca 2+ /CaM in response to Ca 2+ -mobilizing agonists [START_REF] Ju | Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity[END_REF]). In addition to these modulators, phosphorylation of eNOS at key regulatory sites plays an important a role in the regulation of enzyme activity in response to several physiological stimuli [START_REF] Ju | Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity[END_REF]. It has been shown that 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 estrogens [START_REF] Dimmeler | Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases[END_REF][START_REF] Fulton | Regulation of endothelium-derived nitric oxide production by the protein kinase Akt[END_REF]). In addition, eNOS can be also activated on Ser1177 by AMP-activated protein kinase [START_REF] Busse | EDHF: bringing the concepts together[END_REF], protein kinase A (PKA), and protein kinase G (PKG). (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].

The activity of eNOS can also be regulated by post-translational modification. As eNOS is constitutively expressed, phosphorylation plays an important role in regulating the activity of the enzyme. eNOS can be phosphorylated on serine, threonine and tyrosine residues leading to eNOS activation or inactivation. There are various putative phosphorylation sites, but the most extensively studied eNOS residues, the phospho-status of which determines enzyme activity, are a serine residue (human eNOS sequence: Ser1177; bovine sequence Ser1179) in the reductase domain, which positively regulates NO production, and a threonine residue (human eNOS sequence: Thr495; bovine sequence Thr497) within the CaM-binding domain [START_REF] Boo | Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism[END_REF]. Another eNOS regulator is the ischemia-reperfusion injury which leads to eNOS phosphorylation at Ser1177 and Ser 633 through the activation of PKA pathway [START_REF] Li | Tongxinluo reduces myocardial no-reflow and ischemia-reperfusion injury by stimulating the phosphorylation of eNOS via the PKA pathway[END_REF]. Moreover, there are various 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] Chen | Tannin 1-alpha-Ogalloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells[END_REF][START_REF] Michell | The Akt kinase signals directly to endothelial nitric oxide synthase[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].

The endothelium-dependent hyperpolarization (EDH)

The EDH was previously known as the Endothelium-Derived Hyperpolarizing Factor (EDHF) and is another important mechanism of endothelium-dependent relaxation mostly observed in medium to small calibre resistance arteries, small arteries and arterioles such as second and third-branch mesenteric artery [START_REF] Feletou | Endothelium-derived hyperpolarizing factor[END_REF], as well as in coronary arteries. Indeed, the role of EDH is more important in resistance vessels than that of NO and prostacyclin [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 endotheliumdependent relaxations in rat mesenteric circulation[END_REF].

The mechanism of EDH involves the hyperpolarization of endothelial cells induced by activation of both small and intermediate conductance calcium-activated potassium channels (SK Ca and IK Ca , respectively), leading to the exit of potassium ions from the intracellular compartment to the extracellular one [START_REF] Edwards | K+ is an endotheliumderived hyperpolarizing factor in rat arteries[END_REF]. Thereafter in smooth muscle cells, the higher concentration of potassium ions in the extracellular space can activate the inwardly rectifying K + channel (K IR ) and Na + /K + -ATPase to cause hyperpolarization of smooth muscle cells through entry of potassium ions [START_REF] Edwards | K+ is an endotheliumderived hyperpolarizing factor in rat arteries[END_REF][START_REF] Feletou | Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)[END_REF]. Moreover, Edwards et al. have reported that a direct transfer of the hyperpolarization from endothelial cells to smooth muscle cells can also occur via myoendothelial gap junctions [START_REF] Edwards | K+ is an endotheliumderived hyperpolarizing factor in rat arteries[END_REF]). The factors involved in EDH seems to differ depending on the species of the animal and the vascular bed considered, and have been reported to include hydrogen peroxide H 2 O 2 [START_REF] Matoba | Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries[END_REF], and arachidonic acidderived metabolites including epoxyeicosatrienoic acids [START_REF] Quilley | Is EDHF an epoxyeicosatrienoic acid?[END_REF].

Prostacyclin (PGI 2 )

Prostacyclin (PGI 2 ) is a metabolite of arachidonic acid generated by vascular cells and endothelial cells. Arachidonic acid is converted to prostaglandin H 2 (PGH 2 ) by the action of cyclooxygenases, and PGH 2 is then further metabolized into PGI 2 by the prostacyclin synthase [START_REF] Moncada | The role of prostacyclin in vascular tissue[END_REF]. PGI 2 a potent vasodilator that binds the prostaglandin receptor (IP), a G-protein coupled receptor on the vascular smooth muscle cells, leading to an increase in cAMP levels [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF]. Thereafter cAMP can activate the protein kinase A leading to a reduction in the cytosolic Ca 2+ level in smooth muscle cells through Ca 2+ uptake by the endoplasmic reticulum [START_REF] Kukovetz | Prostacyclin increases cAMP in coronary arteries[END_REF]). In addition, PGI 2 is a potent inhibitor of platelet aggregation and adhesion, whereas the inhibition of PGI 2 synthesis promotes thrombus formation in the microcirculation [START_REF] Smith | Prostaglandins and platelet aggregation[END_REF]). Moreover, it has been reported that PGI 2 can enhance the release of NO in endothelial cells [START_REF] Mustard | Prostaglandins and platelets[END_REF]Shimokawa et al. 1988a).

Endothelium-derived contracting factors (EDCFs)

Endothelium-dependent relaxing factors are important for the regulation of vascular homeostasis. However, under various physical and chemical stimuli such as noradrenaline, thrombin, hypoxia, increased transmural pressure and mechanical stretch and circulating hormones (De Mey andVanhoutte 1982, 1983;[START_REF] Harder | Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium[END_REF][START_REF] Katusic | Endothelium-dependent contraction to stretch in canine basilar arteries[END_REF]Shimokawa et al. 1988a), endothelial cells can release contracting factors, which include thromboxane A 2 (TXA 2 ), prostaglandin H 2 (PGH 2 ), reactive oxygen species (ROS), endothelin-1 (ET1), and angiotensin II [START_REF] Taddei | Mechanisms of endothelial dysfunction: clinical significance and preventive non-pharmacological therapeutic strategies[END_REF][START_REF] Vanhoutte | Endothelium-dependent contractions in hypertension[END_REF].

Reactive oxygen species (ROS)

ROS are produced and 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] ), hydroxyl radicals (OH Ɣ-) and hydrogen peroxide (H 2 O 2 ), which can promote vasoconstriction by facilitating the mobilization of cytosolic Ca 2+ and/or by promoting Ca 2+ sensitization of the contractile elements [START_REF] Jin | Reactive oxygen-mediated contraction in pulmonary arterial smooth muscle: cellular mechanisms[END_REF]. In addition, ROS and in particular superoxide anions

(O 2 Ɣ-
) can also act directly or indirectly as potent contracting agents via the reduction of the NO bioavailability or by activating COX S 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 | Impairment of endothelium-dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril[END_REF]). Moreover, ROS can also impair EDH-mediated endothelium-dependent 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] Aubin | Role of probucol on endothelial dysfunction of epicardial coronary arteries associated with left ventricular hypertrophy[END_REF][START_REF] Kanani | Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans[END_REF][START_REF] Liu | Impairment of endothelium-dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril[END_REF]).

Angiotensin II (Ang II)

Angiotensin II is the major bioactive peptide of the renin±angiotensin system (RAS)

and is a multifunctional hormone responsible for the regulation of blood pressure and cardiovascular homeostasis. The liver-synthetized precursor peptide angiotensinogen is cleaved by the kidney-produced enzyme renin, leading to angiotensin I. Angiotensin I, a biologically inactive peptide is further converted by angiotensin-converting enzyme (ACE) to the biologically active octapeptide Ang II. Then, Ang II can be converted to the vasorelaxant peptide Angiotensin (1-7) by plasma or neutral endopeptidases [START_REF] Schindler | Role of the vasodilator peptide angiotensin-(1-7) in cardiovascular drug therapy[END_REF]). Ang II exerts its diverse actions mainly via two G-protein-coupled receptors, Ang II type 1 (AT1R) and type 2 (AT2R) receptors [START_REF] Touyz | Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells[END_REF]. These effects include vasocontraction, induction of growth, apoptosis, fibrosis, matrix metalloproteinase expression and extracellular matrix degradation [START_REF] Griendling | NAD(P)H oxidase: role in cardiovascular biology and disease[END_REF][START_REF] Tomita | Early induction of transforming growth factorbeta via angiotensin II type 1 receptors contributes to cardiac fibrosis induced by longterm blockade of nitric oxide synthesis in rats[END_REF]. It is wellknown that increased Ang II formation is associated with vasoconstriction and vascular structural changes [START_REF] Gibbons | Vasculoprotective and cardioprotective mechanisms of angiotensinconverting enzyme inhibition: the homeostatic balance between angiotensin II and nitric oxide[END_REF]. Such effects are observed in hypertension, atherosclerosis, and portal hypertension [START_REF] Kane | Endothelium-derived contracting factors mediate the Ang II-induced endothelial dysfunction in the rat aorta: preventive effect of red wine polyphenols[END_REF]. The angiotensin II type 1 receptor (AT1R) is a G protein receptor coupled to Gq. Upon the binding of Ang II, AT1R induces the activation of phospholipase C, the mobilization of intracellular Ca 2+ and the activation of mitogenactivated protein kinase in vascular smooth muscle cells, leading to contraction and vascular remodeling [START_REF] Griendling | NADH/NADPH Oxidase and Vascular Function[END_REF]. Such effects may involve production of ROS, particularly O 2 Ɣ-and H 2 O 2 [START_REF] Berry | Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries[END_REF].

Vasocontracting prostanoids

In endothelial cells, the prostaglandin H 2 (PGH 2 ) is generated by the metabolism of arachidonic acid (AA) by COX-1 and COX-2. PGH 2 is further converted to thromboxane A 2 (TXA 2 ) by the action of the thromboxane A synthase in particular in activated platelets. TXA 2 is a potent inducer of platelet aggregation. Moreover, binding of TXA 2 to thromboxane-prostanoid (TP) receptors induces smooth muscle cell contraction and proliferation via increased intracellular Ca 2+ levels [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF][START_REF] Vanhoutte | Endothelium-dependent contractions: when a good guy turns bad![END_REF]. The maintenance of vascular homeostasis needs a fine regulation of the TXA 2 /PGI 2 ratio, due to their opposing functions. The ratio has been suggested to be more important than the individual levels of both prostanoids in pulmonary vascular disease [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].

Endothelins (ET)

Endothelins are a family of peptides, which are potent contracting factors of blood vessels and they can raise blood pressure [START_REF] Schiffrin | Vascular endothelin in hypertension[END_REF]. ET-1 is released by endothelial cells, following the proteolytic process of its precursor, big-endothelin, by endothelin converting enzyme-1 [START_REF] Agapitov | Role of endothelin in cardiovascular disease[END_REF]. Several cytokines, including interleukin-1 and TNF-Į increase the production and release of ET-1, whereas it is inhibited by NO and PGI 2 [START_REF] Alonso | The nitric oxide-endothelin-1 connection[END_REF]. In addition, the activation on smooth muscle cells of endothelin receptors ET A or ET B2 causes the opening of Ca 2+ channels, leading to the entry of extracellular Ca 2+ and subsequent contraction [START_REF] Agapitov | Role of endothelin in cardiovascular disease[END_REF]. In contrast, the activation of ET B1 receptors on endothelial cells causes vasodilatation by inducing the release of NO and PGI 2 [START_REF] Cardillo | Interactions between nitric oxide and endothelin in the regulation of vascular tone of human resistance vessels in vivo[END_REF]. Moreover, studies have shown that a decreased expression of ET B1 receptors on endothelial cells and/or an increased expression of ET B2 receptors on smooth muscle cells contribute to the development of endothelial dysfunction [START_REF] Bohm | Combined endothelin receptor blockade evokes enhanced vasodilatation in patients with atherosclerosis[END_REF].

Arachidonic acid (AA)

Arachidonic acid is metabolized by three distinct enzyme systems including cyclooxygenases, lipoxygenases and cytochrome P450-dependent epoxygenases [START_REF] Fitzpatrick | Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of "epoxygenase"-derived eicosanoids[END_REF]. There are two isoforms of cyclooxygenase, each having the capacity to catalyze the conversion of arachidonic acid to prostaglandins. The first isoform, COX-1, is constitutively expressed in most tissues, whereas the second, COX-2, is inducible (Figure 8).

Studies have shown that the two isoforms are involved in the generation of EDCF [START_REF] Miller | Endothelium-dependent contractions to arachidonic acid are mediated by products of cyclooxygenase[END_REF][START_REF] Tang | Prostanoids and reactive oxygen species: team players in endothelium-dependent contractions[END_REF]. Moreover, a study suggests that COX-1, but not COX-2, is responsible for the cyclooxygenase-derived endothelium-dependent contraction in the spontaneously hypertensive rats [START_REF] Tang | Endotheliumdependent contractions occur in the aorta of wild-type and COX2-/-knockout but not COX1-/-knockout mice[END_REF].

Endothelial dysfunction

Endothelial dysfunction is a pathophysiological condition characterized by impaired endothelium-dependent relaxation and vascular responses [START_REF] Abraham | How does endothelial cell injury start? The role of endothelin in systemic sclerosis[END_REF]. The endothelial dysfunction involves an imbalance between the formation of the endotheliumderived vasodilatating and vasoconstricting substances (Deanfield et al. 2005;Schini-Kerth et al. 2011), as well as growth-promoting and growth-inhibiting factors and also of proatherogenic and anti-atherogenic factors [START_REF] Quyyumi | Endothelial function in health and disease: new insights into the genesis of cardiovascular disease[END_REF]. 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 molecular-1 (VCAM-1) expression [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 eNOS uncoupling.

ROS (O 2.-) 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 (BH 4 ) and the subsequent uncoupling of eNOS thereby further promoting oxidative stress [START_REF] Cai | Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress[END_REF].

Hypertension

Hypertension is a chronic medical condition that affects more than 30% of the adult population in developed countries. This condition is characterized by a disruption of the balance between the endothelium-mediated vasodilatation and vasoconstriction towards the predominance of vasoconstriction [START_REF] Nadar | Endothelial dysfunction: methods of assessment and application to hypertension[END_REF]). Many studies using animal models and hypertensive subjects have demonstrated that hypertension is associated with endothelial dysfunction. Moreover, in spontaneously hypertensive rats (SHR), endothelial dysfunction is associated with an increased vascular oxidative stress, due to an increased expression and activity of NADPH oxidases and uncoupling of eNOS [START_REF] Sanchez | Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats[END_REF]). In addition, Ang II-induced hypertension is associated with an over-production of superoxide anions in both endothelial cells and smooth muscle cells [START_REF] Lee | An ethanolic extract of Lindera obtusiloba stems causes NO-mediated endotheliumdependent relaxations in rat aortic rings and prevents angiotensin II-induced hypertension and endothelial dysfunction in rats[END_REF][START_REF] Rajagopalan | Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone[END_REF].

Moreover, the accumulation of Ang II in the vascular wall has been observed mostly due to an overexpression of Angiotensin-converting enzyme (ACE) in blood vessels of hypertensive animals [START_REF] Harrison | Endothelial function and oxidant stress[END_REF].

Diabetes Mellitus

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 [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 endothelium-dependent vasodilatation in the initiation and development of both macro-vascular and micro-vascular complications of diabetes. Moreover, impaired endothelium-dependent relaxations are also observed in healthy-non diabetic subjects who are first degree relatives of type 2 diabetic patients, and associated with an increased plasma level of markers of endothelial cell activation and systemic pro-inflammatory markers [START_REF] Balletshofer | Endothelial dysfunction is detectable in young normotensive firstdegree relatives of subjects with type 2 diabetes in association with insulin resistance[END_REF][START_REF] Caballero | Microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes[END_REF][START_REF] Tesauro | Vascular, metabolic, and inflammatory abnormalities in normoglycemic offspring of patients with type 2 diabetes mellitus[END_REF]). Increased vascular oxidative stress is another important mechanism of the development of endothelial dysfunction in diabetes, and involves both superoxide anions and hydrogen peroxide, predominantly due to an upregulation of NADPH oxidase, and possibly also to an uncoupling of eNOS [START_REF] Gao | Vascular NAD(P)H oxidase activation in diabetes: a double-edged sword in redox signalling[END_REF]. The increased vascular oxidative stress impairs NO bioavailability in the vascular wall and EDH-mediated relaxation, in part, by reducing the expression the calcium-activated potassium channels (IK ca and SK ca ) [START_REF] Urso | Oxidative stress and endothelial dysfunction[END_REF]. Moreover, the COX-derived arachidonic acid metabolites have been associated with endothelium-dependent contractions in diabetes [START_REF] Feletou | Endothelium-derived vasoactive factors and hypertension: possible roles in pathogenesis and as treatment targets[END_REF]. Taken together, these findings suggest that the endothelial dysfunction associated with diabetes mellitus could be a main contributor to the increased risk of cardiovascular diseases in diabetic patients.

Atherosclerosis

Atherosclerosis is a multifactorial disease that is the major cause of cardiovascular death. It affects arterial blood vessels and is associated with a chronic inflammatory response in the arterial wall [START_REF] Ross | The pathogenesis of atherosclerosis: a perspective for the 1990s[END_REF]. Atherosclerosis is primarily characterized by elevated circulating and vascular levels of low-density lipoprotein (LDL) particles, which can be associated with an increased interaction of monocytes, macrophages, and platelets with endothelial and smooth muscle cells. Atherosclerosis results from several pathological processes including inflammation in the vessel wall in response to retained LDL that become susceptible to oxidation by ROS, leading to oxidized LDL. LDL oxidation is also involved in the induction of arterial damages mediated by the immune system, through the recruitment of macrophages absorbing the oxidized-LDL, leading to the formation of foam cells.

Macrophages are not able to process the oxidized-LDL, and accumulate oxidized particles until they ultimately enter necrosis, depositing a greater amount of oxidized fatty acids and crystalized cholesterol into the arterial wall (Figure 12). The development of this necrotic core in the intima is associated with the migration and proliferation of underlying smooth muscle cells and fibroblasts, leading to the constitution of a fibrous cap, a hard covering of the necrotic core. The development of both necrotic core and fibrous cap causes the narrowing of the lumen of the artery, leading to a reduction and a perturbation of the blood flow [START_REF] Libby | Inflammation in atherosclerosis[END_REF]. Moreover, endothelial dysfunction has been reported to be the result of the accumulation of oxidized lipid derivatives such as lysophosphatidylcholine (LPC), an attenuated eNOS activity, and the stimulation of eicosanoids formation and release by COX, lipoxygenases, and cytochrome P450 (Millanvoye- [START_REF] Brussel | Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2[END_REF]). In addition, endothelial dysfunction is associated with the initiation and progression of atherosclerosis,

and with an increased level of vascular ROS [START_REF] Miller | Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis[END_REF]. The development of the atheroma, constituted of a soft necrotic core under a rigid fibrous cap, can lead to an instable situation prone to rupture or erosion. Such a rupture will expose pro-thrombotic factors that will induce the formation of an intra-arterial thrombus, thus drastically reducing the blood flow. The thrombus causes ischemia in the tissue irrigated by the artery, thus leading to myocardial infarction in the case of a coronary artery, stroke for a cerebral artery, and localised infarction for other vascular beds (mesenteric, renal, splenic, PXVFXODU HWF« [START_REF] Weiss | Deoxycorticosterone acetate salt hypertension in apolipoprotein E-/-mice results in accelerated atherosclerosis: the role of angiotensin II[END_REF]. The mechanism underlying intra-arterial thrombosis might be attributed to platelets adhesion to sub-endothelial glycoproteins exposed to the bloodstream when the endothelium is injured, including collagen, von Willebrand factor (vWF), fibronectin and vitronectin [START_REF] Brass | Thrombin and platelet activation[END_REF][START_REF] Eisenberg | Platelet-dependent and procoagulant mechanisms in arterial thrombosis[END_REF][START_REF] Kroll | Platelets and shear stress[END_REF]. Moreover, the activation of platelets plays a critical role in the development of arterial thrombosis due to their adhesion to vascular cells and generation of lipid peroxides, which lead to the inhibition of the endothelium-dependent relaxation through reduced NO bioavailability and decreased prostacyclin production [START_REF] Eisenberg | Platelet-dependent and procoagulant mechanisms in arterial thrombosis[END_REF].

Nutrition and dietary lipids

Many dietary studies have linked the daily intake of fat, such as polyunsaturated fatty acids, and coronary heart diseases [START_REF] Fiaccavento | Alpha-linolenic acidenriched diet prevents myocardial damage and expands longevity in cardiomyopathic hamsters[END_REF]. The Mediterranean diet which is characterized by a high consumption of fruit and vegetables and moderate consumption of fish and olive oil as major sources of fat has been shown to reduce the risk of cardiovascular diseases in the general population [START_REF] Kourlaba | Dietary quality indices and human health: a review[END_REF]. Findings from the Seven Countries Studies reported a low cardiovascular mortality in Southern Europe compared to Northern Europe and the US [START_REF] Renaud | Cretan Mediterranean diet for prevention of coronary heart disease[END_REF]. Results from meta-analyses have further suggested beneficial effects of the Mediterranean diet on CVD risk [START_REF] Sofi | Accruing evidence on benefits of adherence to the Mediterranean diet on health: an updated systematic review and meta-analysis[END_REF][START_REF] Sofi | Adherence to Mediterranean diet and health status: meta-analysis[END_REF]. The outcome of a meta-analysis including more than 2 million subjects and 50,000 deaths or incident cases from any causes and/or from any cardiovascular diseases, suggest significant protection of the adherence to the Mediterranean diet and the health status. These findings indicated an 8 % reduction of total mortality and 10 % reduction from death and/or incidence of cardio-and cerebrovascular diseases [START_REF] Sofi | Accruing evidence on benefits of adherence to the Mediterranean diet on health: an updated systematic review and meta-analysis[END_REF]).

Dietary omega-3 fatty acids

The inverse association of cardiovascular risk with intake of omega-3 polyunsaturated fatty acids was suspected early in populations that are known to have a high consumption of fish and fish oil. The beneficial effects of omega-3 fatty acid consumption were first reported in Greenland Eskimos who have a high consumption of marine-derived food including seal, walrus and whale [START_REF] Simopoulos | Omega-3 fatty acids in inflammation and autoimmune diseases[END_REF]. Indeed, they have a lower rate of cardiovascular diseases compared to the Danish population, whose diet contains about 22% of saturated fatty acid [START_REF] Bang | The composition of the Eskimo food in north western Greenland[END_REF][START_REF] Dyerberg | Fatty acid composition of the plasma lipids in Greenland Eskimos[END_REF]. Omega-3 fatty acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been reported to have several beneficial effects on cardiovascular risk factors such as reduced blood pressure, heart rate, triglycerides level, inflammation, and an improved endothelial function and cardiac diastolic level [START_REF] Abeywardena | Longchain n-3 polyunsaturated fatty acids and blood vessel function[END_REF][START_REF] Bhatnagar | Omega-3 fatty acids: their role in the prevention and treatment of atherosclerosis related risk factors and complications[END_REF][START_REF] Geelen | Antiarrhythmic effects of n-3 fatty acids: evidence from human studies[END_REF][START_REF] Thies | Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomised controlled trial[END_REF]. A prospective study has shown that consumption of fish or fish oil at least twice a week is associated with a significant reduction by 50% of the relative risk of fatal coronary heart disease [START_REF] Kromhout | The inverse relation between fish consumption and 20-year mortality from coronary heart disease[END_REF]. In 2004, the US Food and Drug Administration (FDA) reviewed the beneficial effects of an increased consumption of omega-3 fatty acids, in particular EPA and DHA from four randomized, controlled, clinical intervention trials. The outcome of the four studies has suggested that an increased intake of 850 mg/d of EPA, 882 mg/d of DHA and alpha-linolenic acid (ALA) is associated with a decreased risk of cardiovascular diseases defined as death, non-fatal myocardial infarction, and non-fatal stroke [START_REF] Burr | Diet and reinfarction[END_REF][START_REF] Ng | GISSI-Prevenzione trial[END_REF][START_REF] Singh | Randomized, double-blind, placebo-controlled trial of fish oil and mustard oil in patients with suspected acute myocardial infarction: the Indian experiment of infarct survival--4. Cardiovascular drugs and therapy / sponsored by the[END_REF][START_REF] Von Schacky | The effect of dietary omega-3 fatty acids on coronary atherosclerosis. A randomized, double-blind, placebo-controlled trial[END_REF]) in patients who had survived a recent myocardial infraction. Additional randomized trials have provided evidence that replacing unsaturated fatty acids by polyunsaturated fatty acids, particularly EPA and DHA, protects against cardiovascular risk, but not against cardiovascular diseases mortality [START_REF] Hooper | Reduced or modified dietary fat for preventing cardiovascular disease[END_REF]). More recently, epidemiological studies have suggested an association between increased consumption of fish and decreased risk for coronary heart diseases and cancer [START_REF] Daviglus | Fish consumption and the 30-year risk of fatal myocardial infarction[END_REF][START_REF] Kromhout | Dietary saturated and trans fatty acids and cholesterol and 25-year mortality from coronary heart disease: the Seven Countries Study[END_REF][START_REF] Lim | Omega-3 polyunsaturated fatty acids inhibit hepatocellular carcinoma cell growth through blocking beta-catenin and cyclooxygenase-2[END_REF], and inflammatory diseases [START_REF] Simopoulos | Omega-3 fatty acids in inflammation and autoimmune diseases[END_REF]). In the DART trial (diet and reinfarction trial), subjects recovering from myocardial infarction and advised to consume more fatty fish (two to three times a week) showed a 29% reduction in total mortality after 2 years compared to patients not advised to do so [START_REF] Burr | Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART)[END_REF]). These observations provide evidence that an increased consumption of omega-3 fatty acids, including EPA, DHA and Į-Linolenic acid (ALA), has a beneficial effect in primary and secondary prevention of cardiovascular diseases [START_REF] Albert | Blood levels of long-chain n-3 fatty acids and the risk of sudden death[END_REF][START_REF] Alter | Letter by Alter and Rupp regarding article, "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][START_REF] Marchioli | Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico (GISSI)-Prevenzione[END_REF]. Moreover, several mechanisms have been suggested to explain the beneficial effects of dietary omega-3 fatty acids on cardiovascular diseases. They include anti-inflammatory and anti-arrhythmic effects, and decreased triglycerides level, blood pressure, thrombosis, and platelet aggregation, as well as plaque stabilization [START_REF] De Lorgeril | Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease[END_REF][START_REF] Peter | A fish a day, keeps the cardiologist away! -A review of the effect of omega-3 fatty acids in the cardiovascular system[END_REF]. The beneficial effects of omega-3 fatty acids on the cardiovascular system may also be attributed, at least in part, to their ability to improve the endothelial function [START_REF] Wang | Effect of omega-3 fatty acids supplementation on endothelial function: a meta-analysis of randomized controlled trials[END_REF]). The omega-3 fatty acids can increase the endothelial release of vasoprotecting factors such as NO and endotheliumdependent hyperpolarization in human atherosclerotic arteries [START_REF] Tagawa | Long-term treatment with eicosapentaenoic acid augments both nitric oxidemediated and non-nitric oxide-mediated endothelium-dependent forearm vasodilatation in patients with coronary artery disease[END_REF], porcine coronary arteries [START_REF] Shimokawa | Effects of dietary supplementation with cod-liver oil on endothelium-dependent responses in porcine coronary arteries[END_REF], and in animal models of hypercholesterolemia [START_REF] Komori | Endothelium-dependent relaxation in response to aggregating platelets in porcine femoral veins and its modulation by diet[END_REF]Shimokawa and Vanhoutte 1988). Moreover, omega-3 fatty acids have also been suggested to increase the NO formation in endothelial cells due, at least in part, to changes in lipid composition in caveolae micro-domains and also changes in the localisation of caveolin-1 that may eventually lead of activation of eNOS [START_REF] Li | Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase[END_REF]).

Structure and classification of fatty acids

Lipids are defined as a group of organic compounds including fatty acids (FA), monoacylglycerols (MG), diacylglycerols (DG), triacylglycerols (TG), phospholipids (PL), and eicosanoids, resolvins, sterols, sterol esters, carotenoids, vitamins A, E, fatty alcohols, hydrocarbons and wax esters. Lipids are insoluble in water but soluble in organic compounds.

In the body, lipids serve as energy storage in adipose tissue, and play a central role in the production of various compounds involved in signalling pathways, including prostaglandins, thromboxanes and leukotrienes. It has been observed that dietary intake of fats such as polyunsaturated fatty acids affect a wide variety of physiological processes by modulating the lipid metabolism in the cell membrane. Moreover, lipids such as triacylglycerols and phospholipids are important for the regulation of membrane functions and activation of membrane-bound enzymes, receptors, and ion channels [START_REF] Clandinin | Relationship between fatty acid accretion, membrane composition, and biologic functions[END_REF]). The diverse biological effects of dietary lipids have been reported as deleterious in the case of excessive consumption of cholesterol and saturated fatty acids (SFAs), while polyunsaturated fatty acids are generally regarded as beneficial for health.

Fatty acids are classically classified into three families based on their degree of unsaturation, namely the saturated fatty acids (SFA), the monounsaturated fatty acids (MUFA) and the polyunsaturated fatty acids (PUFA; Table 1).

Saturated fatty acids (SFA)

Saturated fatty acids consist of an aliphatic chain (straight hydrocarbon chain) without any double bond. They are classified as short, medium and long chain, depending on the number of atoms of carbon in the chain. At room temperature, they are solid or semi-solid.

Saturated fatty acids such as lauric acid (C12:0), myristic acid (C14:0), stearic acid (C18:0), and palmitic acid (C16:0) are present in large quantities in certain food such as whole milk, cocoa butter, palm oil and coconut oil (Figure 13). Many clinical trials have shown the association of an increased consumption of saturated fatty acids with an increased low-density lipoprotein (LDL) cholesterol level in humans and animal models leading to an increasing risk of cardiovascular diseases like coronary heart diseases and cancer [START_REF] Hu | Dietary fat intake and the risk of coronary heart disease in women[END_REF][START_REF] Siri-Tarino | Saturated fat, carbohydrate, and cardiovascular disease[END_REF]).

Monounsaturated fatty acids (MUFA)

Monounsaturated fatty acids have a single double bond in the aliphatic chain (Figure 13). Monounsaturated fatty acids, such as oleic acid (C18:1n-9), palmitoleic acid (C16:1n-7)

and vaccenic acid (C18:1n-11), are found only in plants but in a wide variety of plants including sunflower, corn, soybean, peanut, palm, sesame, and cottonseed, with the highest amount being found in canola acid (rapeseed) oil and olive oil by 75% and 85%, respectively [START_REF] Schwingshackl | Monounsaturated fatty acids, olive oil and health status: a systematic review and meta-analysis of cohort studies[END_REF]. Clinical studies have shown that the consumption of mono or polyunsaturated fatty acids is associated with a reduced risk of cardiovascular diseases [START_REF] Skeaff | Dietary fat and coronary heart disease: summary of evidence from prospective cohort and randomised controlled trials[END_REF] and stroke [START_REF] Samieri | Olive oil consumption, plasma oleic acid, and stroke incidence: the Three-City Study[END_REF], as well as a reduction by 26% of the total mortality in the Spanish branch of the EPIC study [START_REF] Buckland | Olive oil intake and mortality within the Spanish population (EPIC-Spain)[END_REF].

Polyunsaturated fatty acids (PUFA)

Polyunsaturated fatty acids are fatty acids which are characterized by the presence of two or more double bonds in the aliphatic chain (Figure 14). The number following "Omega-"

represents the position of the first double bond, counting from the terminal methyl group on the molecule. Thus, omega-3 fatty acids have their first double bond between the 3 rd and the 4 th carbon atom from the methyl end of the molecule, whereas in omega-6 fatty acids the double bond is between the 6 th and 7 th carbon atom (Figure 14).

Trans-fatty acids

Trans-fatty acids are monounsaturated fatty acids that can be found in certain food items like meat and dairy products (Figure 15). They are also found in higher concentrations in partially hydrogenated vegetable oil, a process used for the conversion of vegetable oil in the agro-food industry. Several studies suggest that an increase intake of trans-fatty acids is associated with increased plasma level of low density lipoprotein (LDL) and total cholesterol, and a reduced levels of high density lipoprotein [START_REF] Lopez-Garcia | Consumption of trans fatty acids is related to plasma biomarkers of inflammation and endothelial dysfunction[END_REF][START_REF] Stampfer | A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction[END_REF], as well as an increased release of eicosanoids [START_REF] Hu | Types of dietary fat and risk of coronary heart disease: a critical review[END_REF], and promotion of inflammation by different mechanisms including increased production of TNF-Į DQG interleukin-6 in humans. All such effects will lead to an increased risk of cardiovascular diseases, endothelial dysfunction and type 2 diabetes mellitus [START_REF] Ascherio | Dietary fat and risk of coronary heart disease in men: cohort follow up study in the United States[END_REF][START_REF] Kasim-Karakas | Effects of dietary fat restriction on particle size of plasma lipoproteins in postmenopausal women[END_REF][START_REF] Kromhout | Dietary saturated and trans fatty acids and cholesterol and 25-year mortality from coronary heart disease: the Seven Countries Study[END_REF][START_REF] Salmeron | Dietary fat intake and risk of type 2 diabetes in women[END_REF][START_REF] Willett | Intake of trans fatty acids and risk of coronary heart disease among women[END_REF]). In

Essential fatty acids

The essential fatty acids are long-chain polyunsaturated fatty acids that play an essential role as structural components of all cell membranes as well as important sources of biologically active metabolites. In humans, there are only two essential fatty acids, namely alpha-linolenic acid and linoleic acid, which are required for survival and must be obtained through diet. Indeed, mammals do not have the enzymes necessary to introduce a double bond at the omega-3 or omega-6 positions in aliphatic chains longer than nine carbons. A study from Evans and Burr reported that mammals can use linoleic acid and alpha-linolenic acid to synthesize respectively the omega-6 fatty acids and omega-3 fatty acids [START_REF] Burr | Lessons from the story of n-3 fatty acids[END_REF], in particular EPA and DHA through elongation and desaturation mechanism. As the rate of conversion of linoleic acid is limited, most of omega-3 fatty acids are conditional essential fatty acids as the intake from diet represents the main source for these compounds. The alphalinolenic acid is mostly found in green leafy vegetables, in vegetable oils such as canola and soybean oils, and nuts such as flaxseed and walnuts. A number of studies suggest that an increased consumption of foods rich in ALA, like flaxseeds that contain about 35 % of ALA, might be associated with a reduced severity of cardiovascular diseases including myocardial infarction, atherosclerosis and hypertension in several animal models [START_REF] Bierenbaum | Reducing atherogenic risk in hyperlipemic humans with flax seed supplementation: a preliminary report[END_REF][START_REF] Bloedon | Flaxseed and cardiovascular risk[END_REF][START_REF] Craig | Phytochemicals: guardians of our health[END_REF][START_REF] Lucas | Flaxseed improves lipid profile without altering biomarkers of bone metabolism in postmenopausal women[END_REF][START_REF] Prasad | Flaxseed and cardiovascular health[END_REF]). It has also been reported that the intake of ALA is associated with a decreased blood pressure in spontaneously hypertensive rats [START_REF] Bacova | Intake of n-3 Polyunsaturated Fatty Acids Increases Omega-3 Index in Aged Male and Female Spontaneously Hypertensive Rats[END_REF]. Moreover, intake of polyunsaturated fatty acids such as ALA has been associated with a decreased platelets clotting activity [START_REF] Austria | Bioavailability of alpha-linolenic acid in subjects after ingestion of three different forms of flaxseed[END_REF][START_REF] Renaud | Dietary lipids and their relation to ischaemic heart disease: from epidemiology to prevention[END_REF][START_REF] Renaud | Influence of long-term diet modification on platelet function and composition in Moselle farmers[END_REF]). Moreover, dietary intake of omega-3 fatty acids, in particular of EPA and DHA, has been associated with a reduced risk of several chronic diseases including cardiovascular diseases [START_REF] He | Accumulated evidence on fish consumption and coronary heart disease mortality: a meta-analysis of cohort studies[END_REF][START_REF] Hu | Fish and omega-3 fatty acid intake and risk of coronary heart disease in women[END_REF]. The main dietary sources of omega-3 fatty acids in humans are the fatty fishes such as herring, mackerel, salmon, albacore tuna, and sardines (Figure 16). The beneficial effect of dietary fish and fish oil is associated with a reduction of cardiovascular diseases risk factors such as a lowering of plasma triglycerides, and blood pressure, and an improvement of the endothelial function [START_REF] Bays | Eicosapentaenoic acid ethyl ester (AMR101) therapy in patients with very high triglyceride levels (from the Multi-center, plAcebo-controlled, Randomized, double-blINd, 12-week study with an open-label Extension [MARINE] trial)[END_REF][START_REF] Gaibazzi | Reversibility of stress-echo induced ST-segment depression by long-term oral n-3 PUFA supplementation in subjects with chest pain syndrome, normal wall motion at stress-echo and normal coronary angiogram[END_REF].

Role of the ratio of omega-6 and omega-3 polyunsaturated fatty acids from our diet

Eicosanoids are 20 carbon molecules derived from omega-3 and omega-6 fatty acids which affect the inflammatory responses and the immune system. Eicosanoids are a family of compounds including prostaglandins, thromboxanes, leukotrienes, lipoxins and eoxins. A balanced intake of both omega-3 and omega-6 fatty acids is important for health because eicosanoids derived from either omega-3 or omega-6 have opposing metabolic properties.

Indeed, ALA is the precursor for the 3-series of prostaglandins and thromboxanes, and the 5series of leukotrienes, which are all associated with anti-inflammatory properties (Figure 17).

Conversely, arachidonic acid (AA) is the precursor for the 2-series of prostaglandins and thromboxanes, and the 4-series of leukotrienes, which are associated with pro-inflammatory properties (Figure 17). Thus, increasing the omega-6/omega-3 fatty acids ratio leads to a shift from a physiological state to a pro-thrombotic and pro-aggregatory state associated with increases in blood viscosity and vasospasms [START_REF] Simopoulos | Omega-6/omega-3 fatty acid ratio and trans fatty acids in non-insulindependent diabetes mellitus[END_REF]. Several governments and organizations have issued recommendations to decrease the omega-6/omega-3 fatty acids ratio through a decreased omega-6 fatty acids intake and an increased omega-3 fatty acids intake [START_REF] Kris-Etherton | Polyunsaturated fatty acids in the food chain in the United States[END_REF][START_REF] Mozaffarian | Fish, n-3 fatty acids, and cardiovascular haemodynamics[END_REF]). In addition, an increased consumption of omega-6 has also been suggested to be directly involved in inhibition of EPA incorporation in cell membrane from dietary fish oil in humans subjects [START_REF] Cleland | Linoleate inhibits EPA incorporation from dietary fish-oil supplements in human subjects[END_REF]). Moreover, reduced intake of linoleic acid has been associated with an increased circulating level of omega-3 fatty acids such as EPA and DHA in the human plasma [START_REF] Stepanova | Gene expression for VEGF-A, VEGF-C, and their receptors in murine lymphocytes and macrophages[END_REF]).

Similarly, it has been observed that the intake of fish oil containing 1.6 g of EPA and 0.32 g of DHA for 4 weeks is associated with a higher incorporation of EPA into the neutrophil membrane phospholipids in healthy male that are maintained on a low-linoleic acid diet for 3 weeks prior to the supplementation, compared to the group maintained on a high linoleic acid diet [START_REF] Cleland | Linoleate inhibits EPA incorporation from dietary fish-oil supplements in human subjects[END_REF]). Moreover, the intake for 4 weeks by healthy Swedish subjects of a Mediterranean-inspired diet containing 3 times the amount of polyunsaturated fatty acids and twice the amount of omega-3 fatty acids was associated with decreased plasma omega-6 to omega-3 fatty acids ratio, platelets aggregation, and serum VEGF levels in comparison to an ordinary Swedish diet [START_REF] Simopoulos | Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases[END_REF].

Beneficial effect of polyunsaturated fatty acids

Biological effects of omega-3 fatty acids in cell membrane

Polyunsaturated fatty acids are the major component of membrane phospholipids, especially in the myocardium, retina, and brain, where they are important for function and growth, and are implicated in many physiological processes [START_REF] Connor | Importance of n-3 fatty acids in health and disease[END_REF]. They are also found in many blood cells including red blood cells and platelets. Dietary consumption of polyunsaturated fatty acids, such as EPA and DHA, has beneficial health effects in many chronic diseases, including heart diseases, diabetes and inflammatory disorders. Many studies have suggested that the beneficial properties of omega-3 fatty acids affect a large range of organs and physiological systems. In vivo studies both in humans and animals have shown that dietary intake of omega-3 fatty acids increases the plasma content of fatty acids such as DHA and EPA [START_REF] Christensen | Marine n-3 fatty acids, wine intake, and heart rate variability in patients referred for coronary angiography[END_REF][START_REF] Harris | Omega-3 fatty acids and coronary heart disease risk: clinical and mechanistic perspectives[END_REF][START_REF] Holub | Fish oils and cardiovascular disease[END_REF][START_REF] Mclennan | Myocardial membrane fatty acids and the antiarrhythmic actions of dietary fish oil in animal models[END_REF], reduces the content of saturated fatty acids in membranes particularly in lipid rafts and calveolae [START_REF] Garattini | Long-chain n-3 fatty acids in lipid rafts: implications for anti-inflammatory effects[END_REF]. Moreover, omega-3 fatty acids can alter the basic properties of cell membrane including fluidity, elastic compressibility, ion permeability, and membrane remodelling [START_REF] Stillwell | Docosahexaenoic acid: membrane properties of a unique fatty acid[END_REF]. Recently, it has been also shown that omega-3 fatty acids, particularly EPA, can induce a change of the lipid environment in caveolae and thus induce the translocation of caveolin-1 outside of caveolae, thereby leading to the subsequent activation of eNOS [START_REF] Li | Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase[END_REF].

Cardiovascular protection

Cardiovascular diseases are the major causes of mortality worldwide and are associated, at least in part, with an increased intake of dietary saturated fats leading to an increased plasma level of LDLs, VLDLs, cholesterol and triglycerides. Since the first studies that reported that the traditional diet of Greenland Eskimos is associated with a decreased risk of cardiovascular diseases, several clinical studies have shown the beneficial effects of the consumption of fish and fish oil rich in omega-3 fatty acids, particularly EPA and DHA, in preventing cardiovascular diseases [START_REF] Kromhout | ) n-3 fatty acids and cardiovascular events after myocardial infarction[END_REF][START_REF] Shaikh | Efficacy of a unique omega-3 formulation on the correction of nutritional deficiency and its effects on cardiovascular disease risk factors in a randomized controlled VASCAZEN((R)) REVEAL Trial[END_REF][START_REF] Svensson | N-3 fatty acids as secondary prevention against cardiovascular events in patients who undergo chronic hemodialysis: a randomized, placebo-controlled intervention trial[END_REF]. Furthermore, several studies have reported that fish oil could exert a beneficial role in primary and secondary prevention of cardiovascular diseases such as myocardial infarction, sudden cardiac death, coronary heart disease, arterial fibrillation and heart failure [START_REF] Kromhout | The inverse relation between fish consumption and 20-year mortality from coronary heart disease[END_REF][START_REF] Liou | The serial changes of ghrelin and leptin levels and their relations to weight loss after laparoscopic minigastric bypass surgery[END_REF][START_REF] Sheard | Fish consumption and risk of sudden cardiac death[END_REF][START_REF] Von Schacky | The effect of dietary omega-3 fatty acids on coronary atherosclerosis. A randomized, double-blind, placebo-controlled trial[END_REF][START_REF] Yokoyama | Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis[END_REF].

Similarly, the GISSI-P (Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico -Prevenzionne) study indicated that dietary supplementation with 850 mg/d of EPA and 882 mg/d of DHA as ethyl esters was associated with a reduction of major cardiovascular events (20% for total deaths, 30% for cardiovascular deaths, and 45% for sudden deaths) in patients surviving a recent myocardial infarction over 3.5 years [START_REF] Von Schacky | The effect of dietary omega-3 fatty acids on coronary atherosclerosis. A randomized, double-blind, placebo-controlled trial[END_REF]). In the Zutphen study a cohort of 1373 males were examined repeatedly after long-term consumption of polyunsaturated fatty acids. This study showed that the strength of the association between long-term fish consumption and CHD death decreased with increasing age. Moreover, fatty-fish consumption lowered sudden coronary death risk while there was no clear dose-response relationship between EPA+DHA intake and sudden coronary death [START_REF] Streppel | Long-term fish consumption and n-3 fatty acid intake in relation to (sudden) coronary heart disease death: the Zutphen study[END_REF]. Further studies reported that the beneficial effects of polyunsaturated fatty acids on the cardiovascular system have been attributed to an inhibition of platelets aggregation and improvement of lipoproteins metabolism including a decreased plasma triglycerides level, decreased inflammation, lowered blood pressure and improved endothelial function [START_REF] Calder | omega 3 polyunsaturated fatty acids, inflammation and immunity[END_REF][START_REF] Connor | Should a low-fat, high-carbohydrate diet be recommended for everyone? The case for a low-fat, high-carbohydrate diet[END_REF][START_REF] Geleijnse | Blood pressure response to fish oil supplementation: metaregression analysis of randomized trials[END_REF][START_REF] Goodfellow | Dietary supplementation with marine omega-3 fatty acids improve systemic large artery endothelial function in subjects with hypercholesterolemia[END_REF][START_REF] Harris | n-3 fatty acids and serum lipoproteins: human studies[END_REF][START_REF] Hornstra | Influence of dietary fat type on arterial thrombosis tendency[END_REF][START_REF] Knapp | Dietary fatty acids in human thrombosis and hemostasis[END_REF][START_REF] Mori | Differential effects of eicosapentaenoic acid and docosahexaenoic acid on vascular reactivity of the forearm microcirculation in hyperlipidemic, overweight men[END_REF][START_REF] Sacks | Randomized clinical trials on the effects of dietary fat and carbohydrate on plasma lipoproteins and cardiovascular disease[END_REF][START_REF] Simopoulos | Omega-3 fatty acids in health and disease and in growth and development[END_REF]).

Hypertension

Hypertension is a complex and common disease with a complex aetiology that could involve several different mechanisms [START_REF] Williams | Hypertension: genetics and nutrition[END_REF]). Many studies have indicated that the dietary consumption of fish, fish oil, and omega-3 fatty acids is associated with a decreased blood pressure in hypertensive subjects. Indeed, the dietary supplementation with fish and fish oil (15 g of omega-3 fatty acids) for four weeks is associated with a reduced blood pressure in mild hypertensive patients (reduction of systolic blood pressure by 6.5 mmHg and of diastolic blood pressure by about 4.4 mmHg) [START_REF] Knapp | The antihypertensive effects of fish oil. A controlled study of polyunsaturated fatty acid supplements in essential hypertension[END_REF].

Moreover, a meta-analysis study has indicated that the supplementation with fish oil is associated with a reduction in the blood pressure values (0.4-0.7 mmHg) in men [START_REF] Appel | Does supplementation of diet with 'fish oil' reduce blood pressure? A meta-analysis of controlled clinical trials[END_REF][START_REF] Morris | Does fish oil lower blood pressure? A meta-analysis of controlled trials[END_REF]). The beneficial effect on blood pressure is, at least in part, due to an improvement of the endothelium function through an increased NO production within the vascular wall [START_REF] Omura | Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation[END_REF]. Human clinical trials suggested that fish oil intake attenuated the vasoconstriction induced by adrenaline and angiotensin II in human peripheral arteries through an improved endothelial function [START_REF] Chin | Marine oils dose-dependently inhibit vasoconstriction of forearm resistance vessels in humans[END_REF][START_REF] Mori | Differential effects of eicosapentaenoic acid and docosahexaenoic acid on vascular reactivity of the forearm microcirculation in hyperlipidemic, overweight men[END_REF].

Metabolic syndrome

Obesity and diabetes mellitus are increasingly common diseases in the world. Indeed, in 2013 about 282 million persons were diagnosed with type 2 diabetes (T2DM) associated with metabolic disorders such as obesity [START_REF] Alberti | Diabetes: a look to the future[END_REF]. T2DM is characterized as an independent risk factor of cardiovascular diseases, hypertension, and hyperlipidaemia.

In overweight and mildly hyperlipidemic men, intake of 4 g of marine oil containing EPA and DHA is associated with an increased endothelium-dependent vasorelaxation and decreased blood pressure and heart rate [START_REF] Stamler | Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial[END_REF]. Similarly, intake of EPA and DHA in T2DM patients was associated with increased fasting glucose, C-peptide levels, and insulin sensitivity [START_REF] Mori | Differential effects of eicosapentaenoic acid and docosahexaenoic acid on vascular reactivity of the forearm microcirculation in hyperlipidemic, overweight men[END_REF][START_REF] Woodman | Effects of purified eicosapentaenoic and docosahexaenoic acids on glycemic control, blood pressure, and serum lipids in type 2 diabetic patients with treated hypertension[END_REF]. In addition, consumption of 3.6 g of purified EPA and DHA decreased the level of urinary F2-isoprostanes, a marker of oxidative stress, in overweight and mildly hyperlipidemic subjects, possibly due to the antiinflammatory properties of omega-3 fatty acids [START_REF] Gopaul | Plasma 8-epi-PGF2 alpha levels are elevated in individuals with non-insulin dependent diabetes mellitus[END_REF]. Since the level of urinary F2-isoprostanes is also elevated in T2DM patients, omega-3 fatty acids may also exert a beneficial anti-inflammatory effect in these subjects [START_REF] Davi | In vivo formation of 8-isoprostaglandin f2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation[END_REF]. Moreover, omega-3 fatty acids intake is associated with an improved endothelial function via the interruption of the insulin-dependent modulation of the PI3-kinase pathway leading to increased endothelial formation of the vasoprotective NO [START_REF] Fulton | Regulation of endothelium-derived nitric oxide production by the protein kinase Akt[END_REF][START_REF] Michell | The Akt kinase signals directly to endothelial nitric oxide synthase[END_REF]).

Improvement of the endothelial function

As described previously, the endothelium plays a pivotal role in the maintenance of vascular homeostasis, in part, by the formation and release of NO. The endothelium-derived NO exerts multiple vascular effects including the relaxation of the vascular smooth muscle, maintenance of the vascular smooth muscle in a quiescent state, the inhibition of platelet adhesion and aggregation, of leucocytes adhesion, and of pro-atherosclerotic and prothrombotic responses, in part, by inhibiting the expression of adhesion molecules, monocyte chemoattractant protein-1, and tissue factor [START_REF] Schini-Kerth | Polyphenol-induced endothelium-dependent relaxations role of NO and EDHF[END_REF]. The intake of omega-3 fatty acids, including EPA and DHA, has been associated with an improvement of the endothelial function and an increased NO bioavailability subsequent of an increased endothelial formation [START_REF] Boulanger | Chronic exposure of cultured endothelial cells to eicosapentaenoic acid potentiates the release of endotheliumderived relaxing factor(s)[END_REF][START_REF] Chin | Marine oils and cardiovascular reactivity[END_REF][START_REF] Chin | How do fish oils affect vascular function?[END_REF][START_REF] Chu | Fish oil feeding selectively attenuates contractile responses to noradrenaline and electrical stimulation in the perfused mesenteric resistance vessels of spontaneously hypertensive rats[END_REF][START_REF] Yin | Blood pressure and vascular reactivity changes in spontaneously hypertensive rats fed fish oil[END_REF]. Moreover, supplementation with omega-3 fatty acids (0.8 g/kg/d) in ovariectomized rats is associated with reduced blood pressure, arterial stiffening and vascular oxidative stress, and increased endothelial function due to increased eNOS expression and activity (Gortan Cappellari et al. 2013;[START_REF] Losurdo | Supplementation of omega-3 polyunsaturated fatty acids prevents increase in arterial stiffness after experimental menopause[END_REF]). In patients with T2DM, the dietary supplementation with 2 g of purified EPA/DHA daily for 6 weeks is associated with a reduction of the postprandial impaired macro-and micro-vascular functions [START_REF] Stirban | Effects of n-3 fatty acids on macro-and microvascular function in subjects with type 2 diabetes mellitus[END_REF]. Similarly, the daily intake of 5 g EPA plus DHA for 3 weeks is associated with an increased vasodilatory response to acetylcholine in the coronary artery of heart transplanted patients [START_REF] Fleischhauer | Fish oil improves endothelium-dependent coronary vasodilation in heart transplant recipients[END_REF]. Moreover, the daily intake of 1.8 g of EPA for 6 weeks by eight patients with documented coronary artery diseases is associated with an improvement of both the NO-dependent and the NO-independent endothelium-dependent forearm vasodilatation [START_REF] Tagawa | Long-term treatment with eicosapentaenoic acid augments both nitric oxidemediated and non-nitric oxide-mediated endothelium-dependent forearm vasodilatation in patients with coronary artery disease[END_REF].

The beneficial effect of omega-3 fatty acids on the endothelial function has been attributed mainly to their ability to increase the endothelium-dependent formation of NO through various mechanisms. Indeed, in primary cultures of human coronary artery endothelial cells, DHA increased the expression and phosphorylation of eNOS as well as the phosphorylation of the Akt [START_REF] Stebbins | Effects of dietary decosahexaenoic acid (DHA) on eNOS in human coronary artery endothelial cells[END_REF].

Moreover, several studies demonstrated that EPA exerted protective actions against the palmitic acid induced endothelial dysfunction through the activation of the AMPK, which the phosphorylation of the Akt/eNOS signalling pathway [START_REF] Lee | Eicosapentaenoic acid protects against palmitic acid-induced endothelial dysfunction via activation of the AMPK/eNOS pathway[END_REF]. On the other side, in cultured endothelial cells, DHA is able to increase the activity of SIRT1 that will catalyse the deacetylaction of lysine 496 and 506 in the calmodulin-binding domain of eNOS, leading to an increase endothelial formation of NO [START_REF] Jung | Docosahexaenoic acid improves vascular function via up-regulation of SIRT1 expression in endothelial cells[END_REF], whereas EPA, but not oleic acid, increased the insulin-stimulated NO production without modulating the insulin-induced Aktdependent phosphorylation of eNOS [START_REF] Lynn | Fatty acids differentially modulate insulin-stimulated endothelial nitric oxide production by an Akt-independent pathway[END_REF]. Similarly, the omega-3 fatty acids induced changes in the membrane organization in caveolae that are accompanied by alterations of caveolin-1 and eNOS interaction, leading an increased phosphorylation and activation of eNOS [START_REF] Li | Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase[END_REF].

The endothelium has an important role in the regulation of the vascular homeostasis, by inducing various vasodilatating mechanisms, which can also inhibit platelet aggregation and reduce the endothelial expression of adhesion molecules, and thus reduce the risk of cardio vascular diseases. Several studies and clinical trials have shown that an endothelial dysfunction has a major role in many cardiovascular diseases and endothelial dysfunction is an early indicator of atherosclerosis. Endothelial dysfunction also occurs as a result of reduced NO bioavailability and increased vascular oxidative stress.

The studies also demonstrate that intake of fish or fish oil rich in polyunsaturated fatty acids, particularly EPA and DHA, improves endothelial function associated with reduced cardiovascular diseases. The improved endothelial function involves an increase NO bioavailability. Such effects have been observed in rat aortic rings and coronary artery rings (Engler and Engler 2000;[START_REF] Omura | Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation[END_REF]. Furthermore, other studies have been shown

that EPA and DHA profoundly alter the lipid composition of caveolae/lipid rafts and are incorporated into the cell membrane, thereby modifying the location and function of proteins in caveolae. This is associated with selective displacement of caveolae-1 and eNOS from caveolae lading enhanced eNOS activation.

The main goal with this thesis was to determine the role of different ratios of omega-3 fatty acids EPA:DHA 1:1, 3:1, 6:1, 1:3, 1:6 and 1:9 to improve endothelium-dependent relaxation by stimulating the endothelial formation of NO and EDH.

More specifically, the aims were:

-The role of EPA:DHA ratio and amount to induce endothelium-dependent relaxation in rings of porcine coronary and human mammary artery.

-To characterize the mechanism underlying EPA:DHA-induced endothelium dependent±relaxation mediated by NO and EDH in coronary artery and human mammary artery rings.

-To characterize the mechanism underlying the ability of omega-3 fatty acids EPA:

DHA 6:1 formulation to stimulate the redox-sensitive Src/PI3-kinase/Akt pathway leading to the phosphorylation of eNOS at Ser1177.

-The ability of omega-3 fatty acids (EPA, DHA and different ratios of EPA:DHA) to cause activation of eNOS in cultured endothelial cells and to determine the underlying signal transduction pathway.

-Evaluate the ability of EPA:DHA to reduce contractile responses in porcine coronary artery and human mammary artery rings.

Article I

Redox In 1953, Hugh Sinclair published the first report indicating that the Inuit diet, which is rich in seal, fish and fish oil, was associated with lower incidence of cardiovascular diseases [START_REF] Sinclair | The Diet of Canadian Indians and Eskimos[END_REF]). He later attributed the beneficial effects of the diet to its high content in long chain omega-3 fatty acids [START_REF] Sinclair | Deficiency of essential fatty acids and atherosclerosis, etcetera[END_REF]). The beneficial cardiovascular effects of omega-3 fatty acids, and especially EPA and DHA, have since been reported in several number of clinical studies, randomized controlled trials and meta-analyses [START_REF] Casula | Long-term effect of high dose omega-3 fatty acid supplementation for secondary prevention of cardiovascular outcomes: A meta-analysis of randomized, placebo controlled trials [corrected][END_REF][START_REF] Mariani | N-3 polyunsaturated fatty acids to prevent atrial fibrillation: updated systematic review and meta-analysis of randomized controlled trials[END_REF][START_REF] Rizos | Association between omega-3 fatty acid supplementation and risk of major cardiovascular disease events: a systematic review and meta-analysis[END_REF][START_REF] Shaikh | Efficacy of a unique omega-3 formulation on the correction of nutritional deficiency and its effects on cardiovascular disease risk factors in a randomized controlled VASCAZEN((R)) REVEAL Trial[END_REF]). Several mechanisms have been suggested to explain the reduction of cardiovascular events associated with the dietary intake of omega-3 fatty acids including the reduction of cardiovascular risk factors such as hypertension, dyslipidemia, inflammation and platelets aggregation [START_REF] Bays | Eicosapentaenoic acid ethyl ester (AMR101) therapy in patients with very high triglyceride levels (from the Multi-center, plAcebo-controlled, Randomized, double-blINd, 12-week study with an open-label Extension [MARINE] trial)[END_REF][START_REF] Geleijnse | Blood pressure response to fish oil supplementation: metaregression analysis of randomized trials[END_REF][START_REF] He | Associations of dietary long-chain n-3 polyunsaturated fatty acids and fish with biomarkers of inflammation and endothelial activation (from the Multi-Ethnic Study of Atherosclerosis [MESA])[END_REF][START_REF] Mozaffarian | Effect of fish oil on heart rate in humans: a meta-analysis of randomized controlled trials[END_REF][START_REF] Theobald | Lowdose docosahexaenoic acid lowers diastolic blood pressure in middle-aged men and women[END_REF]. Another beneficial effect attributed to omega-3 fatty acids is the improvement of the endothelial function [START_REF] Abeywardena | Longchain n-3 polyunsaturated fatty acids and blood vessel function[END_REF][START_REF] He | Associations of dietary long-chain n-3 polyunsaturated fatty acids and fish with biomarkers of inflammation and endothelial activation (from the Multi-Ethnic Study of Atherosclerosis [MESA])[END_REF]. Indeed, several studies have reported that both EPA and DHA can induce potent endothelium-dependent relaxations due, at least in part, to the activation of eNOS and the subsequent formation of NO [START_REF] Komori | Endothelium-dependent relaxation in response to aggregating platelets in porcine femoral veins and its modulation by diet[END_REF]Shimokawa and Vanhoutte 1988;[START_REF] Tagawa | Long-term treatment with eicosapentaenoic acid improves exercise-induced vasodilation in patients with coronary artery disease[END_REF].

However, the beneficial cardiovascular effects of the dietary supplementation in omega-3 fatty acids has been challenged by a meta-analysis by Hooper et al. in 2006 that concluded that there was no clear benefit of omega-3 fatty acids on cardiovascular events [START_REF] Hooper | Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review[END_REF]. Similarly, several randomized double-blind clinical studies did not show a reduction of cardiovascular events after omega-3 fatty acids supplementation [START_REF] Galan | Effects of B vitamins and omega 3 fatty acids on cardiovascular diseases: a randomised placebo controlled trial[END_REF][START_REF] Kromhout | ) n-3 fatty acids and cardiovascular events after myocardial infarction[END_REF].

The discrepancy of these findings has been attributed to several factors including the heterogeneous population studied and parameters determined. In addition, the conflicting results could also be attributed to the vast range of omega-3 fatty acids sources, formulations and doses used in the different studies. Indeed, the supplementation in omega-3 fatty acids has been provided in form of capsules, oils, emulsions, margarines, fatty fish, whereas the control groups were often undefined [START_REF] Hooper | Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review[END_REF]. Moreover, the daily intake dose of omega-3 fatty acids was within a large range going from 0.4 up to 7.0 g containing EPA,

DHA and DPA in very different amounts [START_REF] Hooper | Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review[END_REF]. Similarly, a meta-analysis by Wang et al. evaluating the effects of omega-3 fatty acids on the endothelial function included a large range of omega-3 fatty acids in the 16 randomized clinical studies [START_REF] Wang | Effect of omega-3 fatty acids supplementation on endothelial function: a meta-analysis of randomized controlled trials[END_REF].

Indeed, the daily dose of omega-3 fatty acids ranged from 0.45 to 4.5 g per day in form of fish oil, walnuts and capsules containing various formulations of EPA and/or DHA [START_REF] Wang | Effect of omega-3 fatty acids supplementation on endothelial function: a meta-analysis of randomized controlled trials[END_REF]).

Thus, the aim of our study was to evaluate the role of omega-3 fatty acids formulations on the ability to activate the endothelial function, and in particular to promote the endothelial promotion of NO. Firstly, to identify an optimized formulation of omega-3 fatty acids, we have evaluated the ability of EPA, DHA and formulations with different EPA: DHA ratios ranging from 1:9 to 9:1 to induce potent endothelium-dependent relaxations in porcine coronary artery rings. The influence of the purity was also evaluated by comparing an optimized formulation with a formulation containing the same EPA:DHA ratio, but only with a 50% content in omega-3 fatty acids. Finally, the cellular and molecular mechanisms underlying the omega-3 fatty acid induced relaxation was investigated.

Results and discussion

The main finding of our study indicated that omega-3 fatty acids formulations are able to induce potent endothelium-dependent relaxations in porcine coronary artery rings. This findings is in line with several previously published studies that have shown that omega-3 fatty acids and fish oils are able to induce endothelium-dependent relaxations in various types of isolated arteries including the coronary artery, the pulmonary artery and the cerebral artery (Engler et al. 2000;[START_REF] Omura | Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation[END_REF]Shimokawa et al. 1988b;Shimokawa and Vanhoutte 1988;[START_REF] Vanhoutte | Endothelium and control of vascular function. State of the Art lecture[END_REF]).

The present findings show that while EPA and DHA are both able to induce endotheliumdependent relaxations in porcine coronary artery rings, the combination of the two omega-3 fatty acids in formulations EPA:DHA 6:1 and 9:1 was associated with an increased potency to induce relaxation. The enrichment of the formulation with EPA was associated with a dosedependent increase in the relaxation, with the maximal relaxation obtained with the EPA:DHA 6:1 and 9:1 formulations. The enrichment of the formulations in DHA was only associated with a weak increase in relaxations. The optimized formulation of EPA:DHA was then defined as the EPA:DHA 6:1 ratio, which was subsequently used for further characterization. The role of the purity was assessed by comparing the optimized EPA:DHA 6:1 formulation which is highly purified with more than 90% of EPA and DHA, to a formulation containing EPA:DHA 6:1 with a reduced purity of about 50%. The results have indicated that a reduction of the content in omega-3 fatty acids by 50% was associated with significantly less relaxation. Altogether, these findings indicate that the potency of omega-3 fatty acids formulations to induce endothelium-dependent relaxations is affected by both the EPA:DHA ratio and the purity.

Furthermore, we determined the mechanism underlying the relaxation induced by the optimized formulation. The findings show that the optimized formulation induce an endothelium-dependent relaxation that is reduced by the inhibition of eNOS and abolished by the inhibition of eNOS plus that of EDH-mediated relaxations. These observations indicate that the optimized formulation of omega-3 fatty acids induces endothelium-dependent relaxation mainly due to the formation of NO, and also, to some extent, to the EDH-mediated component of the relaxation. Moreover, the induction of the NO-mediated relaxation was reduced by inhibitors of ether oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. These findings suggest that the optimized formulation induced relaxations in porcine coronary artery rings through a redox-sensitive activation of the Src/PI3-kinase/Akt and MAPKs pathways leading to the activation of eNOS. Such a mechanism is similar to that involved in the endothelium-dependent relaxation induced by polyphenol-rich products, another type of natural compounds. Indeed, a redox-sensitive activation of the Src/PI3kinase/Akt leading to the activation of eNOS and subsequent formation of NO was reported in response to grape-derived products and isolated phenolic compounds (Auger et al. 2010a;Auger et al. 2010b;[START_REF] Kim | Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn[END_REF][START_REF] Schini-Kerth | Polyphenol-induced endothelium-dependent relaxations role of NO and EDHF[END_REF].

In conclusion, the present study has shown that the ability of omega-3 fatty acids products to induce endothelium-dependent relaxations is dependent on the ratio and the amount of the EPA:DHA formulation. Furthermore, the optimized EPA:DHA 6:1 formulation induces endothelium-dependent relaxations via the stimulation of the endothelial formation of NO, and to a lesser extent to the activation of the EDH pathway. The NO-mediated relaxation involves an intracellular oxidative stress triggering the activation by phosphorylation of the Src/PI3-kinase/Akt and MAPKs pathways, ultimately leading to increase eNOS activity.

formation of contracting factors and expression of pro-atherothrombotic factors. Activated platelets are known to induce potent vasoconstriction through the formation and release of several contracting factors including thromboxane A 2 , serotonin and eicosanoids. Moreover, the platelet-induced contractions are more pronounced in blood vessels with an endothelial dysfunction compared with healthy blood vessels.

Thus, the aim of the present study was to evaluate the potency of omega-3 fatty acids products to prevent the contraction induced by platelets in porcine coronary artery rings and, if so, to investigate the underlying mechanism. For this purpose, contractions were induced by human washed platelets in porcine artery rings, and the inhibitory effect of omega-3 fatty acids products was evaluated. The mechanisms involved in the platelet-induced contraction and in the preventive effect of omega-3 fatty acids products were determined. To determine the potential clinical relevance, the beneficial effect of omega-3 fatty acids products as well as the underlying mechanisms were studied in human internal mammary artery rings.

In conclusion, the present findings show that the optimized EPA:DHA 6:1 formulation is able to prevent the 5-HT-mediated platelet-induced contraction in porcine coronary and human internal mammary artery rings through an endothelium-dependent NO-mediated mechanism.

These results suggest that optimized omega-3 products may exert potent beneficial effect on vascular health by activating the endothelial function leading to an increased formation of NO to prevent the deleterious vascular effects of activated platelets.

GENERAL DISCUSSION AND PERSPECTIVES

The endothelium plays a pivotal role in the regulation of vascular homeostasis mainly through the formation and release of vasodilatating and vasocontracting factors. Mumerous studies have shown an association between endothelial dysfunction, an imbalance of the formation of relaxing and contracting factors released by the endothelium, and the development of cardiovascular diseases in the presence of risk factors such as hypertension, diabetes mellitus, atherosclerosis, thrombosis and platelet aggregation [START_REF] Anderson | Assessment and treatment of endothelial dysfunction in humans[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Hadi | Endothelial dysfunction in diabetes mellitus[END_REF][START_REF] Sase | Expression of constitutive endothelial nitric oxide synthase in human blood platelets[END_REF]. The protective effects of the endothelium towards the vascular function has been attributed, at least in part, to its ability to produce vasoprotective dilating factors which are mainly NO, EDH, and PGI 2 .

The impairment of the endothelial function is generally associated with decreased NO bioavailability in many cardiovascular diseases and risk factors such as hypertension, diabetes, and atherosclerosis [START_REF] Davignon | Role of endothelial dysfunction in atherosclerosis[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF].

The beneficial effect of intake of omega-3 fatty acids on the risk of cardiovascular diseases has been reported in numerous studies [START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Lopez-Garcia | Major dietary patterns are related to plasma concentrations of markers of inflammation and endothelial dysfunction[END_REF][START_REF] Nettleton | Associations between dietary patterns and flow cytometry-measured biomarkers of inflammation and cellular activation in the Atherosclerosis Risk in Communities (ARIC) Carotid Artery MRI Study[END_REF][START_REF] Nettleton | A prioridefined dietary patterns and markers of cardiovascular disease risk in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] Nettleton | Associations between markers of subclinical atherosclerosis and dietary patterns derived by principal components analysis and reduced rank regression in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF]). Indeed, the initial studies reported that the high intake of fatty fish and fish oil by the Greenland Eskimos compared to Danes was associated with a reduced mortality from cardiovascular diseases [START_REF] Dyerberg | Haemostatic function and platelet polyunsaturated fatty acids in Eskimos[END_REF][START_REF] Dyerberg | Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis?[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF]. Several further studies have since reported the beneficial effects of the consumption of fatty acids such as EPA and DHA in various diseases such as hypertension, diabetes, and atherosclerosis [START_REF] Albert | Fish consumption and risk of sudden cardiac death[END_REF][START_REF] Burr | Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART)[END_REF][START_REF] De Lorgeril | Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Marchioli | Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico (GISSI)-Prevenzione[END_REF][START_REF] Mozaffarian | Fish intake and risk of incident atrial fibrillation[END_REF][START_REF] Siscovick | Dietary intake and cell membrane levels of long-chain n-3 polyunsaturated fatty acids and the risk of primary cardiac arrest[END_REF].

However, recent meta-analyses of clinical studies have suggested a limited beneficial effect of omega-3 fatty acids supplementation due to conflicting results amongst various studies [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][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Kromhout | Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?[END_REF].

These conflicting results are due, at least in part, to the heterogeneity of omega-3 sources, formulations and doses used in the different studies.

The aim of the present work was firstly to evaluate the role of the formulation of omega-3 fatty acids on the induction of endothelium-dependent relaxations, the contribution of both NO-and EDH-mediated relaxations, and to determine the underlying mechanisms.

The second aim of the study was to evaluate the beneficial vascular effect of omega-3 fatty acids on the platelet-induced coronary artery contraction, and to characterize the role serotonin and vasoconstricting prostanoids.

Therefore, we have evaluated various ratios and different levels of purity of EPA and DHA formulations based on their potency to induce potent endothelium-dependent relaxations in porcine coronary artery rings. Indeed, our findings show that the induction of endothelium-dependent relaxation, and especially the activation of the NO formation, is dependent on both the purity and the ratio of the EPA:DHA formulation. While isolated EPA and DHA are able to induce endothelium-dependent relaxations in porcine coronary artery rings, EPA induced significantly greater relaxations than DHA at the concentration of 0.4% (v/v). Similarly, the evaluation of various ratio of EPA:DHA indicates that the relaxing effect is maximized in presence of an EPA:DHA ratio of 6 or 9 to 1, whereas formulation with lower content of EPA induced significantly weaker relaxations. Moreover, an EPA:DHA 6:1 formulation with a EPA:DHA content reduced by 50% also induced significantly weaker relaxations than the optimized EPA:DHA 6:1 formulation with a high EPA:DHA purity.

These findings thus suggest that the beneficial vascular effects of omega-3 formulations are dependent of the content in term of both total omega-3 contents and EPA:DHA ratio. These parameters should be taken into account when comparing results from both preclinical and clinical studies using different products rich in omega-3 fatty acids. Moreover, the present findings may also help to understand the discrepancies found in several clinical studies assessing the cardiovascular effects of various omega-3 formulations ranging from fish oil to isolated purified EPA or DHA used at different doses.

Indeed, in meta-analyses, the clinical studies selected have used various approaches for omega-3 fatty acids supplementation, including dietary advice to eat 2-3 fish meals/week, consumption of fish oil or supplementation with omega-3 fatty acids formulations [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][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Kromhout | Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?[END_REF]. Moreover, the doses used in the clinical trials are very heterogeneous, with reported omega-3 formulations ranging from fish oil to purified EPA or DHA, with doses from 0.18 up to 10 g/day [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][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF].

Moreover, the various clinical studies published in the literature have focused on different types of patients and measured various clinical parameters. Indeed, the randomized clinical trials published over the years have assessed the effect of omega-3 fatty acids supplementation in patients with documented angina [START_REF] Burr | Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART)[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF], arrhythmia (SOFA), acute myocardial infarction (GISSI-P, DART, OMEGA, Alpha Omega), chronic heart failure (GISSI-HP), hypercholesterolemia (JELIS), and evaluated different primary and secondary outcomes [START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF]). In addition, the discrepancy of the randomized clinical trials could also be due, at least in part, to the fact that the studied population had different medications such as statins, and dietary exposition to omega-3 fatty acids [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF].

The The redox-sensitive activation of the PI3-kinase/Akt/eNOS pathway by EPA:DHA is similar to the pathway activated by other natural products such as the polyphenolic compounds.

Indeed, polyphenol-rich products including red wine extract [START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Ndiaye | Red wine polyphenol-induced, endothelium-dependent NO-mediated relaxation is due to the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of endothelial NOsynthase in the isolated porcine coronary artery[END_REF][START_REF] Ndiaye | Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redoxsensitive activation of the PI3-kinase/Akt pathway[END_REF]), grape skin extract [START_REF] Madeira | eNOS activation induced by a polyphenol-rich grape skin extract in porcine coronary arteries[END_REF]), grape juice [START_REF] Anselm | Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src-and Aktdependent activation of eNOS[END_REF], EGCg (Auger et al. 2010a;[START_REF] Kim | Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn[END_REF]) and plant extracts [START_REF] Rattmann | Natural product extract of Dicksonia sellowiana induces endothelium-dependent relaxations by a redox-sensitive Src-and Akt-dependent activation of eNOS in porcine coronary arteries[END_REF][START_REF] Tokoudagba | Procyanidin-rich fractions from Parkia biglobosa (Mimosaceae) leaves cause redox-sensitive endothelium-dependent relaxation involving NO and EDHF in porcine coronary artery[END_REF]) induced a redox-sensitive endothelium-dependent relaxations.

The intracellular formation of ROS leading to the activation of eNOS induced by polyphenols is dependent of the structure of the compounds (Auger et al. 2010a;Auger et al. 2010b).

Indeed, the presence of hydroxyl groups on the phenolic structure is critical for the formation of ROS most likely through an auto-oxidation process (Auger et al. 2010b;[START_REF] Kurita | Hydroxylation of (-)-epigallocatechin-3-O-gallate at 3'', but not 4'', is essential for the PI3-kinase/Akt-dependent phosphorylation of endothelial NO synthase in endothelial cells and relaxation of coronary artery rings[END_REF].

However, omega-3 fatty acids do not have the hydroxyl moieties needed to undergo autooxidation, and thus should induce the intracellular formation of ROS mostly likely through another mechanism, unless they are converted to hydroxylated metabolites. Indeed, the metabolomics analysis of the eicosanoids founds in human plasma after omega-3 fatty acids supplementation indicates that the levels of CYP450 epoxy metabolites and corresponding dihydroxy metabolites from EPA and DHA were significantly increased [START_REF] Zhang | Systematic metabolomic analysis of eicosanoids after omega-3 polyunsaturated Fatty Acid supplementation by a highly specific liquid chromatography-tandem mass spectrometry-based method[END_REF].

The possibility that these metabolites could be responsible for the endothelial formation of ROS remains to be demonstrated. Another possibility is that the omega-3 fatty acids incorporation into the cell membranes induces the activation of ROS producing enzymes.

Indeed, upon its incorporation in cell membranes, DHA could induce the activation of various membrane proteins such as protein kinase C, cytochrome P450, several transporters and insulin receptor [START_REF] Murphy | Dietary fatty acids and membrane protein function[END_REF][START_REF] Slater | The modulation of protein kinase C activity by membrane lipid bilayer structure[END_REF][START_REF] Stillwell | Docosahexaenoic acid: membrane properties of a unique fatty acid[END_REF]. However, the possibility that such mechanisms contribute to the redox-sensitive endothelium-dependent formation of NO remains to be clarified.

The aim of our second study was to evaluate the potency of the optimized EPA:DHA 6:1 formulation to prevent the platelet-induced vasoconstriction. Indeed, in pathological coronary arteries, platelets can be locally activated and induce vasoconstriction. Similarly, platelets aggregation is associated with contraction of vascular smooth muscles in human and porcine coronary artery rings [START_REF] Chand | Serotonin receptors subserve only contraction in canine and rat pulmonary arteries and veins[END_REF][START_REF] Freeman | Effects of 5-hydroxytryptamine on neuroeffector junction in human pulmonary artery[END_REF][START_REF] Joiner | Contractile responses of canine isolated pulmonary lobar arteries and veins to norepinephrine, serotonin, and tyramine[END_REF]. In arteries with an altered endothelium, such as coronary arteries with atherosclerotic plaques, local activation of platelets and subsequent contraction could lead to ischemia and possibly also vasospasm.

The present findings indicate that in porcine coronary artery rings, human washed platelets induced dose-dependent contractions that were significantly inhibited by the 5-HT 2A receptor antagonist ketanserin and not affected by indomethacin, suggesting that the platelet-induced contraction is mainly due to the release of serotonin from activated platelets. Moreover, the short-term incubation of the rings with the EPA:DHA 6:1 optimized formulation, but not with the EPA:DHA 1:1 formulation, resulted in the abolition of the platelet-induced contraction.

The fact that the prevention of the platelets-induced contraction by EPA:DHA 6:1 0.4% (v/v)

was not observed in endothelium-denuded rings and in the presence of an eNOS inhibitor suggests that the EPA:DHA 6:1 exerts its inhibitory effects through an increased endothelial formation of NO.

Previous in vivo studies have indicated that in dog, the platelets-induced contraction of the large epicardial coronary artery is due both to thromboxane A 2 and serotonin [START_REF] Golino | Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Thromboxane A2 and serotonin are possible mediators[END_REF]). Thus, we have assessed the potency of the omega-3 fatty acids formulation to prevent the contractions induced by both agents. The EPA:DHA 6:1 formulation dose-dependently prevented the 5-HT-induced contraction, whereas it was without effect on that induced by the thromboxane A 2 analogue U46619.

As vascular beds from different species can display different type of reactivity, the extrapolation of the mechanisms observed in the porcine coronary artery to the human artery needs to be validated. Similar results were observed in human internal mammary artery rings, where the EPA:DHA 6:1 formulation at 0.4 % (v/v) significantly prevented the 5-HT-induced contraction and it also induced vasorelaxation through NO-mediated mechanisms. Indeed, 5-HT has been reported as a possible spasmogenic agent in human internal mammary artery [START_REF] Rosenfeldt | Pharmacology of coronary artery bypass grafts[END_REF], and in particular in arteries with an endothelial dysfunction [START_REF] He | Arterial grafts for coronary artery bypass grafting: biological characteristics, functional classification, and clinical choice[END_REF]. In our setting, we were able to observe 5-HT-induced contractions in human internal mammary artery rings, possibly due to the fact that these arteries obtained from patients undergoing coronary artery bypass grafting most likely have an established endothelial dysfunction.

Altogether, these findings indicate that the optimized EPA:DHA 6:1 formulation is able to prevent the 5-HT-mediated platelet-induced contraction in porcine coronary and human internal mammary artery rings through an endothelium-dependent NO-mediated mechanism.

The main limitation of the present findings is the fact that they were obtained ex vivo using artery rings suspended in organ baths. Moreover, we have assessed only the short term effects of the optimized EPA:DHA 6:1 formulation, whereas the beneficial cardiovascular effects of omega-3 fatty acids supplementation reported by clinical trials is most likely due to the chronic impregnation of tissues leading to long-term effects. The present work requires further studies to better characterize the beneficial endothelial effects of omega-3 fatty acids.

Firstly, the mechanism involved in the intracellular formation of ROS in response to the EPA:DHA 6:1 formulation needs to be clarified. Similarly, the long-term effects of the EPA:DHA 6:1 formulation on the endothelium should be evaluated, and in particular its potential ability to induce an up-regulation of eNOS expression leading to an improved basal NO formation in the endothelium, which can be responsible for an improved endothelial function and a decreased risk of developing cardiovascular diseases.

Secondly, the present work on the endothelial effects of EPA:DHA 6:1 formulation needs to be confirmed using in vivo models of endothelial dysfunction. The chronic oral intake of omega-3 has been associated with increased levels of omega-3 fatty acids in plasma, but also of various metabolites which can exert different effects on the endothelium. Thus, the ability of the EPA:DHA 6:1 formulation to prevent the endothelial dysfunction and/or to improve the endothelial function needs to be evaluated in in vivo models of endothelial dysfunction such as the angiotensin II-induced hypertension in rats. Moreover, such studies will also allow the O ¶DWKpURVFOpURVH [START_REF] Davignon | Role of endothelial dysfunction in atherosclerosis[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF].

De nombreuses études ont rapporté les effets bénéfiques de la consommation d ¶acides gras de type oméga-3 sur les risques de développer une maladie cardiovasculaire [START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Lopez-Garcia | Major dietary patterns are related to plasma concentrations of markers of inflammation and endothelial dysfunction[END_REF][START_REF] Nettleton | Associations between dietary patterns and flow cytometry-measured biomarkers of inflammation and cellular activation in the Atherosclerosis Risk in Communities (ARIC) Carotid Artery MRI Study[END_REF][START_REF] Nettleton | A prioridefined dietary patterns and markers of cardiovascular disease risk in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] Nettleton | Associations between markers of subclinical atherosclerosis and dietary patterns derived by principal components analysis and reduced rank regression in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF]) (Q HIIHW OHV SUHPLqUHV pWXGHV RQW PRQWUp TX ¶en comparaison de la population danoise, la consommatiRQ pOHYpH GH SRLVVRQV JUDV HW G ¶huile de poissons par les esquimaux du Groenland était associée avec une réduction de la mortalité cardiovasculaire [START_REF] Dyerberg | Haemostatic function and platelet polyunsaturated fatty acids in Eskimos[END_REF][START_REF] Dyerberg | Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis?[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF]. Par la suite, plusieurs études ont montré les effets bénéfiques de la consommaWLRQ G ¶acides gras oméga-3 comme O ¶EPA et le DHA dans de nombreuses PDODGLHV FRPPH O ¶K\SHUWHQVLRQ OH GLDEqWH HW O ¶DWKpURVFOpURVH [START_REF] Albert | Fish consumption and risk of sudden cardiac death[END_REF][START_REF] Burr | Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART)[END_REF][START_REF] De Lorgeril | Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Marchioli | Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico (GISSI)-Prevenzione[END_REF].

Cependant, les récentes méta-analyses G ¶essais cliniques suggèrent un effet bénéfique limité GH O ¶DSSRUW G ¶RPpJD-3 du fait de résultats contradictoires rapportés par plusieurs études [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][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Kromhout | Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?[END_REF].

Ces résultats contradictoires pourraient en paUWLH V ¶H[SOLTXHU SDU la grande variabilité de pYDOXp O ¶HIIHW G ¶XQ apport en acides gras de type oméga-3 chez des patients ayant soit une angine de poitrine [START_REF] Burr | Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART)[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF], un infarctus du myocarde (GISSI-P, DART, OMEGA, Alpha Omega), une insuffisance cardiaque (GISSI-HP), ou une hypercholestérolémie (JELIS), avec une grande variété G ¶pYpQHPHQWV étudiés en prévention primaire ou secondaire [START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF].

Enfin, les divergences observées dans les résultats des essais cliniques randomisés pourraient également être attribuées, du moins en partie, au fait que les populations incluses dans les essais présentaient des différences dans leurs prises en charge thérapeutique (par exemple prescription de statines) et dans leurs consommations alimentaires en acides gras de type oméga-3 [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF].

La Cette activation redox-sensible de la voie PI3-kinase/Akt/eNOS par la formulation optimisée EPA:DHA 6:1 est similaire à celle induite par G ¶DXWUHV SURGXLWV QDWXUHOV FRPPH OHV FRPSRVpV polyphénoliques. En effet, les produits riches en polyphénols comme un extrait de vin rouge [START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Ndiaye | Red wine polyphenol-induced, endothelium-dependent NO-mediated relaxation is due to the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of endothelial NOsynthase in the isolated porcine coronary artery[END_REF][START_REF] Ndiaye | Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redoxsensitive activation of the PI3-kinase/Akt pathway[END_REF]), de pellicule de raisin [START_REF] Madeira | eNOS activation induced by a polyphenol-rich grape skin extract in porcine coronary arteries[END_REF]), le jus de raisin Concord [START_REF] Anselm | Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src-and Aktdependent activation of eNOS[END_REF], O ¶EGCg (Auger et al. 2010a;[START_REF] Kim | Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn[END_REF]) ou encore des extraits de plantes [START_REF] Rattmann | Natural product extract of Dicksonia sellowiana induces endothelium-dependent relaxations by a redox-sensitive Src-and Akt-dependent activation of eNOS in porcine coronary arteries[END_REF][START_REF] Tokoudagba | Procyanidin-rich fractions from Parkia biglobosa (Mimosaceae) leaves cause redox-sensitive endothelium-dependent relaxation involving NO and EDHF in porcine coronary artery[END_REF], induisent une activation redox-sensible des relaxations dépendantes de O ¶HQGRWKpOLXP /D IRUPDWLRQ LQWUDFHOOXODLUH GH 526 FRQGXLVDQW j O ¶DFWLYDWLRQ Ge la eNOS en réponse aux polyphénols est dépendante de la structure de ces composés (Auger et al. 2010a;Auger et al. 2010b). En effet, la présence de groupements hydroxyles dans la structure phénolique est cruciale pour la formation de ROS, SUREDEOHPHQW SDU XQ SURFHVVXV G ¶DXWRoxydation (Auger et al. 2010b;[START_REF] Kurita | Hydroxylation of (-)-epigallocatechin-3-O-gallate at 3'', but not 4'', is essential for the PI3-kinase/Akt-dependent phosphorylation of endothelial NO synthase in endothelial cells and relaxation of coronary artery rings[END_REF]. Cependant, les acides gras de type oméga- [START_REF] Murphy | Dietary fatty acids and membrane protein function[END_REF][START_REF] Slater | The modulation of protein kinase C activity by membrane lipid bilayer structure[END_REF][START_REF] Stillwell | Docosahexaenoic acid: membrane properties of a unique fatty acid[END_REF].

Q
Le but de la seconde étude a été G ¶pYDOXHU la capacité de la formulation optimisée EPA:DHA

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ahmoud ALHOSIN, Sherzad K. RASHID, Cyril AUGER & Valérie B. SCHINI-KERTH. The highly purified EPA:DHA 6:1 product-evoked endothelium-dependent NO-mediated relaxation in the coronary artery involves a copper-dependent event triggering the redox-sensitive PI3-kinase/Akt pathway to activate eNOS by phosphorylation at Ser 1177, PLOS ONE August 2014 ;9 (8) : e105102 2. Faraj ZGHEEL, Cyril AUGER, Stéphanie PERRIER, Jean-Philippe MAZZUCOTELLI, Olivier MOREL and Valérie B SCHINI-KERTH. The optimized EPA:DHA 6:1 formulation inhibits aggregating platelet-induced 5-HT-mediated contractions in porcine coronary artery and human internal mammary artery rings: role of endothelial NO. En préparation. ORAL PRESENTATION Faraj ZGHEEL, Mahmoud ALHOSIN, Cyril AUGER & Valérie B. SCHINI-KERTH. The highly purified EPA:DHA 6:1 product evokes potent endothelium-dependent relaxations of porcine coronary artery rings via the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of eNOS. European Society of Cardiology (ESC) Congress 2012, Munich, Allemagne, 25-29 août 2012. Eur. Heart J. 33 (Suppl.), 512, 2012 Faraj ZGHEEL, Mahmoud ALHOSIN, Cyril AUGER & Valérie B. SCHINI-KERTH. The omega 3 EPA:DHA 6:1 causes redox-sensitive PI3-kinase/Akt-dependent activation of endothelial NO synthase. 17th World congress on heart disease, Toronto, Canada, 27-30 Juillet 2012 POSTERS 1. Faraj ZGHEEL, Mahmoud ALHOSIN, Cyril AUGER & Valérie B. SCHINI-KERTH. Highly purified omega 3 (EPA:DHA 6:1) product evokes potent endothelium-dependent relaxations of porcine coronary artery rings by phosphorylation of eNOS via a redox-sensitive PI3-NLQDVH$NW SDWKZD\ qPHV -RXUQpHV GX &DPSXV G ¶,OONLUFK ,OONLUFK 2-3 avril 2012 2. Faraj ZGHEEL, Mahmoud ALHOSIN, Sherzad K. RASHID, Cyril AUGER & Valérie B. SCHINI-KERTH. The NO-mediated relaxation induced by the highly purified EPA:DHA 6:1 product involves a copper-dependent redox-sensitive activation of the PI3-kinase/Akt pathway leading to eNOS activation. 11th International symposium on mechanisms of vasodilatation, Zurich, Suisse, 4-6 octobre 2013 3. Faraj ZGHEEL, Mahmoud ALHOSIN, Cyril AUGER & Valérie B. SCHINI-KERTH. The highly purified EPA:DHA 6:1 product evokes potent endothelium-dependent relaxations of porcine coronary artery involves a copper-dependent event triggering the redox-sensitive PI3kinase/Akt-dependent to activate eNOS by phosphorylation. European Society of Cardiology (ESC) Congress 2013, Amsterdam, Pays-Bas, 31 aout ± 4 septembre 2013.

Figure 1 .

 1 Figure 1. Structure of the blood vessel ...................................................................................

Figure 2 .

 2 Figure 2. Endothelium-dependent release of vasocontracting and vasorelaxing factors. .......

Figure 3 .

 3 Figure 3. Nitric oxide synthesis pathway in the endothelial cell and its actions in the vascular smooth muscle cell. ..................................................................................................................Figure 4. Regulation of eNOS activity.. ..................................................................................

Figure 4 .

 4 Regulation of eNOS activity.. ..................................................................................

Figure 5 .

 5 Figure 5. The activation and inhibition sites of eNOS.. ..........................................................

Figure 6 .

 6 Figure 6. Hypothesis describing the endothelium-derived hyperpolarizing pathway. ............

Figure 7 .

 7 Figure 7. Physicochemical stimuli, can evoke endothelium-derived contractile factors (EDCF) in certain blood vessels ............................................................................................... Figure 8. Arachidonic acid metabolism pathways ..................................................................

Figure 9 .

 9 Figure 9. Endothelial dysfunction in cardio-and cerebro-vascular diseases. ........................

Figure 10 .

 10 Figure 10. Endothelium-derived contracting factors (EDCF) pathways in hypertension. ......

Figure 11 .

 11 Figure 11. Pathological pathways of endothelial dysfunction associated with insulin resistance and hyperinsulinemia ............................................................................................... Figure 12. Role of low-density lipoprotein (LDL) in the atherosclerotic process. .................

Figure 13 .

 13 Figure 13. Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA). .....................

Figure 14 .

 14 Figure 14. Polyunsaturated fatty acids (PUFA). .....................................................................

Figure 15 .

 15 Figure 15. Trans fatty acids structure of oleic acid and elaidic acid .......................................

Figure 16 .

 16 Figure 16. Content of omega-3 PUFA in fishes ......................................................................

Figure 17 .

 17 Figure 17. Eicosanoid metabolites derived from omega-3 and omega-6 polyunsaturated fatty acids .......................................................................................................................................... Figure 18. Synthesis pathway of omega-6 and omega-3 fatty acids in mammals. ................

Figure 19 . 2 (

 192 Figure 19. Incorporation of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenic acid (DHA) into lipid rafts and caveolae . ....................................................Figure 20. Synthesis and actions of lipid mediators produced from arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). ............................................

2 (

 2 permeability and blood homeostasis. The endothelium exerts its function, at least in part, by releasing several factors that control vascular tone. The key endothelium-derived relaxing factors are the nitric oxide (NO), the endothelium-dependent hyperpolarization (EDH) and prostacyclin (PGI 2 ). Endothelium-derived vasocontracting factors include thromboxane A TXA 2 ), angiotensin II, prostaglandin H 2 (PGH 2 ), reactive oxygen species (ROS), and endothelins.Evidence suggests that dietary factors play an important role in the modulation of the endothelial function. Hence, relations between diet and vascular structure and function (vascular health) have been the subject of several studies, both epidemiological and interventional. Improvements of endothelial function have been associated with several kind of foods and beverages including fruit, vegetables, fatty fish, tea and red wine, and with micronutrients such as omega-3 fatty acids, vitamins C, D and E, and carotenoids.In addition, 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). 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. 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 activation of the endothelium vasoprotective factors NO and EDH.The aim of the present work was firstly to evaluate the role of the formulation of omega-3 fatty acids on the induction of endothelium-dependent relaxations, the contribution of both NO-and EDH-mediated relaxations, and to determine the underlying mechanisms. The second aim of the study was to evaluate the beneficial vascular effect of omega-3 fatty acids on the platelet-induced coronary artery contraction, and to characterize the role of serotonin and vasoconstricting prostanoids.The first study assessed the role of the formulation of omega-3 fatty acids on the induction of endothelium-dependent relaxations. The effects of different omega-3 fatty acid products were studied either on porcine coronary artery rings or in cultured porcine coronary artery endothelial cells.

Figure 1 .

 1 Figure 1 . Structure of the blood vessel (adapted from Boston University School of Public Health).

Figure 2 .

 2 Figure 2. Endothelium-dependent release of vasocontracting and vasorelaxing factors. NO, nitric oxide; PGI 2 , prostacyclin; EDHF, endothelium-derived hyperpolarizing factor; A-II, angiotensin II; ET, endothelin; TxA 2 /PGH 2 , thromboxaneA 2 /prostaglandin H 2 O 2, hydrogen peroxide (Abeywardena and Head 2001).

Figure 3 .

 3 Figure 3. 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 3-kinase; Akt, Protein kinase B; Ca/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 4 .

 4 Figure 4. 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 5 .

 5 Figure 5. 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 6 .

 6 Figure 6. 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; EDHF, endothelium-derived hyperpolarizing factor; EETs, epoxyeicosatrienoic acids; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; BK Ca , large conductance Ca 2+ -activated K+ channel; SK Ca , small-conductance Ca 2+ -activated K + channel subtype 3; IK Ca , intermediate-conductance Ca 2+ -activated K+ channel; Kir, inwardly rectifying K + channel; meGJ, myoendothelial gap-junction; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; VDCC, voltage-dependent 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 7 .

 7 Figure 7. Physicochemical stimuli can evoke endothelium-derived contractile factors (EDCFs) in certain blood vessels, Ach; acetylcholine , AA; arachidonic acid, ADP; adenosine diphosphate, 5-HT; serotonin (Vanhoutte et al. 2005).

  . Several enzymes have been implicated in the production of ROS in the vascular wall, including cytochrome P450, cyclooxygenases (COX-1 and COX-2), lipoxygenases, NADPH oxidase, xanthine oxidase and the mitochondrial respiratory chain complexes (Griendling and Ushio-Fukai 1997). The major species of ROS involved in vascular oxidative stress include superoxide anions (O 2 Ɣ-

Figure 8 .

 8 Figure 8. Arachidonic acid metabolism pathways (Tang and Vanhoutte 2009).

Figure 9 .

 9 Figure 9. Endothelial dysfunction in cardio-and cerebro-vascular diseases. Risk factors associated with endothelium dysfunction are hypertension, hyperlipidemia, diabetes mellitus, chronic smoking (Abeywardena and Head 2001).

Figure 10 .

 10 Figure 10. Endothelium-derived contracting factors (EDCF) pathways in hypertension. 5-HT, serotonin; ACE, angiotensin-converting enzyme; Ach, acetylcholine; ADMA, asymmetric dimethyl arginine; AI, angiotensin I; AII, angiotensin II; AT1, angiotensin receptor type 1; bET, big endothelin; BK, bradykinin; cAMP, cyclic adenosine ¶5 ¶ monophosphate; cGMP, cyclic guanosine monophosphate; ECE, endothelinconverting enzyme; EDCF, endothelial-derived contracting factors; ET-1, endothelin-1; ETA, endothelin receptor-A; ETB, endothelin receptor-B; M, muscarinic receptor; NADPH-Ox, NADPH oxidase; NO, nitric oxide; NOS, nitric oxide synthase; O 2 , oxygen; PGH 2 , prostaglandin H 2 ; PGI 2 , prostacyclin; S 1 , serotonin UHFHSWRU W\SH 7 WKURPELQ UHFHSWRU 7*)ȕ WUDQVIRUPLQJ JURZWK IDFWRU-ȕ 7+% WHWUDK\GURELRSWHULQ 7KU thrombin; TXA 2 , thromboxane A 2 . (Spieker et al. 2006).

Figure 11 .

 11 Figure 11. Pathological pathways of endothelial dysfunction associated with insulin resistance and hyperinsulinemia. Endothelial dysfunction characterised by increased expression of adhesion molecules, stimulates macrophage infiltration and release of proinflammatory cytokines (green spheres), induces proliferation of VSMC, and increases platelet aggregation. VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule; eNOS, endothelial NO synthase; ROS, reactive oxygen species. (Potenza et al. 2009).

Figure 12 .

 12 Figure12. Role of low-density lipoprotein (LDL) in the atherosclerotic process. Oxidative stress increases free radical production. These radicals provoke an oxidative modification from low-density lipoprotein (LDL) to oxidized low-density lipoproteins (ox-LDL). Circulating monocytes migrate to the sub endothelial space in response to ox-LDL and causes endothelial cell injury. The modified LDL is taken up by macrophages which become foam cells, leading to the formation of atherosclerotic plaque.[START_REF] Bonomini | Atherosclerosis and oxidative stress[END_REF]).

Figure 13 .

 13 Figure 13 . Saturated fatty acids (SFA), Monounsaturated fatty acids (MUFA).

Figure 14 .

 14 Figure 14. Polyunsaturated fatty acids (PUFA). ALA, Į-linolenic acid and; LA, Linoleic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid (Nair et al. 1997).

  January 2006, the US FDA noted that foods containing trans-fatty acids must have a label sharing this information (Food and Drug Administration 2006).

Figure 15

 15 Figure 15 Trans fatty acids structure of oleic acid and elaidic acid (Mensink and Katan 1990).

Figure 16 .

 16 Figure 16. Content of omega-3-PUFA in fishes (Feskens et al. 1993).

Figure 17 .

 17 Figure 17. Eicosanoid metabolites derived from omega-3 and omega-6 polyunsaturated fatty acids. COX, cyclooxygenase; LOX, lipoxygenase; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; AA, arachidonic acid; LT, leukotriens; PG, prostaglandin; TXA 2 , thromboxane (Abeywardena and Head 2001).

Figure 18 .

 18 Figure 18. Synthesis pathway of omega-6 and omega-3 fatty acids in mammals (Kidd 2007).

Figure 19 .

 19 Figure 19. Incorporation of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into lipid rafts and caveolae. Modulation of the microdomain lipid composition, resulting in the modulation of downstream cellular signaling events[START_REF] Ma | n-3 PUFA and membrane microdomains: a new frontier in bioactive lipid research[END_REF]).

Figure 20 .

 20 Figure 20. Synthesis and actions of lipid mediators produced from arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). COX, cyclooxygenase; LOX, Lipoxygenase; LT, Leukotriene; PG, prostaglandin; 5-LOX, Lipoxygenases; LTs, Leukotrienes (Calder 2010).

  characterization of the relaxation induced by the optimized EPA:DHA 6:1 formulation shows that it is able to induce endothelium-dependent relaxation involving mainly the NOand EDH-mediated components of the relaxation. Moreover, the relaxation induced by the optimized EPA:DHA 6:1 formulation was significantly reduced in the presence of inhibitors of Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK as well as by intracellular ROS chelators. Altogether, the results indicate that the EPA:DHA 6:1 formulation induces an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways leading to the activation of eNOS.

  identification of cellular and molecular mechanisms involved in the beneficial effects of omega-3 fatty acids on the endothelium. Moreover, these in vivo studies could lead to the identification of pertinent biological markers of the EPA:DHA effects on the endothelial function. Such markers would allow a better evaluation of the beneficial effects of the EPA:DHA 6:1 formulation during future randomized clinical trials in patients with cardiovascular diseases or risk factors associated with endothelial dysfunction. In conclusion, the present work has assessed the potency of EPA:DHA formulations to protect the endothelial function ex vivo. The ability of EPA:DHA formulation to induce endothelium-dependent relaxations is dependent on both the ratio and the purity of the formulation, with the maximal effect obtained with a highly purified EPA:DHA 6:1 ratio. The endothelium-dependent relaxation induced by the optimized EPA:DHA 6:1 formulation is mainly mediated by a redox-sensitive activation of PI3-kinase/Akt and/or MAPKs pathways leading to the activation of eNOS by phosphorylation. Moreover, the optimized EPA:DHA 6:1 formulation is able to effectively inhibit the platelet-induced serotonin-mediated contraction of porcine coronary artery rings, but not the thromboxane A 2 -mediated contraction. The inhibition by the optimized EPA:DHA 6:1 formulation of the 5-HT-induced contraction in both porcine coronary artery and human internal mammary artery rings is mainly due to an increased endothelial formation of NO. Taken together, the present findings indicate that the optimized EPA:DHA 6:1 formulation is able to exert potent beneficial cardiovascular effects through the activation of the endothelial function leading to an increased formation of NO. Resume / ¶HQGRWKpOLXP MRXH XQ U{OH FOp GDQV OD UpJXODWLRQ GH O ¶KRPpRstasie vasculaire principalement via la formation et la libération de facteurs vasodilatateurs ou vasoconstricteurs. Plusieurs études ont montré une association entre la SUpVHQFH G ¶XQH dysfonction endothéliale, caractérisée par un déséquilibre dans la formation endothéliale des facteurs vasorelaxants et des facteurs vasoconstricteurs, et le développement des maladies cardiovasculaires comme O ¶K\SHUWHQVLRQ HW O ¶DWKpURVFOpURVH (Anderson 1999; Forstermann et al. 1994; Hadi and Suwaidi 2007; Sase and Michel 1995) /HV HIIHWV SURWHFWHXUV GH O ¶HQGRWKpOLXP vis-à-vis de la fonction vasculaire ont été en partie attribués à sa capacité à produire plusieurs facteurs vasoprotecteurs qui sont principalement le NO, O ¶EDH, et le PGI 2 . La détérioration de la fonction endothéliale est généralement associée à une diminution de la biodisponibilité du NO dans de nombreuses maladies chroniques FRPPH O ¶K\SHUWHQVLRQ OH GLDEqWH ou

  caractérisation de la relaxation indique que la formulation optimisée EPA:DHA 6:1 induit une relaxation GpSHQGDQWH GH O ¶endothélium impliquant principalement les composantes NO et EDH de la relaxation. De plus, la relaxation induite par la formulation optimisée EPA:DHA 6:1 est réduite significativement en présence des inhibiteurs de la Src kinase, PI3-kinase, p38 MAPK, MEK, ou JNK, aussi bien que par les chélateurs de ROS intracellulaires. Ces résultats indiquent que formulation EPA:DHA 6:1 induit une activation redox-sensible des voies PI3kinase/Akt and MAPKs conduisant à une activation de la eNOS.

  ¶RQW SDV GH IRQFWLRQV K\GUR[\OHV pouvant conduire à une auto-oxydation, et par conséquent induisent vraisemblablement la formation intracellulaire de ROS par G ¶DXWUHV PpFDQLVPHV j moins TX ¶ils ne soient transformés en métabolites hydroxylés. En effet, des analyses métabolomiques des eicosanoïdes trouvés dans le plasma humain indiquent que les concentrations des métabolites époxy G ¶(3$ HW GH '+$ SURYHQDQW GX CYP450 et des métabolites dihydroxy correspondants sont significativement augmentées après ingestion G ¶DFLGHV JUDV RPpJD-3 (Zhang et al. 2015). La possibilité que ces métabolites soient responsables de la formation endothéliale de ROS reste cependant à démontrer. Alternativement, O ¶LQFRUSRUDWLRQ GHV DFLGHV JUDV RPpJD-3 dans les membranes cellulaires pourrait induire O ¶DFWLYDWLRQ GHV HQ]\PHV SUR-oxydantes menant à la formation de ROS. En effet, après incorporation dans les membranes cellulaires, le DHA a été impliqué dans O ¶DFWLYDWLRQ GH QRPEUHXVHV SURWpLQHV PHPEUDQDLUHV FRPPH OD SURWpLQH NLQDVH & OH cytochrome P450, plusieurs transporteurs et les réFHSWHXUV GH O ¶LQVXOLQH

6

  :1 à prévenir la vasoconstriction induite par les plaquettes. Dans les artères coronaires pathologiques, les plaquettes peuvent être activées localement et induire une vasoconstriction. En effet O ¶DJUpJDWLRQ SODTXHWWDLUH HVW associée avec la contraction du muscle lisse vasculaire FKH] O ¶KRPPH HW GDQV O ¶DUWqUH FRURQDLUH GH SRUF Chand and Altura 1980; Freeman et al. 1981; Joiner et al. 1975). Dans les artères présentant une altération GH O ¶endothélium, comme les artères coronaires ayant GHV SODTXHV G ¶DWKpURVFOpURVH O ¶DFWLYDWLRQ ORFDOH GHV SODTXHWWHV HW la contraction consécutive peuvent FRQGXLUH j O ¶LVFKpPLH, voire à un possible vasospasme. Nos résultats indiquent que dans dHV DQQHDX[ G ¶DUWqUHV FRURQDLUHV GH SRUF OHV SODTXHWWHV humaines lavées induisent des contractions dépendantes de la dose qui sont significativement inhibées en présence de kétansérine, un antagoniste du récepteur de la sérotonine 5-HT 2A , ce qui suggère que la contraction induite par les plaquettes est principalement due à une libération de sérotonine VHFRQGDLUH j O ¶DFWLvation plaquettaire. 'H SOXV O ¶LQFXEDWLRQ j FRXUW terme des anneaux avec la formulation optimisée EPA:DHA 6:1 conduit à la suppression de la contraction induite par les plaquettes, alors que la formulation EPA:DHA 1:1 est sans effet. Le fait que la prévention de la contraction par la formulation optimisée EPA:DHA 6:1 à 0,4% (v/v) Q ¶HVW SDV REVHUYpH HQ O ¶DEVHQFH G ¶HQGRWKpOLXP IRQFWLRQQHO RX HQ SUpVHQFH G ¶un inhibiteur de la eNOS suggère que la formulation optimisée EPA:DHA 6:1 exerce ses effets inhibiteurs par une augmentation de la formation endothéliale de NO.De précédentes études réalisées in vivo ont montré que la contraction induite par les plaquettes dans des artères coronaires canines est due à la fois à la libération plaquettaire de thromboxane A 2 et de 5-HT[START_REF] Golino | Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Thromboxane A2 and serotonin are possible mediators[END_REF]. Ainsi, nous avons évalué le potentiel de la formulation en acides gras oméga-3 à empêcher les contractions induites par ces deux vasoconstricteurs. La formulation EPA:DHA 6:1 supprime de façon dépendante de la dose la contraction induite par la 5-HT WDQGLV TX ¶HOOH est sans effet sur celle induite par un analogue du thromboxane A 2 (U46619). Comme les réponses des lits vasculaires peuvent varier VHORQ O ¶HVSqFH FRQVLGpUpH, O ¶H[WUDSRODWLRQ GHV PpFDQLVPHV REVHUYpV SRXU O ¶DUWqUH FRURQDLUH GH SRUF QpFHVVLWH G ¶rWUH validée dans une artère humaine. Ainsi, nous avons pu observer des résultats similaires dans dHV DQQHDX[ G ¶DUWqUHV PDPPDLUHV LQWHUQHV KXPDLQHV, la formulation EPA:DHA 6:1 à 0,4% (v/v) inhibant significativement la contraction induite par la 5-HT et provoquant une vasorelaxation par des mécanismes dépendants du NO endothélial. Toutefois, la 5-HT est décrite comme un agent pouvant induire des spasmes GDQV O ¶DUWqUH PDPPDLUH interne humaine (Rosenfeldt et al. 1999) et en particulier dans les artères ayant une dysfonction endothéliale (He 1999). Lors de notre étude, nous avons pu observer la contraction induite par la 5-HT GDQV OHV DQQHDX[ G ¶DUWqUHV PDPPDLUHV LQWHUQHV KXPDLQHV ce qui peut être dû au fait que ces artères proviennent de patients ayant subis un pontage aorto-coronarien présentant probablement aussi une dysfonction endothéliale avérée. 'DQV O ¶HQVHPEOH FHV UpVXOWDWV LQGLTXHQW TXH OD formulation optimisée EPA:DHA 6:1 est capable de supprimer les contractions induites par la 5-HT plaquettaire dans la coronaire de porc et l ¶DUWqUH PDPPDLUH LQWHUQH KXPDLQH via des mécanismes dépendants GH O ¶endothélium et impliquant le NO. Une principale limitation de nos résultats vient GX IDLW TX ¶LOV VRQW REWHQXV ex vivo en utilisant GHV DQQHDX[ G ¶DUWqUHV VXVSHQGXV GDQV GHV FXYHV à organes isolés. De plus, nous avons évalué seulement les effets à court terme induits par la formulation optimisée EPA:DHA 6:1, tandis que les effets bénéfiqXHV G ¶XQ DSSRUW HQ DFLGHV JUDV GH W\SH RPpJD-3 sur le système cardiovasculaire décrits dans les essais cliniques sont peut-être dû à une modulation à longterme induite par O ¶LPSUpJQDWLRQ FKURQLTXH GHV WLVVXV Le travail présent demande donc des recherches compléPHQWDLUHV SRXU PLHX[ FDUDFWpULVHU O ¶HIIHW EpQpILTXH GHV DFLGHV JUDV GH W\SH oméga-3 VXU O ¶HQGRWKpOLXP ' ¶DERUG OHV PpFDQLVPHV LPSOLTXpV GDQV OD IRUPDWLRQ intracellulaire de ROS en réponse à la formulation EPA:DHA 6:1 QpFHVVLWHQW G ¶être clarifiés. De même, les effets à long terme de la formulation EPA:DHA 6:1 doivent être évalués sur O ¶HQGRWKpOLXP, et en particulier OD SRVVLELOLWp G ¶LQGXLUH XQH expression de la eNOS conduisant à une augmentation de la formation basale GH 12 GDQV O ¶HQGRWKpOLXP, ce qui peut à terme conduire à O ¶DPpOLRUDWLRQ GH OD IRQFWLRQ HQGRWKpOLDOH HW à une diminution du risque de développer une maladie cardiovasculaire. De plus, le travail actuel sur les effets endothéliaux de la formulation EPA:DHA 6:1 doivent être confirmés sur des modèles de dysfonction endothéliale in vivo. La prise orale chronique G ¶RPpJD-3 a été associée à une augmentation plasmatique GX WDX[ G ¶DFLGHV JUDV GH W\SH oméga-3, DLQVL TX ¶j la présence de nombreux métabolites circulants pouvant exercer des effets VXU O ¶HQGRWKpOLXP Ainsi, la capacité de la formulation EPA:DHA 6:1 à empêcher la dysfonction endothéliale et/ou améliorer la fonction endothéliale doit être évaluée sur des modèles in vivo GH G\VIRQFWLRQ HQGRWKpOLDOH FRPPH O ¶K\SHUWHQVLRQ LQGXLWH SDU O ¶DQgiotensine-II chez le rat. De plus, de telles études pourraient également permettre G ¶identifier les mécanismes cellulaires et moléculaires impliqués dans les effets bénéfiques des acides gras oméga-VXU O ¶HQGRWKpOLXP. En outre, ces études in vivo pourront conduire à O ¶identification de nouveaux marqueurs biologiques pertinents des effets de la formulation EPA:DHA 6:1 sur la fonction endothéliale. Ces marqueurs pourront in fine permettre une meilleure évaluation des effets bénéfiques de la formulation EPA:DHA 6:1 lors de futurs essais cliniques randomisés chez des patients ayant des maladies cardiovasculaires ou des facteurs à risques associés à une dysfonction endothéliale. (Q FRQFOXVLRQ OH WUDYDLO DFWXHO D pYDOXp O ¶DFWLYLWp GH IRUPXODWLRQV (3$'+$ à protéger la fonction endothéliale ex vivo. La capacité de la formulation EPA:DHA à induire des relaxations dépendantes GH O ¶endothélium est liée à la fois à la teneur et à la pureté de la formulation, avec un effet maximal obtenu avec un rapport EPA:DHA de 6:1 hautement purifiés. La relaxation GpSHQGDQWH GH O ¶HQGRWKpOLXP induite par la formulation optimisée EPA:DHA 6:1 est principalement due à une activation redox-sensible des voies PI3kinase/Akt et/ou MAPKs conduisant à une phosphorylation activatrice de la eNOS. De plus, la formulation optimisée EPA:DHA 6 HVW FDSDEOH G ¶LQKLEHU fortement la contraction induite par la 5-HT plaquettaire dans les anneaux d ¶DUWqUHV FRURQDLUHV GH SRUF PDLV Q ¶D SDV G ¶HIIHW sur la contraction induite par un analogue du thromboxane A 2 / ¶LQKLELWLRQ GH OD formulation optimisée EPA:DHA 6:1 sur la contraction induite par la 5-HT dans les artères coronaires de SRUF HW VXU OHV DQQHDX[ G ¶DUWqUHV PDPPDLUHV LQWHUQHV KXPDLQHV HVW SULQFLSDOHPHQW due à une augmentation de la formation endothéliale de NO. 'DQV O ¶Hnsemble, ces résultats indiquent que la formulation optimisée EPA:DHA 6 HVW FDSDEOH G ¶H[HUFHU GH SXLVVDQWV HIIHWV bénéfiques sur le système cardiovasculaire via O ¶DFWLYDWLRQ GH OD IRQFWLRQ HQGRWKpOLDOH

Chapter one: Physiology of the endothelium Introduction 1. Cardiovascular diseases

	formulation seems to be a promising novel strategy against the endothelial dysfunction
	characterized, at least in part, by a reduced endothelial formation of NO, and associated with
	the development of 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. Cardiovascular diseases are the leading cause of mortality and morbidity
	worldwide, with an estimated 17 million annual deaths (WHO 2011). Amongst cardiovascular
	diseases, CHD accounts for 7.2 million deaths and 5.7 million are due to stroke. It is now well
	established that endothelial dysfunction is an early hallmark of major cardiovascular diseases,
	The
	endothelium-dependent relaxation induced by the optimized EPA:DHA 6:1 formulation is
	mainly mediated by a redox-sensitive activation of PI3-kinase/Akt and/or MAPKs pathways
	leading to the activating phosphorylation of eNOS. Moreover, the optimized EPA:DHA 6:1
	formulation is able to effectively inhibit the platelet-induced serotonin-mediated contraction
	of porcine coronary rings, but has no effect on the thromboxane A 2 -mediated contraction. The
	inhibition by the optimized EPA:DHA 6:1 formulation of the 5HT-induced contraction in
	both porcine coronary artery and human internal mammary artery rings is mainly due to an
	increased endothelial formation of NO. Taken together, the present findings indicate that the
	optimized EPA:DHA 6:1 formulation is able to exert potent beneficial cardiovascular effects
	through the activation of the endothelial function. The omega-3 EPA:DHA 6:1 optimized

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

 1

  -Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in

	Response to EPA:DHA 6:1.
	Faraj Zgheel, Mahmoud Alhosin, Sherzad Rashid, Mélanie Burban, Cyril Auger, Valérie B.
	Schini-Kerth.
	PLOS ONE 9, 2014; (8): 2014 e105102. doi:10.1371/journal.pone.0105102.

  VRXUFHV G ¶RPpJD-3, des formulations et des dosages utilisés dans les différentes études. L ¶REMHFWLI du présent travail de thèse a été dans un premier temps G ¶pYDOXHU OH rôle de la formulation en oméga-VXU O ¶LQGXFWLRQ GHV UHOD[DWLRQV dépendantes de O ¶endothélium, ainsi que la contribution respectives des composantes NO et EDH de la relaxation, et également de déterminer les mécanismes sous-MDFHQWV /H VHFRQG REMHFWLI D pWp G ¶pYDOXHU O ¶HIIHW YDVFXODLUH bénéfique des oméga-3 sur la contraction des artères coronaires induite par les plaquettes et de FDUDFWpULVHU O ¶LPSOLFDWLRQ de la sérotonine et des prostanoïdes vasoconstricteurs. Pour cela, nous avons évalué la capacité de formulations présentant différents ratios et degrés de pureté en EPA et DHA à inGXLUH GHV UHOD[DWLRQV GpSHQGDQWHV GH O ¶HQGRWKpOLXP GDQV des DQQHDX[ G ¶artères coronaires de porc. Ainsi, nRV UpVXOWDWV PRQWUHQW TXH O ¶LQGXFWLRQ GH OD fonction endothéliale, HW HQ SDUWLFXOLHU O ¶DFWLYDWLRQ GH OD IRUPDWLRQ endothéliale de NO, est dépendante à la fois du ratio EPA:DHA et de la pureté des formulations en oméga-3. L ¶(3$ ou le DHA isolés VRQW FDSDEOHV G ¶LQGXLUH GHV UHOD[DWLRQV dépendantes GH O ¶endothélium dans GHV DQQHDX[ G ¶DUWqUH FRURQDLUH de porc, la relaxation en réponse à O ¶(3$ à une concentration de 0,4% (v/v) étant significativement plus importante que celle du '+$ 'H PrPH O ¶pWXGH des différents ratios (3$'+$ LQGLTXH TXH O ¶HIIHW UHOD[DQW HVW PD[LPDO SRXU XQ UDSSRUW EPA:DHA de 6 :1 ou 9 :1, tandis que les formulations avec des ratios EPA:DHA inférieurs En outre, les divers essais cliniques publiés dans la littérature montrent une grande variabilité GDQV OHV SDUDPqWUHV G ¶LQFOXVLRQ GHV SDWLHQWV HW GHV volontaires, ainsi que dans les événements étudiés et les paramètres cliniques déterminés. En effet, les essais cliniques randomisés ont

induisent des relaxations plus faibles. De plus, une formulation EPA:DHA 6 :1 avec une pureté relative en omega-3 de 50 % induit une relaxation significativement plus faible que celle obtenue avec une formulation EPA:DHA de grande pureté (> 90 %). Ces résultats suggèrent que les effets bénéfiques des formulations en oméga-3 vis-à-vis du système vasculaire sont dépendants à la fois de la composition en oméga-3 totaux et du rapport EPA:DHA. Ces paramètres devraient donc être pris en compte SRXU O ¶LQWHUSUpWDWLRQ HW OD comparaison des résultats des essais précliniques et cliniques utilisant différents produits riches en oméga-3. De plus, ces travaux peuvent aussi aider à comprendre les différences trouvés dans les résultats de méta-analyses évaluant les effets cardiovasculaires de plusieurs formulations allant GH O ¶KXLOH GH SRLVVRQV j des omégas-3 (EPA et/ou DHA) extraits et purifiés à différents ratios et doses. En effet, dans plusieurs méta-analyses, les essais cliniques sélectionnés UDSSRUWHQW O ¶XWLOLVDWLRQ de plusieurs approches pour O ¶apport en acides gras de type oméga-3 incluant des conseils nutritionnels (comme manger du poisson 2 à 3 fois par semaine) XQH FRQVRPPDWLRQ G ¶KXLOH de poissons ou une supplémentation avec diverses formulations G ¶oméga-3 (Delgado-Lista etal. 2012;[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][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Kromhout | Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?[END_REF]. De plus, les doses G ¶RPpJD-3 utilisées dans les essais cliniques sont très hétérogènes avec des teneurs allant de 0,18 à 10 g par jour sous forme d ¶KXLOHs de poissons ou de formulations d ¶(3$ et/ou de DHA purifiés[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][START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF][START_REF] Kromhout | Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?[END_REF]).

Chapter Two:

Omega-3 fatty acids and cardiovascular diseases

Lowering triglyceride levels

Several epidemiological studies have reported the positive association between an increased level of triglycerides in plasma and an increased development of cardiovascular diseases [START_REF] Harris | n-3 fatty acids and serum lipoproteins: human studies[END_REF][START_REF] Sacks | Randomized clinical trials on the effects of dietary fat and carbohydrate on plasma lipoproteins and cardiovascular disease[END_REF]. Recent studies suggest that omega-3 fatty acids play an important role in the regulation and reduction of triglycerides concentration via inhibition of the hepatic secretion of VLDL and triglycerides synthesis [START_REF] Harris | Why do omega-3 fatty acids lower serum triglycerides?[END_REF][START_REF] Sampath | Polyunsaturated fatty acid regulation of genes of lipid metabolism[END_REF]. Moreover, a study has shown that consumption of 3.4 g/day of EPA and DHA reduces the plasma triglycerides level by 17 % [START_REF] Eritsland | Effects of highly concentrated omega-3 polyunsaturated fatty acids and acetylsalicylic acid, alone and combined, on bleeding time and serum lipid profile[END_REF]. Moreover the beneficial effect of omega-3 fatty acids on the plasma TG level has been reflected to inhibition an increased intracellular degradation of apolipoprotein B-100.

Anti-inflammatory effects of omega-3 fatty acids

Studies on the effects of lipids derived from polyunsaturated fatty acids in human have attracted much attention in recent years. Both omega-6 and omega-3 fatty acids have been associated with the activation and resolution of inflammatory responses, respectively. Arachidonic acid (AA) derived from omega-6 fatty acids is the most important precursor which is converted by different enzymes, such as COX and lipoxygenases (LOX), to different eicosanoid families including the prostaglandins, thromboxanes, leukotrienes, lipoxins, hydroxy and hydroperoxy-eicosatetraenoic acids [START_REF] Calder | Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms and clinical relevance[END_REF]. Arachidonic acid metabolites are implicated in various pathological processes such as platelet aggregation, haemorrhage, and vasoconstriction [START_REF] Rizos | Psychotropic drug interactions in the patient with late-onset depression or psychosis. Part 2[END_REF]. Conversely, dietary omega-3 fatty acids, and particularly EPA, are the main sources of the 3-series of prostaglandins (PGs) and thromboxanes, and 5-series leukotrienes (LTs), all having anti-inflammatory and vasorelaxant effects. In addition, anti-inflammatory DHA-derived metabolites have been identified as dseries resolvins and protectins [START_REF] Hong | Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation[END_REF]). Cells culture and animal feeding studies have shown that the EPA-and DHA-derived resolvins and related compounds, such as resolvin E1, resolvin D1 and protectin D1, have anti-inflammatory and pro-resolution effects [START_REF] Calder | Omega-3 fatty acids and inflammatory processes[END_REF]. These effects are mediated, at least in part, through down-regulation of TNF expression, which plays a major role in inflammatory gene expression [START_REF] Serhan | Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators[END_REF].

Moreover, studies have suggested that omega-3 fatty acids may down-regulate the nuclear factor (NF)-kB pathway by the activation of peroxisome proliferators-activated receptors [START_REF] Adkins | Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids[END_REF][START_REF] De Winther | Nuclear factor kappaB signaling in atherogenesis[END_REF].

AIM OF THE STUDY

RESULTS

Article I

Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1.

Article II

The omega-3 EPA:DHA 6:1 formulation prevents the platelets-induced arterial contraction by abolishing the serotonin-mediated contraction in porcine coronary and human internal mammary artery rings.

Article II

The omega-3 EPA:DHA 6:1 formulation prevents the platelets-induced arterial contraction by abolishing the serotonin-mediated contraction in porcine coronary and human internal mammary artery rings Faraj ZGHEEL 1 , Stéphanie PERRIER

In preparation

Our previous work has indicated that various formulations of omega-3 fatty acids products induce potent relaxations in porcine coronary artery rings depending on the EPA:DHA ratio and degree of purity. Indeed, the EPA:DHA 6:1 ratio with a high purity rate was amongst the most potent product tested. Moreover, the relaxation induced in porcine coronary artery rings by omega-3 fatty acids products is an endothelium-dependent event mediated mainly by the activation of eNOS but also, to a lesser extent, to the EDH component. The EPA:DHA 6:1 ratio has been shown to induce the endothelium-dependent NO-mediated relaxation through redox activation of Src/PI3-kinase/Akt and MAPKs with the subsequent activation of eNOS.

While these findings indicate that omega-3 fatty acids products activate the endothelial function in healthy porcine coronary artery rings, the relevance of the beneficial effects of omega-3 products in pathophysiological conditions remains to be determined. Indeed, in pathophysiological conditions such as atherosclerosis, hypertension, metabolic syndrome or diabetes, blood vessels often present an endothelial dysfunction which is associated a reduced formation of endothelium-derived relaxing factors and increased

Results and discussion

The present study indicates that human washed platelets can induce dose-dependent contractions in porcine coronary artery rings. The platelet-induced contraction is significantly inhibited in the presence of the EPA:DHA 6:1 formulation, whereas the EPA:DHA 1:1 formulation had little effect. Moreover, the EPA:DHA 6:1 formulation dose-dependently prevented the serotonin (5-HT)-induced contraction, whereas it was without effect on the thromboxane A 2 analogue (U46619)-induced contraction. The latter, associated to the fact that the platelet-induced contraction was markedly reduced by ketanserin, a 5-HT 2A receptor antagonist, strongly suggests that human washed platelets induced the contraction in porcine coronary artery rings mainly through serotonin released from activated platelets. The EPA:DHA 6:1 at 0.4 % (v/v) was able to prevent both the platelet-induced and the 5HTinduced contractions only in the presence of a functional endothelium, whereas it was without effect in endothelium-denuded rings or in the presence of an eNOS inhibitor. These findings suggest that the preventive effect of EPA:DHA 6:1 on platelet-induced serotonin-mediated contraction is mainly due to its ability to induce the endothelium-dependent formation of NO. This hypothesis is reinforced by the fact that a low concentration SNP, a NO donor, significantly inhibited the contraction induced by 5-HT, but not that by U46619. In addition, the EPA:DHA 6:1 formulation at 0.4 % (v/v) also significantly prevented the 5-HT-induced contraction in human mammary artery rings through NO-mediated mechanisms.

Abstract

The aim of the present work was to evaluate both the potency of omega-3 formulations to induce the endothelial function and the effect of omega-3 on the platelet-induced coronary artery contraction.

The endothelium-dependent relaxations induced by EPA:DHA formulations is dependent on both the ratio and the purity of the formulation, with the maximal effect obtained with a highly purified EPA:DHA 6:1 ratio through a redox-sensitive activation of PI3-kinase/Akt and/or MAPKs pathways and subsequent activation of eNOS. The EPA:DHA 6:1 formulation inhibits the platelet-induced serotonin-mediated contraction of porcine coronary rings and the 5HT-induced contraction in both porcine coronary artery and human internal mammary artery rings, mainly due to an increased endothelial formation of NO. Taken together, the present findings indicate that the optimized EPA:DHA 6:1 formulation is able to exert potent beneficial cardiovascular effects through the activation of the endothelial function.