K Sherzad

Noureddine Idris Rashid

Cyril Khodja

Mahmoud Auger

Nelly Alhosin

Monique Boehm

Valérie B Oswald-Mammosser

Schini-Kerth

Blackcurrant

Noureddine Rashid

Cyril Idris-Khodja

Probiotics

3 Oswald-Mammosser

Monique

Sherzad K Rashid

Boehm Nelly

Agin Arnaud

Geny Bernard

Schini-Kerth Valérie
email: valerie.schini-kerth@unistra.fr

Anne Charloux

Effect

Boisramé-Helms Amissi Said

Burban Julie

Rashid Mélanie

Cyril Auger

Eric Anselm

Jong Hun Kim

Mahmoud 7 Faraj Zgheel

Sherzad K Alhosin

Cyril Rashid

Valérie B Auger

Israa Mahmoud Alhosin

Agnès Dandache

Sherzad K Lelay

Luc-Matthieu Rashid

Laurent Fornecker

Raoul Mauvieux

Valérie B Herbrecht

Bilberry

Noureddine Rashid

The

Noureddine Rashid

Mahmoud Alhosin

Noureddine Rashid

Sherzad K Mélanie

Florence Auger

Ferhat Toti

Valérie B Meziani

Sherzad 2 Monique K Oswald-Mammosser

Nelly Rashid

Arnaud Boehm

Bernard Agin

Linus Pauling

Noureddine Idris Khodja

Nelly Boehm

Monique Oswald-Mammosser
email: monique.oswald@chru-strasbourg.fr

Vale ´rie

B Schini-Kerth

mesenteric artery of rats with portal hypertension, soumis pour publication

Keywords: Portal Hypertension, Endothelial Dysfunction, Blackcurrant, Polyphenols, Angiotensin II, Oxidative Stress, Inflammatory Cytokines

The highly purified EPA:DHA 6:1 product-evoked endotheliumdependent NO-mediated relaxation in the coronary artery involves a copper-dependent event triggering the redox-sensitive

ABBRAVATIONS

abnormalities are known as the hepatopulmonary syndrome (HPS): arterial de-oxygenation due to pulmonary vascular dilatation in the context of liver disease. It is important to diagnose HPS because it is an independent prognostic factor of survival and because it also affects postoperative mortality in liver transplantation. Till now the only available treatment for HPS is liver transplantation which leads to a regression and often a disappearance of the HPS.

Nevertheless, having an efficient medical treatment is necessary for two reasons. First, given that there is no close correlation between the occurrence of hypoxemia and the severity of liver disease, there are cases where severe hypoxemia requires liver transplantation although liver disease is only mild and would otherwise not be an indication for transplantation.

Improving hypoxemia would therefore allow delaying transplantation in such cases. Second, since HPS is associated with an increased transplantation morbidity and mortality, improving hypoxemia could ameliorate survival in patients with a HPS during and after transplantation.

The mechanisms leading to vasodilation due to portal hypertension are not fully understood. Till now, nitric oxide (NO) has been thought to play a major role. It is a well known vasodilator the production of which has been shown to be increased in the rat model of biliary cirrhosis. Moreover, in animal studies chronic treatment with N G -nitro-L-arginine methyl ester (L-NAME), a non-specific NO synthase inhibitor attenuated or prevented the occurrence of HPS. However clinical trials of treatment with inhaled L-NAME led to contradictory results. This can be explained by the fact that other factors play a role. It has been suggested that the renin-angiotensin-aldosterone system associated to oxidative stress may be important. Oxidative stress is linked in part to bacterial translocation that is present in a significant proportion of patients with advanced cirrhosis. Finally, other factors have been implicated: the carbon monoxide which is a vasodilator produced by the heme-oxygenase and it has also been suggested that estrogens (high levels in some patients) could lead to pulmonary vascular dilatation.

The aims of our work were thus to estimate the effects of a probiotic which could act on bacterial translocation, the effects of polyphenols on oxidative stress and the possible improvement of HPS by the estrogen receptor antagonist fulvestrant in a rat model of biliary cirrhosis. The first two studies have been devoted to the effects of a probiotic and a polyphenol rich compound on the splanchnic vessels and the last study to the effects of fulvestrant on the lung. Biliary cirrhosis with portal hypertension has been induced by bile duct ligation in wistar rats under general anesthesia. This leads to a hyperdynamic circulatory state and a hepatopulmonary syndrome. The rats were examined 4 weeks after surgery.

It was shown in an experimental model of partial portal vein ligation in the rat that the initial event because of portal hypertension is an up-regulation in the expression of VEGF (vascular endothelial growth factor) and eNOS (endothelial nitric oxide synthase) in the intestinal microcirculation. Thereafter, a systemic vasodilation occurs, and in particular in the lung leading to a HPS.

The effects of the probiotic and polyphenols were mainly studied on the mesenteric artery and the aorta:

-Vascular reactivity studies were performed in isolated organ chambers in the presence of indomethacin to prevent the formation of vasoactive prostanoids. The NO component of relaxation was studied in the presence of charybdotoxin and apamin (inhibitors of the EDHF response). The EDHF (endothelial derived hyperpolarizing factor) component was studied in the presence of N G -nitro-L-arginine.

-The formation of reactive oxygen species (ROS) was estimated using the fluorescent probe dihydroethidine before the determination of different sources of ROS.

-Immunofluorescence and western blot analyses were used to quantify the expression of different components of the vascular reactivity and of the renin-angiotensin-aldosterone system (RAAS).

-Inflammatory cytokines levels were measured in the plasma

The effects of fulvestrant were studied on the lungs:

-Immunohistochemistry on lung sections allowed to quantify the intravascular macrophages and the small vessels diameter.

-Western blot analyses was used to quantify the expression of eNOS, iNOS, nitrotyrosine and p-VASP, and VEGF and p-Akt.

-Nitrites and hormones levels were measured in the plasma and COHb was measured in arterial blood samples.

The first study showed that treatment with probiotics improved the NO-and EDHF-dependent components of relaxation in the mesenteric vascular bed. This was accompanied by a decreased production of ROS and expression of RAAS factors and a decrease in proinflammatory cytokines in the plasma. Bacterial translocation and oxidative stress leading to the production of pro-inflammatory cytokines and to the stimulation of local RAAS is probably an important factor in the development of endothelial dysfunction in cirrhotic rats.

The administration of probiotics has improved these abnormalities.

The second study showed that the administration of polyphenols improves endothelial dysfunction in cirrhotic rats: improved EDHF-mediated relaxations, decreased expression of eNOS, ROS production, and some components of the RAAS and decreased levels of proinflammatory cytokines. Oxidative stress, part of which may be due to bacterial translocation, but other factors are also involved, is probably a key factor in the endothelial dysfunction in cirrhotic rats. The relation between the RAAS and oxidative stress is again highlighted in this study which confirms our previous studies where treatment with losartan (angiotensin type 1 receptor antagonist) improved endothelial dysfunction in rats with biliary cirrhosis.

In the third study, treatment with fulvestrant (an estrogen receptor antagonist) decreased the expression of eNOS and nitrotyrosines production in the lung. However, the expression of p-VASP which is a marker of the action of NO was reduced in cirrhotic rat lungs and fulvestrant had no effect on this marker. The hypothesis is that despite the increase in eNOS expression, the produced NO is captured by ROS which would prevent its activity.

This could also explain the impaired NO component of relaxation in the two earlier studies.

Fulvestrant had no effect neither on the expression of VEGF, nor on the number of macrophages in the pulmonary vessels, nor on the diameter of small pulmonary vessels. In conclusion, either fuvestrant is not effective in cirrhotic rats or the relative increase in estradiol in male rat has no role in the occurrence of a HPS

The important role of oxidative stress in the development of endothelial dysfunction in rats with biliary cirrhosis is emphasized in our studies. Oxidative stress is associated with the stimulation of the RAAS and bacterial translocation. Treatment with antioxidants (polyphenols) and probiotics have clearly demonstrated their beneficial effects on the endothelial dysfunction in our cirrhotic rats. The role of NO as a pure vasodilator is perhaps not essential which may explain the disappointing clinical results published by some authors.

Résumé

Les atteintes hépatiques chroniques se caractérisent par une vasodilatation progressive qui touche en particulier les lits vasculaires splanchnique et pulmonaire. La vasodilatation est due à l'hypertension portale que celle-ci soit la conséquence d'une cirrhose hépatique ou qu'elle soit primitive.

Au niveau pulmonaire la vasodilatation peut entraîner un shunt pouvant conduire à une hypoxémie. On parle alors de syndrome hépato-pulmonaire (SHP) dont la définition est une

CHAPTER I

INTRODUCTION Chapter 1

Introduction:

Vascular endothelium

The vascular endothelium plays a major role in controlling the vascular tone through different mechanisms. The vessel's wall consists of three individualized layers (Figure 1)

from the lumen to the periphery: the tunica intima (comprising the endothelium with a basement membrane and the internal elastic lamina), the media (smooth muscle cells and collagen fibers limited by the external elastic lamina) and the adventitia (where are nerve endings, vasa vasorum, fibroblast and macrophages in connective tissue) [START_REF] Mulvany | Structure and function of small arteries in hypertension[END_REF]).

The importance of each layer depends on the size of the arteries: more elastic fibers in the media in large conducting arteries, more smooth muscles in muscular arteries, less smooth muscle cells with only an internal elastic lamina in arterioles and only endothelium and basement membrane and connective tissue in capillaries). The endothelium covers the whole entire wall of all vessels from the heart to small vessels. Hence the endothelium has many function, this enables it to be called the chief regulator of body homeostasis. Due to their position and their large surface area, endothelial cells (ECs) assume a large variety of functions: synthesis and secretion of various molecules, including vasodilator and vasoconstrictor factors, control of smooth muscle cell (SMC) proliferation, exchanges of molecules between the plasma and the interstitial fluid; they have also a role in the balance between pro-and anticoagulant factors and in immunity.

The major endothelial functions

Endothelial cells 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][START_REF] Moncada | Nitric oxide and the vascular endothelium[END_REF] and respond also to other stimuli like microparticles and pro-inflammatory mediators. In response to stimuli the endothelium has the capacity to regulate local vascular homeostasis by maintaining the balance between vasodilation and vasoconstriction, by modulating the proliferation and migration of smooth muscle cells and by acting on thrombosis and fibrinolysis by the release of various factors [START_REF] Davignon | Role of endothelial dysfunction in atherosclerosis[END_REF]. In case of endothelial dysfunction, imbalance of these different mechanisms occurs and may lead to serious cardiovascular diseases. One of the endothelial dysfunction may be due to imbalance between vasodilators and vasoconstrictors. The principal vasoconstrictors are: thromboxane A 2, prostaglandin H 2 (PGH 2 ), endothelin-1, angiotensin II and the superoxide anions, [START_REF] Furchgott | Endothelium-derived relaxing and contracting factors[END_REF][START_REF] Touyz | Angiotensin II and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells[END_REF]). Vasodilator factors are : nitric oxide (NO), the prostacyclin or prostaglandin I 2 (PGI 2 ) and the endothelium derived hyperpolarizing (EDH) [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]Moncada and Higgs 2006) (Table1). NO is an important cellular signaling molecule involved in different physiological and pathological processes. NO was first known as a ligand capable of activating the soluble guanylyl cyclase and responsible for vascular smooth muscle cells relaxation [START_REF] Katsuki | Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine[END_REF]. Soon after, [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF] discovered that the endothelium is responsible for vascular smooth muscle relaxation through production of an endothelium-derived relaxing factor, EDRF. Finally, NO was identified as being the EDRF described earlier [START_REF] Palmer | Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor[END_REF][START_REF] Palmer | Vascular endothelial cells synthesize nitric oxide from L-arginine[END_REF][START_REF] Palmer | A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells[END_REF][START_REF] Moncada | Nitric oxide in the vasculature: physiology and pathophysiology[END_REF]. The endothelium constitutively expresses a NO synthase (endothelial NO synthase, eNOS) which is one of the three isoforms of NOS [START_REF] Mombouli | Endothelial dysfunction: from physiology to therapy[END_REF][START_REF] Stuehr | Mammalian nitric oxide synthases[END_REF]) (Table 2). Under normal conditions, inactive eNOS is bound to the protein caveolin and is located in microdomains in the cell membrane called caveolae [START_REF] Bucci | In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation[END_REF]. When intracellular Ca 2+ levels increase, (in calcium-dependent activation of eNOS) (Figure 3) calmodulin detaches eNOS from caveolin permitting the enzyme to become active. Apart from the increases in intracellular [Ca 2+ ] ,

other mechanisms (calcium-independent activation of eNOS), leading to the phosphorylation of eNOS (especially phosphorylation of Ser1177) and hence to the activation of eNOS occur via protein kinases pathways, such as protein kinase A (PKA) and cGMP dependent protein kinase II (PKG). The relative contributions of different kinase pathways remain under active investigation, but it is clear that extra-cellular stimuli (shear stress, estrogen, ET-1 and VEGF) activate distinct kinase pathways leading to eNOS phosphorylation and regulating the eNOS state at transcriptional level and stability [START_REF] Dimmeler | Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation[END_REF][START_REF] Haynes | Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells[END_REF][START_REF] Sandoo | The endothelium and its role in regulating vascular tone[END_REF]) (Table 3). eNOS generates NO upon the conversion of L-arginine to L-citrulline in the endothelium (Figure 2). NO has the ability to diffuse towards the underlying vascular smooth muscle to reduce vascular tone through activation of soluble guanylyl cyclase (sGC), and to prevent smooth muscle cell proliferation and migration thereby, maintaining the arterial wall in a quiescent state [START_REF] Murad | Guanylate cyclase: activation by azide, nitro compounds, nitric oxide, and hydroxyl radical and inhibition by hemoglobin and myoglobin[END_REF]. Apart from its action on the smooth muscles, NO has another effect: NO has also been shown to prevent the expression of numerous pro-inflammatory and pro-atherothrombotic mediators such as monocyte chemoattractant protein-1 (MCP-1), tissue factors and adhesion molecules. Moreover, NO helps maintaining blood fluidity by preventing the adhesion and aggregation of platelets and the adhesion of monocytes [START_REF] Lee | An ethanolic extract of Lindera obtusiloba stems causes NO-mediated endothelium-dependent relaxations in rat aortic rings and prevents angiotensin II-induced hypertension and endothelial dysfunction in rats[END_REF]. Diminished NO production or bioavailability has been implicated in the pathogenesis of essential and pulmonary hypertension and in multiple other vascular disorders including atherosclerosis [START_REF] Voetsch | Nitric oxide insufficiency and atherothrombosis[END_REF]). In portal hypertension: NO is diminished in the liver and increased in the splanchnic and systemic vascular beds. Many factors contribute to intra-hepatic reduced eNOS activity including increased oxidative stress, increased binding ability of caveolin-1 to eNOS and increased activity of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS causing un-coupling of NOS and leading to the generation of peroxynitrate [START_REF] Shah | Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat[END_REF][START_REF] Laleman | A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis[END_REF]. Other factors play a role like decreased Akt activity and tetra-hydrobiopterin (BH4) level (BH4 acts as important catalytic agent in the oxidation of L-arginine by NO synthases) [START_REF] Alp | Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease[END_REF]) (Figure 2), and impairment of the antioxidant system [START_REF] Hu | Current concepts on the role of nitric oxide in portal hypertension[END_REF]. In contrast to hypoactive hepatic endothelial cells, endothelial cells in the splanchnic and systemic circulation have been shown to produce NO which could modulate the vascular changes observed in cirrhosis. Increased activity of vascular Akt signaling [START_REF] Fernandez-Varo | Inactivation of extrahepatic vascular Akt improves systemic hemodynamics and sodium excretion in cirrhotic rats[END_REF], VEGF-induced NO production have been also implicated (Abraldes et al, 2006). Likewise, in portal hypertensive rats, NO production is increased in response to shear stress [START_REF] Tazi | Role of shear stress in aortic eNOS up-regulation in rats with biliary cirrhosis[END_REF] and finally the role of bacterial lipopolysaccharide (LPS) induced overproduction of pro-inflammatory cytokines like TNF-and subsequent induction of inducible NOS (iNOS) in extrahepatic vasculature have been reported [START_REF] Kajita | iNOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats[END_REF][START_REF] Moreau | Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis[END_REF][START_REF] Huang | Evolution of portal-systemic collateral vasopressin response in endotoxemic portal hypertensive rats[END_REF]. It is worthy to mention, that the iNOS induced in the endothelial and other inflammatory cells following immunological activation [START_REF] Geller | Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes[END_REF], is calcium independent, mostly transcriptionally regulated and is not normally produced in most cell [START_REF] Forstermann | Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions[END_REF], iNOS generates NO in 100-1000 fold more than eNOS and the NO activity persists for many hours [START_REF] Morris | New insights into the regulation of inducible nitric oxide synthesis[END_REF][START_REF] Nathan | Nitric oxide synthases: roles, tolls, and controls[END_REF].

Endothelium-derived hyperpolarizing factor (EDHF)

Beside NO and prostacyclin, EDHF plays an important role in endothelium-dependent relaxation in most medium-to small sized arteries [START_REF] Feletou | Endothelium-derived hyperpolarizing factor[END_REF]. EDHF is an important relaxing factor in the coronary artery as well as in small arteries and arterioles such as second and third-order mesenteric arteries [START_REF] Shimokawa | The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation[END_REF]. After its discovery in 1987, the identity of EDHF is still in the center of controversy because it differs depending on the animal species and the type of blood vessel. In 1996, Shimokawa et al. reported that EDHF is more important in resistance vessels than NO and prostacyclin [START_REF] Shimokawa | The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation[END_REF]. Also, in human arteries, endothelium-dependent vasodilation involves EDHF [START_REF] Nakashima | Endothelium-dependent hyperpolarization caused by bradykinin in human coronary arteries[END_REF]. EDHF is defined as a hyperpolarization of endothelial origin that is transmitted to the vascular smooth muscle leading to its relaxation. The EDHF component of the relaxation is evaluated in the presence of the combination of inhibitors of eNOS like L-NAME and COXs like indomethacin. To block also the EDHF mediated relaxation, we need to add the inhibitors, apamin and charybdotoxin for both calcium-dependent potassium channels of intermediate (IK Ca ) and small conductance (SK Ca ). In the EDHF-mediated response, IK Ca and SK Ca are activated so that potassium ions move from the intracellular compartment to the extracellular space of endothelial cells, which leads to their hyperpolarization (Figure 4). Thereafter, low concentrations of potassium ions in the extracellular space can activate inwardly rectifying K + (K IR ) channels and Na+/K+-ATPase to cause hyperpolarization of smooth muscle cells through the movement of potassium ions out of the smooth muscle cells [START_REF] Edwards | K+ is an endothelium-derived hyperpolarizing factor in rat arteries[END_REF][START_REF] Feletou | Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)[END_REF][START_REF] Feletou | EDHF and endothelial potassiun channels: IKCa and SKCa[END_REF]. Another pathway leading to a direct transfer of the hyperpolarization from endothelial cells to smooth muscle cells occurs via myoendothelial gap junctions (meGJ) as reported previously by [START_REF] Edwards | K+ is an endothelium-derived hyperpolarizing factor in rat arteries[END_REF][START_REF] Edwards | K+ is an endothelium-derived hyperpolarizing factor in rat arteries[END_REF]. Gap junctions are intracellular channels which can transfer signals from the endothelial cells to the underling smooth muscle cells [START_REF] Sandoo | The endothelium and its role in regulating vascular tone[END_REF] In addition, in some tissues, the hyperpolarization of the endothelial cells might be regulated by hydrogen peroxide (H 2 O 2 ) [START_REF] Matoba | Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries[END_REF] or the activation of cytochrome P450 and the resulting generation of epoxyeicosatrienoic acids (EET), which are metabolites of arachidonic acid [START_REF] Quilley | Is EDHF an epoxyeicosatrienoic acid?[END_REF]. The EET results in increasing K + efflux from the smooth muscle cells resulting in hyperpolarization and relaxation (Figure 4). Hyperpolarization of smooth muscle cells leads to a decrease in cytosolic calcium concentration with subsequent relaxation.

Prostacyclin (PGI 2 )

Another important factor of endothelium-dependant relaxation is PGI 2 . PGI 2 is a member of prostanoids (together with other prostaglandins and thromboxanes) which are products from the arachidonic acid metabolism (Figure 5). PGI 2 is a potent vasodilator, and effective endogenous inhibitor of platelet aggregation [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF][START_REF] Moncada | The role of prostacyclin in vascular tissue[END_REF]. PGI2 is produced in the endothelium from prostaglandin H 2 (PGH 2 ) by the action of the enzyme prostacyclin synthase. PGI 2, PGG 2 and PGH 2 are major products of vascular cycloxygenase (COX). There are two isoforms of COX encoded by two separate genes. COX-1 is constitutively expressed and is present in many tissues, including endothelial cells. COX-2 is not constitutively expressed, but can be induced rapidly and transiently in many cells, including vascular endothelial cells and smooth muscle cells, under the effect of physical stimuli and pro-inflammatory agents [START_REF] Marnett | Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition[END_REF][START_REF] Hong | Activation of phospholipases A2 and C in pig aortic endothelial cells synthesizing prostacyclin[END_REF][START_REF] Topper | Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress[END_REF]. PGI 2 elicits smooth muscle relaxation by stimulating adenylyl cyclase and formation of cyclic adenosine -3', 5'-monophosphate. Its vasodilator activity is determined by the expression of specific receptors which are prostaglandin I2 receptors of the G-protein coupled receptor family in vascular smooth muscle cells [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF]. The binding of PGI 2 to its receptors causes a change of conformation of the receptors, which will lead to an increase of cyclic adenosine-3', 5'-monophosphate (cAMP) levels in the vascular smooth muscle. cAMP then activates protein kinase A, which reduces intracellular Ca 2+ by decreasing Ca 2+ release from the endoplasmic reticulum and by stimulating its uptake by it [START_REF] Kukovetz | Prostacyclin increases cAMP in coronary arteries[END_REF]. Furthermore, PGI 2 is a potent inhibitor of platelet adhesion, aggregation, and degranulation [START_REF] Mustard | Prostaglandins and platelets[END_REF]. In addition, PGI 2 facilitates the release of NO by endothelial cells [START_REF] Shimokawa | Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig[END_REF] and in turn, the action of PGI 2 in vascular smooth muscle cells and platelets is potentiated by NO [START_REF] Delpy | Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3[END_REF].

Endothelium-derived contracting factors (EDCF)

There is large heterogeneity in the formation of EDCF, depending on the stimuli, vascular bed and the age of the experimental animal used. Among the different contracting factors that are produced by the endothelial cells, we retain: thromboxane A 2 (TXA 2 ), prostaglandin H 2 (PGH 2 ) and also reactive oxygen species, endothelin-1 (ET1) and angiotensin II. (Figure 8) summarizes the different EDCF factors.

Reactive oxygen species (ROS)

Under normal conditions ROS are produced and released from endothelial cells for vascular homeostasis. However, in different pathological conditions like hypertension, diabetes mellitus, atherosclerosis and in acute and chronic inflammatory diseases there is over-production of ROS causing vascular oxidative stress [START_REF] Eisenberg | Platelet-dependent and procoagulant mechanisms in arterial thrombosis[END_REF]; [START_REF] Mathis | Oxidative stress promotes hypertension and albuminuria during the autoimmune disease systemic lupus erythematosus[END_REF][START_REF] Sedeek | Oxidative stress, Nox isoforms and complications of diabetes--potential targets for novel therapies[END_REF][START_REF] Xi | Potent free radical scavenger, edaravone, suppresses oxidative stress-induced endothelial damage and early atherosclerosis[END_REF] sensitization of the contractile elements [START_REF] Jin | Reactive oxygen-mediated contraction in pulmonary arterial smooth muscle: cellular mechanisms[END_REF]; [START_REF] Suzuki | Superoxide stimulates IP3-induced Ca2+ release from vascular smooth muscle sarcoplasmic reticulum[END_REF]. In addition, ROS in particular O 2 -can also potentiate the contractile responses by reducing the bioavailability of NO [START_REF] Rubanyi | Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle[END_REF] or by activating COXs in vascular smooth muscle cells [START_REF] Hibino | Oxygen-derived free radical-induced vasoconstriction by thromboxane A2 in aorta of the spontaneously hypertensive rat[END_REF] or associated with impairment of endothelium-dependent relaxation [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]. The effect of oxidative stress on EDHF pathway occur through reducing the activity of calcium dependent potassium channels (SK Ca and IK Ca ) [START_REF] Kusama | Reduced hyperpolarization in endothelial cells of rabbit aortic valve following chronic nitroglycerine administration[END_REF] and also via modifying the passage of hyperpolarization from endothelial cells to underling smooth muscle cells through myoendothelial gap junctions [START_REF] Griffith | 5-Methyltetrahydrofolate and tetrahydrobiopterin can modulate electrotonically mediated endothelium-dependent vascular relaxation[END_REF] [START_REF] Thorin-Trescases | Age-dependent oxidative stress: toward an irreversible failure in endothelial maintenance[END_REF]. This has been supported by earlier works where it has been shown that the antioxidants are able to improve the deleterious effect of oxidant stress on vascular endothelial function in vitro and in vivo in animals [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] and as well as in humans [START_REF] Kanani | Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans[END_REF][START_REF] Holowatz | Altered mechanisms of vasodilation in aged human skin[END_REF].

Thromboxane A 2 & prostaglandin H 2 (T X A 2 & PGH 2 )

T X A 2 and PGH 2 are generated subsequently to the metabolism of arachidonic acid (AA) by COX-1 and COX-2 in endothelial cells [START_REF] Oates | Clinical implications of prostaglandin and thromboxane A2 formation (1)[END_REF]. PGH 2 is the precursor of the prostaglandins E 2 (PGE 2 ), F 2 (PGF 2 ) and T X A 2 . T X A 2 is synthesised from PGH 2 by thromboxane synthase in platelets, and produced during platelet activation to promote platelet aggregation, vasocontraction, and smooth muscle proliferation. In smooth muscle cells, it causes an increase of intracellular Ca 2+ levels via binding to thromboxane-prostanoid (TP) receptors to induce vasocontraction [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] (Figure 3). In normal vessels, the regulation of T X A 2 and PGI 2 is important, and has essential but opposing functions in the maintenance of vascular homeostasis. Especially, the ratio of both seems to be more important than the absolute amounts of both 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].

Angiotensin II (Ang II)

In the endothelial cells there is angiotensin converting enzyme (ACE) which is responsible for the conversion of Ang I to Ang II. The vast pharmacological effects of Ang II observed are mediated through ATI receptors. Ang II is a multifunctional hormone responsible for regulation of blood pressure and cardiovascular homeostasis. It regulates endothelial function and stimulates inflammatory, proliferative, fibrotic and thrombotic processes in the vasculature. It has potent effects on vascular tone, constricts smooth muscle cells, regulates vascular cell growth, apoptosis, fibrosis, matrix metalloproteinase production and extracellular matrix degradation (Griendling et al, 1997;[START_REF] Tomita | Early induction of transforming growth factor-beta via angiotensin II type 1 receptors contributes to cardiac fibrosis induced by long-term blockade of nitric oxide synthesis in rats[END_REF][START_REF] Yoo | Tissue-specific regulation of growth factors and clusterin by angiotensin II[END_REF]. It is well-known that Ang II leads to vasoconstriction and structural changes [START_REF] Gibbons | Vasculoprotective and cardioprotective mechanisms of angiotensinconverting enzyme inhibition: the homeostatic balance between angiotensin II and nitric oxide[END_REF]). These effects are observed often 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]) (Figure 7). The Ang II is produced from the biologically inactive decapeptide, Ang I, the metabolic product of the liver-synthesized angiotensinogen and the enzyme renin. Ang I has a half-life of a few seconds, as it is quickly converted mostly by angiotensin-converting enzyme (ACE) to the biologically active octapeptide, Ang II. In addition, Ang I can be converted to the biologically active Ang (1-7) by plasma or neutral endopeptidases or to Ang (1)(2)(3)(4)(5)(6)(7)(8)(9) by ACE2 and subsequently to Ang (1)(2)(3)(4)(5)(6)(7). Ang II can also be converted to Ang (1)(2)(3)(4)(5)(6)(7) by plasma and neutral endopeptidases. There are three subtypes of Ang II receptors: type 1 (AT1R), type 2 (AT2R) and type 4 (AT4R). AT1R are localised on cardiomyocytes, vascular smooth muscle and endothelial cells, nerve endings and conductive tissues. AT2R are present in endothelial and vascular smooth muscle cells and in fibrous tissue of the heart [START_REF] Regitz-Zagrosek | Myocardial angiotensin receptors in human hearts[END_REF]. Effects of each receptor activation are summarized in (Figure 7). Now it is recognized that, in addition to the classical reninangiotensin system RAS pathway in which ACE generates the powerful vasoconstrictor Ang II, there is an alternate arm of the RAS through which Ang II is cleaved by ACE2 to Ang (1)(2)(3)(4)(5)(6)(7), which stimulate Mas-receptor (MasR) (G-protein-coupled receptor) leading to cause vasodilatation (Figure 7, [START_REF] Grace | Activation of the MAS receptor by angiotensin-(1-7) in the renin-angiotensin system mediates mesenteric vasodilatation in cirrhosis[END_REF].

Endothelin-1 (ET-1)

Endothelin (ET) are proteins that constrict the blood vessels and raise blood pressure.

There are different ET isoforms expressed in the body, ET-1, ET-2 and ET-3 [START_REF] Kedzierski | Endothelin system: the double-edged sword in health and disease[END_REF], the endothelial cells only release ET-1. ET-1 is produced by converting big ET-1 to ET-1 by the endothelin converting enzyme [START_REF] Yanagisawa | A novel potent vasoconstrictor peptide produced by vascular endothelial cells[END_REF][START_REF] Alonso | The nitric oxide-endothelin-1 connection[END_REF]. Furthermore, the ET-1 production as well as its release is stimulated by proinflammatory cytokines such as interleukins and TNF-and decreased by NO and PGI 2 [START_REF] Alonso | The nitric oxide-endothelin-1 connection[END_REF]. ET-1 receptors subtypes have been identified both on endothelial cells (ET-B1 ) and smooth muscle cells (ET-A and ET-B2 ) [START_REF] Sakurai | Molecular characterization of endothelin receptors[END_REF].

Moreover, the distribution of ET-1 receptors is dependent on the type of vascular bed : in veins ET-A to ET-B receptor ratio is reduced in comparison to arteries [START_REF] Kedzierski | Endothelin system: the double-edged sword in health and disease[END_REF]. Regarding the function of ET-1, when it binds to ET-A and ET-B2 receptors, smooth muscle Ca 2+ channels open allowing extracellular Ca 2+ to enter into the cell.

This causes vasoconstriction in a similar way as T X A 2 . Activation of ET-B1 receptors on the endothelium causes vasodilatation by inducing the release of NO and PGI 2 [START_REF] De Nucci | Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor[END_REF][START_REF] Cardillo | Interactions between nitric oxide and endothelin in the regulation of vascular tone of human resistance vessels in vivo[END_REF]. In endothelial dysfunction due to atherosclerotic arteries, endothelial cells ET-B1 receptors are down-regulated, while smooth muscle cells ET-B2 receptors are up-regulated, thus enhancing vasoconstriction [START_REF] Dagassan | Up-regulation of endothelin-B receptors in atherosclerotic human coronary arteries[END_REF][START_REF] Sandoo | The endothelium and its role in regulating vascular tone[END_REF][START_REF] Bohm | Combined endothelin receptor blockade evokes enhanced vasodilatation in patients with atherosclerosis[END_REF]. In portal hypertension it has been reported from animal studies an over-production of ET-1 by the liver, which binds to ET-B1 receptors which are upregulated in the lung vascular endothelial cells, leading to an increase of eNOS expression and activity, and finally causing pulmonary vasodilation (Zhang et al, 2009). [START_REF] Buob | Portal hypertension: pathophysiology, diagnosis, and treatment[END_REF]. It has been reported that hepatic and non-hepatic endothelial dysfunction is a key factor that causes and worsens portal hypertension [START_REF] Iwakiri | Vascular endothelial dysfunction in cirrhosis[END_REF]). In the hepatic microcirculation, hypoactive endothelial cells (ECs), contribute to an increase in intra-hepatic resistance mainly by decreasing production of nitric oxide (NO), which in turn leads to portal hypertension [START_REF] Bosch | Vascular deterioration in cirrhosis: the big picture[END_REF][START_REF] Iwakiri | The hyperdynamic circulation of chronic liver diseases: from the patient to the molecule[END_REF]. Portal hypertension is associated with splanchnic and systemic and a hyperdynamic syndrome which in turn leads to an increase in portal vein blood flow further increasing portal hypertension. [START_REF] Bosch | Vascular deterioration in cirrhosis: the big picture[END_REF][START_REF] Iwakiri | The hyperdynamic circulation of chronic liver diseases: from the patient to the molecule[END_REF]. While the endothelial cells are hypoactive in the intra-hepatic microcirculation, endothelial cells in the splanchnic and systemic circulation are hyperactive and have been shown to over produce NO. These two different phenotypic endothelial cells found in the intra-vs. extra-hepatic circulation contribute to the development and exacerbation of portal hypertension [START_REF] Iwakiri | Vascular endothelial dysfunction in cirrhosis[END_REF]) (Figure 9).

An up-regulation of vascular endothelial growth factor (VEGF) and of endothelial nitric oxide synthase (eNOS) has been reported to be the initial event in portal hypertension (Abraldes et al, 2006). The mechanisms involved in the occurrence of the subsequent generalized vasodilatation and hyperdynamic syndrome are incompletely understood. Several lines of evidence support a role for an increased formation of nitric oxide (NO) in the occurrence of the general vasodilatation in chronic liver diseases in patients and also in the chronic bile duct ligated (CBDL) rat model (Nunes et al, 2001;Zhang et al, 2003;Chabot et al, 1996;Gomez et al, 2006). Other vasodilators have been reported to play a role: carbon monoxide (CO) produced as consequence of macrophage activation [START_REF] Iwakiri | The hyperdynamic circulation of chronic liver diseases: from the patient to the molecule[END_REF][START_REF] Iwakiri | Vascular endothelial dysfunction in cirrhosis[END_REF], EDHF [START_REF] Cahill | Endothelial dysfunction in cirrhosis and portal hypertension[END_REF][START_REF] Heinemann | Effect of terlipressin on in vitro vascular hyporeactivity of portal hypertensive rats[END_REF][START_REF] Mathie | Mesenteric vasodilator responses in cirrhotic rats: a role for nitric oxide?[END_REF] may also be implicated and possibly prostacyclin. In addition to vasodilatation which has been related to the production of vasodilators (NO, CO, EDHF, prostacyclin) chronic activation of the renin-angiotensin-aldosterone system (RAS) has been observed in cirrhotic patients [START_REF] Newby | The L-arginine/nitric oxide pathway contributes to the acute release of tissue plasminogen activator in vivo in man[END_REF]Helmy et al, 2003) and in cirrhotic rats (Yang et al, 2002;Dal-Ros et al, 2010). Finally, chronic liver diseases are also characterized by an increased formation of reactive oxygen species (ROS).

Thus chronic liver diseases are characterized by an endothelial dysfunction related to a generalized vasodilation and a hyperdynamic syndrome with an increased cardiac output. The generalized vasodilation leads to complications and especially to a hepatopulmonary syndrome in approximately 10%-15% of patients awaiting liver transplantation (Rodriguez-Roisin et al, 2004). The hepatopulmonary syndrome (HPS) is characterized by arterial deoxygenation because of intrapulmonary vascular dilatation in the context of liver disorder.

Its occurrence in patients with liver diseases is of prognostic value for their survival before and also after liver transplantation (Rodriguez-Roisin et al, 2004;Arguedas et al, 2003). It resolves in most cases with liver transplantation, but having an efficient approach to improve it is of great clinical importance because this could ameliorate survival or even delay liver transplantation in some cases. Thus the aim of our studies was to assess treatments which could ameliorate endothelial dysfunction in a rat model of biliary cirrhosis.

Mechanisms of endothelial dysfunction in portal hypertension

Role of vasodilators

Nitric oxide

It has been suggested that NO has a major role in the pathogenesis of vasodilation and vascular hypocontractility associated with portal hypertension [START_REF] Groszmann | Nitric oxide and hemodynamic impairment[END_REF]. In a rat model of portal vein ligation leading to mild portal hypertension a significant increase in the expression of intestinal vascular endothelial growth factor (VEGF) with a subsequent increase in the expression of eNOS in the intestinal microcirculation (Abraldes et al, 2006) has been observed. The model of mild portal hypertension is most likely representative of the portal pressure changes observed in early stages of liver cirrhosis during which portal hypertension generally progresses slowly. Thereafter, with the worsening of portal hypertension, vasodilation in the arterial splanchnic circulation and hyperdynamic circulation occurs and other mechanisms such as shear stress may lead to an increased NO production. (Abraldes et al, 2006;[START_REF] Tsai | Mesenteric vasoconstriction triggers nitric oxide overproduction in the superior mesenteric artery of portal hypertensive rats[END_REF][START_REF] Iwakiri | Endothelial dysfunction in the regulation of cirrhosis and portal hypertension[END_REF]. From animal studies, it has also been shown that an increase in the expression of eNOS occurs in the lung (Nunes et al, 2001). Pulmonary eNOS expression is closely related to the increased level of plasma ET-1. Over production of ET-1 occurs in the liver of cirrhotic rat. ET-1 binds further to the ET-B receptors whose expression is increased in the pulmonary vascular bed leading to an increase in eNOS expression and activity with subsequent pulmonary vasodilation (Ling et al, 2004). Thus in portal hypertension the general vasodilation has been in part related to the increased production of NO by eNOS in the splanchnic and the lung vascular beds.

Moreover, inducible NO synthase (iNOS) is also stimulated. This has been observed in the splanchnic circulation where iNOS is expressed by resident macrophages in rats with CBDL [START_REF] Moreau | Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis[END_REF][START_REF] Morales-Ruiz | Increased nitric oxide synthase expression in arterial vessels of cirrhotic rats with ascites[END_REF]. While the liver detoxification function is weakened, endotoxins and cytokines, especially TNF-, can activate iNOS, leading to increased local or systemic NO level (Zhang et al, 2007;[START_REF] Umeda | Hepatopulmonary syndrome and portopulmonary hypertension[END_REF][START_REF] Ferguson | Inducible nitric oxide synthase activity contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites[END_REF][START_REF] Kajita | iNOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats[END_REF][START_REF] Theodorakis | The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice[END_REF][START_REF] Wei | Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct-ligated rats[END_REF]. Finally, it has also been demonstrated that intravascular lung macrophages probably play a key role in the occurrence of the hepatopulmonary syndrome and in part by their ability to produce NO by iNOS stimulation (Thenappan et al, 2011).

Thus in portal hypertension an increased production in NO has been reported either related to the increased expression of eNOS and/or due to the stimulation of iNOS.

Supporting this concept, Ferguson et al, observed that a selective iNOS inhibition by N- [3-(aminomethyl) benzyl] acetamidine, resulted in peripheral vasoconstriction in patients with cirrhosis [START_REF] Ferguson | Inducible nitric oxide synthase activity contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites[END_REF]. It has also been observed that cirrhotic patients with a hepatopulmonary syndrome had increased exhaled NO levels, a measure of pulmonary NO production, which normalized after orthotopic liver transplantation. In addition, methylene blue, an inhibitor of soluble guanylyl cyclase, has been reported to transiently improve hypoxemia in patients with hepatopulmonary syndrome [START_REF] Tumgor | Mcp-1, eNOS, tPA and PAI-1 gene polymorphism and correlation of genotypes and phenotypes in hepatopulmonary syndrome[END_REF][START_REF] Zhang | Experimental study on the role of endotoxin in the development of hepatopulmonary syndrome[END_REF].

Thus from animal and human studies it has been reported that portal hypertension is related to increased production of NO thought to lead to a general vasodilation after the initial increased expression of eNOS in the intestinal microcirculation.

Prostacyclin

Shear stress [START_REF] Topper | Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress[END_REF] and pro-inflammatory substances [START_REF] Marnett | Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition[END_REF][START_REF] Hong | Activation of phospholipases A2 and C in pig aortic endothelial cells synthesizing prostacyclin[END_REF] are factors regulating in the local release of PGI 2 . Few studies have been devoted to the possible role of an increased prostacyclin (PGI 2 ) level in the occurrence of hemodynamic abnormalities in cirrhosis. A pathogenesis role for PGI 2 in the hyperdynamic circulatory syndrome has been suggested in an experimental portal hypertension in rats [START_REF] Munoz | Factors mediating the hemodynamic effects of tumor necrosis factor-alpha in portal hypertensive rats[END_REF]. In a rabbit model of chronic portal hypertension, elevated levels of PGI 2 have been observed probably due to an increased splanchnic production of the vasodilator (Sitzmann et al, 1994). In patients increased plasma levels of PGI 2 have been reported in cirrhotic patients with portal hypertension [START_REF] Yin | Plasma prostacyclin (PGI2) levels in peripheral venous, arterial and portal venous blood in cirrhotic patients with portal hypertension and their clinical implication[END_REF] as well as increased levels of 6-keto-PGF1 (a stable metabolite of PGI 2 ) in the plasma and the gastric mucosa of cirrhotic patients with portal hypertensive gastropathy [START_REF] Ohta | Increased prostacyclin content in gastric mucosa of cirrhotic patients with portal hypertensive gastropathy[END_REF].

Moreover, in the study done by Hou et al., in rats subjected to portal vein ligation increased plasma levels of PGI2 were observed along with an enhanced COX-1 (but not COX-2) expression in the superior mesenteric artery (Figure 10).

Carbon monoxide (CO)

CO is a vasodilator which has been shown to be increased in cirrhosis especially in the lungs of cirrhotic rats. The main pathway which produces CO is the heme metabolism. Heme is oxidized by the heme-oxygenase (HO) producing CO and biliverdin, the latter being reduced to bilirubin (Figure 11). Two isoforms of HO have been identified: HO-1 and HO-2.

HO-1 is inducible by multiple agents like pro-inflammatory cytokines from inflammatory macrophage, whereas HO-2 is constitutively expressed [START_REF] Maines | Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible[END_REF]. Like NO, CO is an endogenously produced gas molecule that activates soluble guanylyl cyclase, resulting in increased production of cGMP (Morita et al, 1995) and regulates vascular tone in a manner similar to NO [START_REF] Morita | Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide[END_REF][START_REF] Cook | Heme oxygenase activity in the adult rat aorta and liver as measured by carbon monoxide formation[END_REF]. CO has a great affinity for hemoglobin, resulting in the formation of carboxyhemoglobin which was used by authors to measure the CO generation [START_REF] De Las Heras | Increased carbon monoxide production in patients with cirrhosis with and without spontaneous bacterial peritonitis[END_REF]. A progressive increase in the expression of HO-1 in the aorta and mesenteric arteries of rats with biliary cirrhosis has been reported, whereas no change was observed in HO-2 expression. Moreover, the acute intraperitoneal injection of zinc protoporphyrin, a selective inhibitor for HO to cirrhotic rats improved the hyperdynamic circulatory syndrome, suggesting the role of CO in arterial vasodilatation in portal hypertension [START_REF] Chen | Increased vascular heme oxygenase-1 expression contributes to arterial vasodilation in experimental cirrhosis in rats[END_REF]. It has been reported that HO-1 expression was increased in pulmonary intravascular macrophages (Zhang et al, 2003) in rats and an increased CO generation was observed in patients presenting with a hepatopulmonary syndrome compared with those without [START_REF] Arguedas | Carboxyhemoglobin levels in cirrhotic patients with and without hepatopulmonary syndrome[END_REF][START_REF] Van Landeghem | Carbon monoxide produced by intrasinusoidally located haem-oxygenase-1 regulates the vascular tone in cirrhotic rat liver[END_REF].

Administration of HO-1 inhibitors partially improved vascular contraction to hypoxia, while administration of the NOS inhibitor, L-nitro-L-arginine methylester (L-NAME), brought HO-1 expression and vascular hypoxic vasoconstriction back to normal (Zhang et al, 2003;Carter et al, 2002). These evidences suggest that NO causes hypoxic contraction insensitivity, and induces the production of HO-1, thereby enabling an increase in CO production, which synergistically contributes to the pathogenesis of hepatopulmonary syndrome. The endogenous NOS/NO and HO-1/CO systems may relatively independently or synergistically contribute to the pathogenesis of hepatopulmonary syndrome [START_REF] Umeda | Hepatopulmonary syndrome and portopulmonary hypertension[END_REF]Carter et al, 2002).

EDHF

Role of EDHF in the occurrence of vasodilation and hemodynamic abnormalities was suggested in rats with biliary cirrhosis (Barriere et al, 2000). These studies showed an EDHFdependent inhibition of contraction in mesenteric arteries (Barriere et al, 2000) and in an isolated perfused lung (Carter et al, 2002). It has been reported that in normal resistance artery there may be a balance between EDHF and NO, with for example an up-regulation of EDHFdependent relaxation in eNOS knockout mice [START_REF] Waldron | Acetylcholine-induced relaxation of peripheral arteries isolated from mice lacking endothelial nitric oxide synthase[END_REF] or a decrease in EDHF [START_REF] Bauersachs | Nitric oxide attenuates the release of endotheliumderived hyperpolarizing factor[END_REF]. On the contrary to these previous studies we find both a decreased NO-and EDHF -dependent relaxation in our studies and this will be discussed in chapter four.

Intestinal endotoxemia and role of monocyte /macrophage activation

Bacterial translocation (BT) is defined as the migration of viable micro-organsims or bacterial endotoxins from the intestinal lumen to mesenteric lymph nodes and other extraintestinal sites [START_REF] Wiest | Bacterial translocation up-regulates GTPcyclohydrolase I in mesenteric vasculature of cirrhotic rats[END_REF]. BT has been postulated as an important mechanism in the development of portal hypertension and hyperdynamic circulatory state (HCS) [START_REF] Pinzone | Microbial translocation in chronic liver diseases[END_REF]. The hyperdynamic circulatory state occurs as a consequence of splanchnic and systemic vasodilatation which leads to the subsequent activation of compensatory mechanisms (i.e., renin-angiotensin aldosterone, sympathetic nervous system, and antidiuretic hormone) with an increased plasma volume and cardiac output. The hyperdynamic circulatory state contributes further to the worsening of portal hypertension, and is associated with systemic vascular complications of cirrhosis (Garcia-Tsao and Wiest 2004). The consequence of BT is an increased production of endotoxins, which results in an increase in inflammatory cytokines thought in part to stimulate iNOS in vessel walls, causing NO overproduction, vasodilatation and hyperdynamic circulatory state [START_REF] Vallance | Hyperdynamic circulation in cirrhosis: a role for nitric oxide?[END_REF]. Among the inflammatory cytokines, TNF-plays probably a role in the development of hyperdynamic circulatory state as suggested by a study by [START_REF] Lopez-Talavera | Thalidomide inhibits tumor necrosis factor alpha, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive rats[END_REF] in which the inhibition of TNF-ameliorated hyperdynamic circulatory state in cirrhotic rats. Moreover, in addition to BT, there is evidence of monocyte/macrophage activation in studies of liver cirrhosis and portal hypertension in experimental animals (Sztrymf et al, 2005;Zhang et al, 2007;[START_REF] Zhang | Experimental study on the role of endotoxin in the development of hepatopulmonary syndrome[END_REF][START_REF] Neugebauer | Bacterial translocation increases phagocytic activity of polymorphonuclear leucocytes in portal hypertension: priming independent of liver cirrhosis[END_REF][START_REF] Lopez-Talavera | Thalidomide inhibits tumor necrosis factor alpha, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive rats[END_REF]. Endotoxin induces and activates the monocyte/macrophage system in vivo, including liver Kupffer cells (specialized liver macrophages), splenic macrophages, pulmonary intravascular macrophages (PIMs), and blood mononuclear cells [START_REF] Li | Expression and significance of Toll-like receptor 4 of splenic macrophage in patients with hypersplenism due to portal hypertension[END_REF][START_REF] Gill | Role of pulmonary intravascular macrophages in endotoxin-induced lung inflammation and mortality in a rat model[END_REF]. As far as the cirrhotic liver losses the bacterial detoxification capacity and due to portosystemic shunt the bacteria and toxins probably pass to lung in compensation.

Subsequently, blood mononuclear cells, accumulate in the pulmonary vascular endothelium, and then differentiate into pulmonary intravascular macrophages [START_REF] Zhang | Progress in investigating the pathogenesis of hepatopulmonary syndrome[END_REF]. The function of pulmonary intravascular macrophages phagocytosis is enhanced to remove the bacteria and toxins from the blood. Upon presence of foreign bodies macrophages are activated and secrete a variety of active substances such as NO, CO, TNF-, IL-1, IL-6, IL-8, and other inflammatory mediators. These mediators play an important role in the occurrence and development of hepatopulmonary syndrome and hyperdynamic circulatory state. It has been shown, especially in the lung of cirrhotic rats that the administration of norfloxacin (an antibiotic) inhibits intestinal BT and reduces endotoxin injury to the lung and improves the symptoms of hepatopulmonary syndrome and hyperdynamic circulatory state [START_REF] Wang | Reduction of endotoxin may protect lung from injury in rats with hepatopulmonary syndrome[END_REF]Wiest et al, 1999). Moreover, inhibiting TNF-by pentoxifylline has been reported to reduce the number and activity of pulmonary intravascular macrophages, and to improve hepatopulmonary syndrome in cirrhotic rats along with decreased pulmonary intravascular macrophages adhesion and phagocytosis and iNOS expression in lung tissue (Sztrymf et al, 2004, Figure 12).

Angiogenesis in portal hypertension

In addition to the arterial vasodilatation in the splanchnic and systemic circulation, the development of collateral vessels as an adaptive response to an increase in portal pressure leads to worsening of portal hypertension and its complications [START_REF] Bosch | Vascular deterioration in cirrhosis: the big picture[END_REF]. These collateral vessels are formed by the enlargement of pre-existing vessels and by angiogenesis [START_REF] Sumanovski | Increased angiogenesis in portal hypertensive rats: role of nitric oxide[END_REF]. The process of angiogenesis is modulated by growth factors like VEGF. Studies using portal hypertensive rats by Abraldes et al. and Fernandez et al., reported that a sudden increase in portal pressure is sensed at the intestinal microcirculation and induces VEGF expression (Abraldes et al, 2006;[START_REF] Fernandez | Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats[END_REF]. Furthermore, antiangiogenic agents such as blockers of VEGF receptor-2 and inhibitors of tyrosine kinases (Sorafenib and Sunitinib) lead to decrease in collateral circulation and reduce portal hypertension [START_REF] Fernandez | Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice[END_REF][START_REF] Mejias | Beneficial effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats[END_REF]. More recently, angiogenesis, has been attributed to both intrahepatic and extrahepatic component of portal hypertension [START_REF] Thabut | Intrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease: new targets for the treatment of portal hypertension?[END_REF]. Angiogenesis has also been reported in the lung of experimental hepatopulmonary syndrome (Figure 13) (Zhang et al, 2009). In this latter study, increased numbers of lung microvessels, increased lung monocytes accumulation and activation of Akt and eNOS have been observed after CBDL in rats. Pentoxifylline treatment reduced the number of microvessels, lung monocytes accumulation, down-regulated pulmonary angiogenic factors, and improved the symptoms of hepatopulmonary syndrome (Zhang et al, 2009;[START_REF] Gill | Role of pulmonary intravascular macrophages in endotoxin-induced lung inflammation and mortality in a rat model[END_REF]Sztrymf et al, 2004). In addition, angiogenesis has been reported to be stimulated via VEGF-dependent pathways (Zhang et al, 2009) and especially VEGF-A (Zhang et al, 2009) produced in alveolar macrophages and blood vessel endothelial cells.

VEGF angiogenic signaling occurs through interaction with specific VEGF receptors (VEGFR-1 and VEGFR-2). Subsequently, VEGFR-2 activation triggers downstream signaling in part through Akt and eNOS (Zhang et al, 2009). ), which reacts with NO to form peroxynitrite (ONOO -). Thus, the bioavailability of intra-hepatic NO is decreased [START_REF] Gracia-Sancho | Increased oxidative stress in cirrhotic rat livers: A potential mechanism contributing to reduced nitric oxide bioavailability[END_REF], favoring vasoconstriction which leads to an increased vascular resistance as an initiating step in portal hypertension [START_REF] Iwakiri | Endothelial dysfunction in the regulation of cirrhosis and portal hypertension[END_REF]. It was demonstrated that administration of vitamin C as an antioxidant to cirrhotic patients markedly attenuates portal pressure, and significantly reduces MDA levels, suggesting that increased oxidative stress in cirrhotic patients contributes to portal hypertension [START_REF] Hernandez-Guerra | Ascorbic acid improves the intrahepatic endothelial dysfunction of patients with cirrhosis and portal hypertension[END_REF]. Furthermore, in CBDL-as well as partial portal vein ligated rats, N-acetylcysteine prevented the development of the hyperdynamic circulation. Moreover, by the using of antioxidant therapy a reduction in the increased urinary F 2 -isoprostanes which were measured as a marker of oxidative stress, and increased SOD activity measured in lung homogenates have been observed (Fernando et al, 1998;Vercelino et al, 2008).

Renin-angiotensin system in portal hypertension

Up to date, the current understanding is that there is activation of the classical pathway of renin-angiotensin system (RAS) in patients with advanced liver disease and portal hypertension in response to vasodilatation and systemic hypotension [START_REF] Schrier | Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis[END_REF].

Angiotensin II (Ang II), the main effector of the RAS, regulates key steps in the tissue remodeling process through angiotensin type 1 (AT1R) receptors [START_REF] Mezzano | Angiotensin II and renal fibrosis[END_REF][START_REF] Mezzano | Angiotensin II and renal fibrosis[END_REF]. Recent research showed that, there is hepatic up-regulation of RAS, mediating fibrosis and stellate cell contraction in response to oxidative stress and systemic activation of RAS [START_REF] Bataller | Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct-ligated rats[END_REF][START_REF] Grace | Activation of the MAS receptor by angiotensin-(1-7) in the renin-angiotensin system mediates mesenteric vasodilatation in cirrhosis[END_REF][START_REF] Asbert | Assessment of the renin-angiotensin system in cirrhotic patients. Comparison between plasma renin activity and direct measurement of immunoreactive renin[END_REF][START_REF] Paizis | Up-regulation of components of the renin-angiotensin system in the bile duct-ligated rat liver[END_REF][START_REF] Bataller | Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II[END_REF]. The blockade of Ang II synthesis or its binding to AT1 receptors markedly ameliorates hepatic fibrosis in rats with CBDL [START_REF] Ramalho | Effect of losartan, an angiotensin II antagonist, on secondary biliary cirrhosis[END_REF][START_REF] Yoshiji | Inhibition of renin-angiotensin system attenuates liver enzyme-altered preneoplastic lesions and fibrosis development in rats[END_REF][START_REF] Paizis | Effect of angiotensin II type 1 receptor blockade on experimental hepatic fibrogenesis[END_REF]. In an earlier study performed in our laboratory the angiotensin II receptor 1 (AT1) antagonist losartan improved endothelial dysfunction possibly by its antioxidant effect (Dal-Ros et al, 2010).

CHAPTER III POLYPHENOLS

Chapter three

Polyphenols

Polyphenols, constitute of group of natural products, represent a secondary metabolite of plants and that have a great variability of chemical structures. One of the fundamental structural element is the presence of hydroxyl groups which can be found in free state or bound to other forms such as; ether, ester or heteroside. The polyphenols are abundant in vegetables, and are important part of human and animal nutrition. We can find polyphenols in large quantity in red wine, also in fruits, cereals, chocolate, legumes and the drinks like tea, and coffee [START_REF] Bravo | Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance[END_REF]).

Classification of polyphenols

Polyphenols composed of multiples hydroxyl groups (-OH) found on aromatic rings related to benzene (phenyl C6H5). They are mainly classified according to the chemical structure of the aglycons (the polyphenol deprived from its sugar moieties) into flavonoids, phenolic acids, stilbenes, and lignans. Anthocyanins are widely distributed in human diet including red wine, certain varieties of cereals and certain vegetables (cabbage, beans, onions and radishes), but most abundant especially in fruit like berries. There are six main anthocyanidins distributed throughout the plant kingdom: cyanidin, malvidin, delphinidin, peonidin, petunidin and pelargonidin. Among them cyanidin is the most common anthocyanidin in food. Food contents of anthocyanins are generally proportional to color intensity and increase as the fruit ripens up to 2-4 g/kg fresh wt in blackcurrant or blackberries [START_REF] Manach | Polyphenols: food sources and bioavailability[END_REF].

Phenolic acids are further divided into benzoic acid and cinnamic acid and consisting of a C1-C6 and C3-C6 backbones, respectively. They contain protocatechuic acid, vanillic acid, gallic acid and syringic acid as examples of phenolic acids in the former; and pcoumaric acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, ferulic acid and sinapic acid in the latter (Figure 14).

Stilbenes are diphenylethene barely present in human diet, a main member is resveratrol.

Lignans consists of two phenylpropane units. This group of polyphenol is further metabolized by the intestinal microflora into enterodiol and enterolactone (Figure 14).

Absorption, metabolism and excretion of polyphenols

The metabolism of polyphenols is not yet well clarified. However, several studies were conducted in an effort to understand the metabolic and absorption processes of polyphenols in vivo.

In this respect, the metabolism of flavonoids is the most studied. This group is mainly characterized their presence in a glycosylated form [START_REF] Crozier | Antioxidant flavonols from fruits, vegetables and beverages: measurements and bioavailability[END_REF].

However, these flavonoids undergo deglycosylation in the intestinal lumen by lactase phlorizin hydrolase, an enzyme found in the brush border of the small intestine, yielding free aglycones. The latter compounds are hydrophobic and according to their solubility coefficients in the lumen, they can diffuse through the intestinal epithelial cells either passively or by facilitated diffusion [START_REF] Azuma | Combination of lipids and emulsifiers enhances the absorption of orally administered quercetin in rats[END_REF][START_REF] Sriram | Loss of zinc and selenium does not occur through peritoneal dialysis[END_REF].

However, flavonoids can also be absorbed in the enterocytes of the small intestine via membrane transporters such as sodium/D-glucose cotransporter 1 (SGLT1) without occurrence of any metabolism changes. Once within the cell, deglycosylation takes place by the action of cytosolic -glucosidase to release aglycones [START_REF] Day | Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity[END_REF]. Within the small intestine, most flavonoids undergo conjugation mainly by glucoronosyl transferases (UGTs)

to form glucouromide conjugates. In small intestine models, such conjugate forms seem to be important for flavonoids to pass from the mucosal compartment to the blood compartment [START_REF] Andlauer | Absorption and metabolism of genistin in the isolated rat small intestine[END_REF]. Nevertheless, flavonoids can undergo further metabolic steps characterized by methylation or the replacement of glucoronide residues with sulfate groups, since the presence of both form were reported in peripheral blood [START_REF] Donovan | Catechin is metabolized by both the small intestine and liver of rats[END_REF]).

These processes were shown to take place in the hepatic cells of the liver and to play a role in the stabilization of flavonoids [START_REF] Monfardini | Stabilization of substances in circulation[END_REF][START_REF] O'leary | Metabolism of quercetin-7-and quercetin-3glucuronides by an in vitro hepatic model: the role of human beta-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism[END_REF].

However, all conjugate flavonoids are then exported into the bile and eventually reenter the small intestinal lumen. At this stage, if flavonoids fail to undergo deconjugation steps, they will be transported to the colon.

The colon microorganisms have catalytic potential. Thereby, flavonoids could undergo deglucuronidation and subsequent sulfation. The resulting flavonoids are then reabsorbed thus leading to a continuous cycle referred to as the enterohepatic circulation [START_REF] Franke | Inhibition of neoplastic transformation and bioavailability of dietary flavonoid agents[END_REF] Flavonoids are then delivered to different tissues by blood. Within tissues, flavonoids will be further deconjugated by the actions of -glucuronidase and sulfatase activities. However, regarding anthocyanin the major polyphenolic composant of blackcurrant juice we used in our study, Passamonti et al., have proposed that the intact glycosides are the major circulating forms of anthocyanins, and the glycosides of anthocyanins may be transported by bilitranslocase at the gastric level, because the glycosides are good ligands for this carrier [START_REF] Passamonti | The interaction of anthocyanins with bilitranslocase[END_REF]. Some more recent studies show that, in rats and mice, anthocyanins are absorbed from the stomach [START_REF] Passamonti | Fast access of some grape pigments to the brain[END_REF][START_REF] Matuschek | The jejunum is the main site of absorption for anthocyanins in mice[END_REF].

The excretion of flavonoids in urine varies following the type of flavonoids, but the yield from urine cannot be considered as a useful indicator of their bioavailability.

Other subtypes of polyphenols undergo similar metabolic pathways with some differences.

For instance, unlike flavonoids, catechins and proanthocyanidins are mainly unglycosylated and therefore cannot get absorbed in the stomach but reach the small intestine intact [START_REF] Rios | Cocoa procyanidins are stable during gastric transit in humans[END_REF] Figure 15, shows an example of the pharmacokinetic properties of the flavanol.

Beneficial effect of polyphenols in vascular function

Polyphenols are well known to protect the cardiovascular system by a variety of actions, including their ability to improve the lipid profile; dilate blood vessels by stimulating the endothelial formation of NO, and also in some blood vessels EDHF, and by their antioxidant properties, which will prevent the degradation of NO by superoxide anions [START_REF] Ndiaye | Red wine polyphenols cause endotheliumdependent EDHF-mediated relaxations in porcine coronary arteries via a redoxsensitive mechanism[END_REF][START_REF] Frankel | Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine[END_REF][START_REF] Fitzpatrick | Isolation and characterization of endotheliumdependent vasorelaxing compounds from grape seeds[END_REF][START_REF] Madeira | eNOS activation induced by a polyphenol-rich grape skin extract in porcine coronary arteries[END_REF][START_REF] Mann | Activation of endothelial nitric oxide synthase by dietary isoflavones: role of NO in Nrf2-mediated antioxidant gene expression[END_REF]. In addition, polyphenol-rich sources such as a red wine extract have been shown to reduce systolic blood pressure and to improve endothelial dysfunction in several experimental models of hypertension (Sarr et al, 2006;[START_REF] Lopez-Sepulveda | Wine polyphenols improve endothelial function in large vessels of female spontaneously hypertensive rats[END_REF][START_REF] Madeira | eNOS activation induced by a polyphenol-rich grape skin extract in porcine coronary arteries[END_REF][START_REF] Bernatova | Wine polyphenols improve cardiovascular remodeling and vascular function in NO-deficient hypertension[END_REF][START_REF] Lee | An ethanolic extract of Lindera obtusiloba stems causes NO-mediated endothelium-dependent relaxations in rat aortic rings and prevents angiotensin II-induced hypertension and endothelial dysfunction in rats[END_REF]. Altogether, these findings support the view that polyphenol-rich natural products may protect the cardiovascular system, in part, by improving the endothelial function and, hence, retarding the development of cardiovascular diseases.

Polyphenols and tea polyphenols inhibit the expression of several NADPH oxidase subunits including NOX 1 and p22phox (Yu et al, 2010;[START_REF] Dal-Ros | Chronic intake of red wine polyphenols by young rats prevents aging-induced endothelial dysfunction and decline in physical performance: role of NADPH oxidase[END_REF], and tea-polyphenols upregulated the expression of catalase in vascular cells and the arterial wall [START_REF] Stangl | Molecular targets of tea polyphenols in the cardiovascular system[END_REF]. Atherosclerosis is characterized by the development of plaques which predominantly occur at the aortic roots, arch and abdominal aortas, and around the renal artery branches [START_REF] Nakashima | ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree[END_REF]. In addition, endothelial cells at sites of atherosclerotic plaques have been shown to have high level of inflammatory responses [START_REF] Feletou | Endothelium-derived vasoactive factors and hypertension: possible roles in pathogenesis and as treatment targets[END_REF]. Previous studies have shown that natural products such as a garlic extract and a grape seed extract decrease atherosclerotic plaque size in animal models [START_REF] Jimenez | Polyphenols restore endothelial function in DOCA-salt hypertension: role of endothelin-1 and NADPH oxidase[END_REF][START_REF] Chang | Possible superoxide radical-induced alteration of vascular reactivity in aortas from streptozotocin-treated rats[END_REF].

The blackcurrant and its health benefit

The blackcurrant (Ribes nigrum L., Grossulariaceae), a small, perennial shrub native to central Europe and northern Asia, is cultivated throughout the world, including the United

Phytochemical constituents of blackcurrant

Biochemical profiling of blackcurrants has revealed a myriad of constituents: flavonoids, polyunsaturated fatty acids (PUFA), structural and nonstructural carbohydrates, non-volatile organic acids, tannins and stilbenoids [START_REF] Bordonaba | Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars[END_REF][START_REF] Jaworska | Qualitative comparison of blackcurrant and blackcurrant--whey beverages[END_REF][START_REF] Tabart | Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum[END_REF]. Blackcurrant flavonoids, a group of polyphenolic compounds with a diphenylpropane skeleton, include anthocyanins and flavonols [START_REF] Mikkonen | Flavonol content varies among black currant cultivars[END_REF].

Blackcurrants are an important source of anthocyanins with concentrations up to four-fold greater than other common fruits. Studies in various genotypes of blackcurrants reveal a great variation in the anthocyanin content of 80-280 mg per 100 g of fruit with maximum levels observed up to 300 mg per 100 g of fruit [START_REF] Nour | Anthocyanins profile, total phenolics and antioxidant activity of black currant ethanolic extracts as influenced by genotype and ethanol concentration[END_REF][START_REF] Moyer | Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus, and ribes[END_REF]. The aglycones and cyanidin-3-O-rutinoside (Figure 17) (Maatta et al, 2003;[START_REF] Delazar | Isolation and free-radical-scavenging properties of cyanidin 3-O-glycosides from the fruits of Ribes biebersteinii Berl[END_REF]. Among PUFAs, g-linolenic acid (Figure 18) is an essential polyunsaturated fatty acid found in blackcurrants. g-linolenic acid has been isolated from the blackcurrant seed oil [START_REF] Watson | Cytokine and prostaglandin production by monocytes of volunteers and rheumatoid arthritis patients treated with dietary supplements of blackcurrant seed oil[END_REF][START_REF] Leventhal | Treatment of rheumatoid arthritis with blackcurrant seed oil[END_REF].

Antioxidant properties

Multiple studies support the antioxidant ability of blackcurrants [START_REF] Rubanyi | Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes[END_REF]. Data from additional studies demonstrate the degradation of hydrogen peroxide (H 2 O 2 )

and NO, as well as impeding the propagation of lipid and protein peroxidation [START_REF] Yoshiki | Superoxide-and 1,1-diphenyl-2-picrylhydrazyl radicalscavenging activities of soyasaponin beta g related to gallic acid[END_REF][START_REF] Viljanen | Inhibition of protein and lipid oxidation in liposomes by berry phenolics[END_REF]. Furthermore, antioxidant enzymes, such as glutathione (GSH) peroxidase and superoxide dismutase, were also shown to increase significantly by the blackcurrant components (using commercially available antioxidant status kit) through an unknown mechanism [START_REF] Nielsen | Absorption and excretion of black currant anthocyanins in humans and watanabe heritable hyperlipidemic rabbits[END_REF]. The specific antioxidant capacities of the blackcurrant fruit as a whole are mostly attributed to its phenolic and anthocyan content [START_REF] Moyer | Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus, and ribes[END_REF][START_REF] Ehala | Characterization of phenolic profiles of Northern European berries by capillary electrophoresis and determination of their antioxidant activity[END_REF]. This content can vary widely depending on the cultivar, season it was grown in, state of ripening and the particular part of the plant used [START_REF] Tabart | Antioxidant capacity of black currant varies with organ, season, and cultivar[END_REF]. Although the phenolic content is considered to contribute a more potent antioxidant activity than natural vitamins, high levels of vitamin C confers the additional inherent antioxidant properties to blackcurrants [START_REF] Mikkonen | Flavonol content varies among black currant cultivars[END_REF]Borges et al, 2010).

Following oral consumption, the antioxidant activity of anthocyanins in blackcurrants is affected by the pH of its environment, potentially due to a shift in its prototropic equilibrium [START_REF] Estevez | A computational study on the acidity dependence of radical-scavenging mechanisms of anthocyanidins[END_REF]. Free radical scavenging activity was noted to attain maximum levels at a pH between 6.0 and 7.0, slightly more acidic than human serum [START_REF] Matsumoto | Antioxidant activity of black currant anthocyanin aglycons and their glycosides measured by chemiluminescence in a neutral pH region and in human plasma[END_REF]; [START_REF] Young | Effect of fruit juice intake on urinary quercetin excretion and biomarkers of antioxidative status[END_REF]. This suggests that the anthocyanin antioxidant activity potentially varies depending on its location in the human body. Studies involving human subjects drinking blackcurrant juice revealed an increase in serum sulfahydryl group levels within two hours of consumption [START_REF] Gopalan | The health benefits of blackcurrants[END_REF]. Additional in vitro studies quantified a 94% inhibition of copper ion-induced low density lipoprotein (LDL) oxidation [START_REF] Rosenblat | Consumption of polyphenolic-rich beverages (mostly pomegranate and black currant juices) by healthy subjects for a short term increased serum antioxidant status, and the serum's ability to attenuate macrophage cholesterol accumulation[END_REF]. 1-As recent studies suggested that abnormal intestinal flora (pathogenic) contributes to a lot of deleterious complications of chronic liver diseases by enhancing intestinal inflammatory cell-dependent production of pro-inflammatory cytokines.

CHAPTER IV AIM OF THE STUDY

The translocation of these pathogenic bacteria lead to recruitment of pulmonary intravascular macrophages which themselves produce more local inflammatory cytokines, which over all has also effect on vascular endothelial function. The possibility that probiotics (VSL#3) therapy may improve vascular reactivity of the mesenteric artery of rats with portal hypertension and, in particular, the endothelial function was examined. 3-Previous studies have suggested that an increased estradiol level in CBDL rats might contribute to pulmonary vasodilatation via excessive formation of the potent vasodilator NO (Aller et al, 2001;Aller et al, 2002;Yol et al, 2005). The last part of our study was to investigate the effect of fulvestrant, which is an estrogen receptor antagonist with no agonistic effect, in CBDL rat model of biliary cirrhosis with portal hypertension. Indeed, fulvestrant is expected to prevent the increased estradiol-stimulated NO formation and, thus may improve the abnormalities observed in cirrhotic rat lungs.

EXPERIMENTAL PROTOCOLS

Various animal models of portal hypertension (PH), the main feature leading to the splanchnic and systematic vascular abnormalities in chronic liver diseases, exist. The common bile duct ligation (CBDL) [START_REF] Fallon | Common bile duct ligation in the rat: a model of intrapulmonary vasodilatation and hepatopulmonary syndrome[END_REF]) model leads to liver biliary cirrhosis with a hyperdynamic state and a hepatopulmonary syndrome. The latter is not induced by the use of carbon tetrachloride CCl 4 [START_REF] Cervinkova | Structural and functional changes after the administration of tetrachlormethane in the liver of rats fed on diets with different protein contents[END_REF] to obtain liver cirrhosis. Other studies were devoted to the effect of PH induced by partial portal vein ligation which induces the same abnormalities than the CBDL model but without liver cirrhosis.

In our work we were interested to assess the endothelial dysfunction in portal hypertension and especially with its consequences on the splanchnic and lung vascular bed.

Thus, we have chosen the CBDL rat model for our studies. CBDL leads to an acute obstructive jaundice in two weeks, and a progression to cirrhosis in about 4 weeks. CBDL stimulates the proliferation of biliary epithelial cells and oval cells (which are hepatocyte progenitors), resulting in proliferating bile ductules with an accompanying portal inflammation and fibrosis. Cholangiocyte proliferation starts after CBDL at the edge of the portal tract. During the first week of CBDL the hepatic microcirculation does not show any alterations with respect to the normal liver (Figure 19), [START_REF] Chambliss | Estrogen modulation of endothelial nitric oxide synthase[END_REF].

Vascular reactivity study in isolated organ chambers

The main superior mesenteric artery and the aorta were cleaned of connective tissue and cut into rings (2-3 mm in length). The mesenteric artery has been selected due to the fact that endothelium dependent relaxations involve both NO and EDHF, these vessels reflecting also the consequences of PH in the splanchnic circulation. In some preparations, the endothelium was removed by rubbing the intimal surface of rings with a pair of forceps. Left: from the mesenteric circulation arrowed we take segments from main mesenteric artery.

Right: organ bath used and segment of artery put in physiological tension with electrode attached and read changes in relaxation and constriction curve. We assessed in our first study the VSL#3 probiotics as an alternative to selective intestinal decontamination. By decreasing bacterial translocation, probiotics may play a role in normalizing the upregulated pro-inflammatory cytokines and endothelial dysfunction. It is known that probiotics can alter the composition of intestinal bacteria and minimize the progression of certain illnesses, for example, the probiotics can be used to prevent relapse of pouchitis, and ulcerative colitis [START_REF] Bibiloni | VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis[END_REF] and prevent antibiotics-induced diarrhea as well as Crohn's disease [START_REF] Borruel | Increased mucosal tumour necrosis factor alpha production in Crohn's disease can be downregulated ex vivo by probiotic bacteria[END_REF][START_REF] Gupta | Is lactobacillus GG helpful in children with Crohn's disease? Results of a preliminary, open-label study[END_REF] see (Figure 23) which demonstrate the possible effect of probiotics on commensal bacteria. Probiotic mixture of VSL#3 contains 8 bacterial strains Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii subsp. Bulgaricus (Rehman et al, 2012). Hence, it is hypothesized that probiotics preserve epithelial barrier function. In vitro studies on epithelial monolayers showed that probiotics improve barrier function following Escherichia coli infection or incubation with pro-inflammatory cytokines [START_REF] Bai | Assessment of a bioactive compound for its potential antiinflammatory property by tight junction permeability[END_REF][START_REF] Parassol | Lactobacillus casei DN-114 001 inhibits the increase in paracellular permeability of enteropathogenic Escherichia coli-infected T84 cells[END_REF][START_REF] Resta-Lenert | Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC)[END_REF]. Probiotics also preserve the intestinal epithelial barrier in several in vivo models of mice, such as the IL-10 knockout colitis [START_REF] Madsen | Probiotic bacteria enhance murine and human intestinal epithelial barrier function[END_REF] or models of sepsis [START_REF] Ewaschuk | Probiotic bacteria prevent hepatic damage and maintain colonic barrier function in a mouse model of sepsis[END_REF]; [START_REF] Qin | Effect of lactobacillus on the gut microflora and barrier function of the rats with abdominal infection[END_REF]. Pathological bacterial translocation to mesenteric lymph nodes as a marker of impaired barrier function can also be effectively reduced by probiotic therapy [START_REF] Ewaschuk | Probiotic bacteria prevent hepatic damage and maintain colonic barrier function in a mouse model of sepsis[END_REF][START_REF] Osman | Modulation of the effect of dextran sulfate sodiuminduced acute colitis by the administration of different probiotic strains of Lactobacillus and Bifidobacterium[END_REF][START_REF] Qin | Effect of lactobacillus on the gut microflora and barrier function of the rats with abdominal infection[END_REF][START_REF] Schmitz | Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis[END_REF].

Main branches of superior mesenteric artery

RESULTS

RESULTS

PART

Since

Introduction

Advanced chronic liver diseases are characterized by generalized progressive vasodilatation, related to portal hypertension (PH), which is especially observed in the splanchnic and pulmonary beds. In a rat model of partial portal vein ligation, Albrades et al. [1] reported that the initial event in response to PH is an up-regulation of vascular endothelial growth factor and endothelial nitric oxide synthase (eNOS) in the intestinal microcirculation. Although animal models have led to a number of hypotheses, the exact mechanism involved in the occurrence of the generalized vasodilatation and hyperdynamic syndrome is incompletely understood [2][3][4][5]. Several lines of evidence support a role for an increased formation of nitric oxide (NO) in the general vasodilatation in chronic liver diseases in patients [6] and also in the chronic bile duct ligated (CBDL) rat model [2,4]. In addition, chronic liver diseases in patients and also in experimental PH are characterized by an increased vascular formation of reactive oxygen species (ROS) [7,8] and the activation of the renal reninangiotensin-aldosterone system and the local angiotensin system [9][10][11]. Moreover, it has been suggested that bacterial translocation (BT) is an important step in the occurrence of the general vasodilatation and especially in the pulmonary bed [3,12]. In cirrhosis, BT is defined as the migration of bacteria and/or their products from the intestinal lumen to mesenteric lymph nodes [13] as a consequence of intestinal bacterial overgrowth, impaired host defenses, and/or disruption of the gut mucosal barrier [14,15]. In cirrhotic rats, BT has been reported to lead to endotoxaemia, which stimulates the expression of TNF-a leading to an enhanced formation of the potent vasodilators NO and carbon monoxide [3]. Thus, reducing BT to improve the excessive oxidative stress and/or to reduce the increased vasodilator factors is an interesting target to improve the general vascular dysfunction in PH. Earlier studies have reported that treatment with either antiobiotics or pentoxifylline, an inhibitor of TNF-a, to prevent BT and/or its consequences improves the vasodilatation in CBDL rats [12,16] and also in patients with liver cirrhosis [17,18]. Due to the poor tolerance of pentoxifylline [18] and the increased risk of bacterial resistance with antibiotics, probiotics such as VSL#3 which are well tolerated [19,20] may be of interest. Thus, the aim of the present study was to evaluate the effect of the probiotic VSL#3 formulation on the vascular dysfunction in an animal model of biliary cirrhosis with PH with a special attention to oxidative stress and the local angiotensin system.

Materials and Methods

Ethics statement

This study conforms to the Guide of Care and the Use of laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996)

Animal model

Male Wistar rats (10-12 week-old) were anaesthetized with an intraperitoneal mixture of ketamine (80 mg.kg body wt 21 ) and xylazine (4 mg.kg body wt 21 ). Biliary cirrhosis was induced by CBDL. After laparotomy, the bile duct was isolated, double ligated and resected between the two ligatures. Sham rats had laparotomy, underwent mobilization of the common bile duct without ligation. After surgery, rats were housed in a thermo-neutral environment, on a 12:12 h photoperiod and were provided food and drinking water at libitum. Eight CBDL rats and seven sham rats were treated three weeks before and 4 weeks following surgery with the probiotic formulation VSL#3 (VSL Pharmaceuticals, Inc, Towson, MD, USA; Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus bulgaricus) at a dose of 50 billion bacteria.kg body wt 21 daily, given in the drinking water. Seven CBDL rats and five sham rats receiving vehicle served as controls.

Four weeks after surgery, rats were sacrificed. The liver and spleen were removed and weighted. Liver samples (taken from the median lobe) from each rat were incubated in the Bouin-Holland fixative and subsequently embedded in paraffin for histological analysis. Segments of the main superior mesenteric artery and thoracic aorta were cleaned, embedded in Tissu-Tek O.C.T. compound (Sakura Finetek France SAS, Villeneuve d'Ascq, France) and snap-frozen for immunofluorescence studies and the determination of the formation of ROS. Samples of blood were taken by heart puncture, and, thereafter, plasma was stored at -80uC for subsequent serological analysis.

Assessment of liver cirrhosis and portal hypertension

Sections of liver samples (5 mm thick) were stained with hematoxylin, eosin and Masson's trichrome stain and evaluated by light microscopy. Histologically, liver of chronic bile duct ligation showed evidence of intrahepatic tubular duct hypertrophy with severe fibrosis. Portal hypertension is reflected by an increase in the spleen weight.

Vascular reactivity studies

Vascular reactivity studies on the main superior mesenteric artery were performed as described previously [11]. Briefly, the main superior mesenteric artery was cleaned of connective tissue, cut into rings (2-3 mm in length) and suspended in organ baths containing oxygenated Krebs bicarbonate solution for the determination of changes in isometric tension. After equilibration and functional tests, rings were contracted with phenylephrine (PE, 1 mM) before construction of concentration-response curves in response to acetylcholine (ACh), sodium nitroprusside or levcromakalim. In some experiments, rings were exposed to an inhibitor for 30 min before contraction with PE. Relaxations were expressed as percentage of the contraction induced by PE.

Immunofluorescence studies

Frozen arteries were cryosectioned at 14 mm. Sections were airdried for 15 min and stored at 280uC until use. Sections were first fixed with paraformaldehyde at 4%, washed and treated with 10% milk or 5% goat serum in PBS containing 0.1% Triton X-100 for 1 h at room temperature to block non-specific binding. Mesenteric artery sections were then incubated overnight at 4uC with an antibody directed against either eNOS (1/100), small and intermediate conductance calcium-activated potassium channels (SK Ca , IK Ca , 1/200), or connexins (Cx37, Cx40 and Cx43; 1/100 to 1/200). Aortic sections were incubated with an antibody directed against either angiotensin II (1/500), AT1 receptors (1/ 400), angiotensin-converting enzyme (ACE, 1/200), nitrotyrosine (1/200), p47phox (1/200), p22phox (1/200) or cyclooxygenase-1 or -2 (COX-1, COX-2, 1/200). Sections were then washed with PBS, incubated with the secondary antibody (1/300, Alexa 488-or 637-conjugated goat anti-rabbit IgG) for 2 h at room temperature in the dark before being washed with PBS and mounted in Dako fluorescence mounting medium (Dako France SAS, Les Ulis, France) and cover-slipped. For negative controls, primary antibodies were omitted.

Western blot analysis

Mesenteric artery and aortic segments were homogenized in extraction buffer (composition in mM: Tris/HCl 20 (pH 7.5; Q Biogene), NaCl 150, Na 3 VO 4 1, sodium pyrophosphate 10, NaF 20, okadaic acid 0.01 (Sigma), a tablet of protease inhibitor (Complete Roche) and 1% Triton X-100 (Euromedex)). Total proteins (10 mg) were separated on SDS-polyacrylamide gels and transferred electrophoretically onto polyvinylidine difluoride membranes (Amersham). Membranes were blocked with blocking buffer containing 5% bovine serum albumin, Tris-buffered saline solution (Biorad) and 0.1% Tween 20 (Sigma) (TBS-T) for 1 h. Membranes were then incubated overnight at 4uC with an antibody directed against either eNOS (1/10000), SK Ca (1/200), AT1 receptors (1/1000), p47phox (1/1000), p22phox (1/1000) or COX-2, (1/1000). Thereafter, membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody and signals were detected using enhanced chemiluminescence (Amersham).

Determination of vascular and mitochondrial ROS Formation

In situ formation of ROS was performed using the method previously described [21]. The redox-sensitive fluorescent dye dihydroethidium (DHE, 2.5 mM) was applied onto 25 mm unfixed cryosections of aorta for 30 min at 37uC in a light-protected humidified chamber. To determine the sources of ROS, sections were incubated with either apocynin (NADPH oxidase inhibitor and antioxidant, 300 mM), L-NA (NO synthase inhibitor, 300 mM), sulfaphenazol (cytochrome P450 inhibitor, 100 mM), indomethacin (cyclooxygenase inhibitor, 10 mM) or inhibitors of the mitochondrial respiration chain (myxothiazol, 0.5 mM+rote- none, 1 mM+KCN, 1 mM) for 30 min at 37uC before the addition of dihydroethidium. The in situ formation of mitochondrial ROS was performed using MitoSox (Life Technologies SAS, Saint Aubin, France), a mitochondrion-specific dihydroethidium-derivative fluorescent dye. Briefly, 14 mm unfixed cryosections of aorta were incubated with MitoSox (5 mM at 37uC for 60 min) in a light-protected humidified chamber. Sections were then washed three times, mounted in DAKO and cover-slipped.

Image analysis

All samples from immunofluorescence and in situ ROS formation studies were observed using a confocal laser-scanning microscope (Leica SP2 UV DM IRBE). Quantification of fluorescence levels was performed using FIJI GPL v2 software (http://fiji.sc/Fiji).

Determination of plasma inflammatory cytokine levels

The determination of the plasma level of TNF-a, IL-1a, MCP-1 and IL-4 was performed using a commercial rat cytokine Kit Flowcytomix (eBioscience SAS, Paris, France), multiple analyte detection kit, according to the protocol supplied by the manufacturer. The raw data were analysed using ''The FlowCytomixPro software''.

Statistical analysis

Values are expressed as means 6 SEM. Statistical analysis was performed using either Student's t test or an analysis of variance (ANOVA) followed by the Bonferroni post-hoc test as appropriate using GraphPad Prism (version 5 for Microsoft windows, GraphPad software, Inc, San Diego, CA, USA). A P value less than 0.05 was considered to be statistically significant.

Results

Characteristics and histologic findings

As shown in Table 1, four weeks after surgery, CBDL rats had a significantly increased spleen weight, and an enhanced bile duct proliferation as assessed by histological analysis of liver sections. Chronic intake of VSL#3 for seven weeks starting three weeks before the surgery reduced slightly but significantly the CBDLinduced increase in spleen weight whereas the liver weight was unaffected. The VSL#3 treatment alone had no effect on liver and spleen weight.

VSL#3 treatment prevents the CBDL-induced impaired EDH but not NO component of the relaxation to acetylcholine in mesenteric artery rings

ACh caused similar concentration-dependent relaxations in mesenteric artery rings from control and CBDL rats (maximal relaxations amounted to 97.461.4% and 94.962.4%, respectively, n = 6-8). However, the NO component of the relaxation as assessed in the presence of indomethacin and charybdotoxin plus apamin to prevent the formation of vasoactive prostanoids and EDH, respectively, was significantly impaired in the CBDL group (Figure 1A). In addition, the EDH-mediated component of the relaxation as assessed in the presence of indomethacin plus N Gnitro-L-arginine (an inhibitor of eNOS), was also significantly reduced in the CBDL group (Figure 1B). Although the VSL#3 treatment did not affect the CBDL-induced impaired NO component, it significantly improved the EDH component (Figures 1A andB). In addition, both the sodium nitroprusside (an NO donor) and the levcromakalim (an ATP-sensitive K channel opener) induced endothelium-independent relaxations were similar in all groups (Figures 1C andD).

VSL#3 treatment prevents CBDL-induced up-regulation of eNOS and down-regulation of SK Ca , IK Ca , Cx37, Cx40, and Cx43 in the mesenteric artery Immunofluorescence studies indicated that the eNOS signal is observed predominantly at the luminal surface of mesenteric artery sections in the control group, and that this signal is significantly increased in the CBDL group (Figure 2A). Pronounced fluorescence signals for SK Ca and IK Ca, two calciumactivated potassium channels involved in EDH [11], are observed throughout the arterial wall in the control group whereas both signals are significantly reduced in the CBDL group (Figure 2A). In addition, the fluorescence signal of connexins Cx37, Cx40 and Cx43, which form myoendothelial gap junctions transmitting the hyperpolarization from endothelial cells to the underlying smooth muscle cells to cause relaxation [11], is observed predominantly at the luminal surface and also to some extent in the vascular smooth muscle (Figure 2B). The fluorescence signals of Cx37, Cx40 and Cx43 are significantly reduced in the CBDL group (Figure 2B). The VSL#3 treatment prevented partially but significantly the CBDL-induced up-regulation of eNOS, and the down-regulation of SK Ca and IK Ca , and of Cx37, Cx40 and Cx43 (Figure 2). The VSL#3 treatment of sham rats was without effect except for SK Ca , which was significantly reduced (Figure 2). An increased eNOS protein level and a decreased SK Ca level were also observed in the CBDL group as assessed by Western blot analysis (Figure 2C). These effects were significantly prevented by the VSL#3 treatment (Figure 2C). In addition, IK Ca and Cx37 protein levels were below the detection level in all groups.

VSL#3 treatment reduces the CBDL-induced vascular oxidative stress involving several sources in the aorta CBDL markedly increased the DHE fluorescence signal, the MitoSOX fluorescence signal and the nitrotyrosine fluorescence signal throughout the aortic wall, and the eNOS fluorescence signal at the luminal surface and eNOS protein level as assessed by Western blot analysis (Figures 3A and4B). All these effects were significantly prevented by the ingestion of VSL#3 (Figures 3A and4B). The VSL#3 treatment of sham rats affected only minimally these signals (Figures 3A and4B).

The characterization of the cellular sources of oxidative stress has indicated that the CBDL-induced increased DHE fluorescence signal was markedly reduced by apocynin (an NADPH oxidase inhibitor and antioxidant), L-NA (an eNOS inhibitor), sulfaphenazol (a cytochrome P450 inhibitor), indomethacin (a cyclooxygenase inhibitor) and by a combination of inhibitors of the mitochondrial respiration chain (KCN, myxothiazol and rotenone) suggesting the involvement of NADPH oxidase, COXs, uncoupled eNOS, cytochrome P450, and the mitochondrial respiration chain (Figure 3C).

To obtain further evidence for a role of NADPH oxidase and COXs, their expression level was determined by immunofluorescence staining and Western blot analysis in the aorta. A significantly increased immunofluorescence signal of the NAPDH oxidase subunits p22phox and p47phox, and of COX-1 and COX-2 was observed in the aorta of the CBDL group compared to the sham group (Figure 4A). The VSL#3 treatment significantly reduced the CBDL-induced stimulatory effect for p22phox, p47phox and COX-2 whereas that for COX-1 was minimally affected (Figure 4A). The VSL#3 treatment of sham rats affected minimally all these signals (Figure 4A). In addition, an upregulation of p22phox and p47phox protein levels was also observed in the CBDL group as assessed by Western blot analysis whereas no such effect was observed in the CBDL+VSL#3 group (Figure 4B). In contrast, COX-2 protein levels were similar in all groups, and COX-1 protein levels were below the detection level (Figure 4B). An increased immunofluorescence level of angiotensin II, AT1 receptors and ACE throughout the aortic wall was observed in the CBDL group (Figure 5). The stimulatory effect of CBDL was significantly prevented by the VSL#3 treatment (Figure 5). The VSL#3 treatment alone affected minimally all these signals (Figure 5). An upregulation of the AT1 receptor protein level was also observed in the CBDL group by Western blot analysis whereas no such effect was observed in the CBDL+VSL#3 group (Figure 4B).

VSL#3 treatment prevents the CBDL-induced increase in plasma pro-inflammatory cytokines

A significant increase in the plasma level of pro-inflammatory cytokines including IL-1a, MCP-1 and TNF-a was observed in the CBDL group, and this effect was significantly prevented by the VSL#3 treatment (Figure 6). In contrast, the plasma level of IL-4, which is a potent anti-inflammatory cytokine, was markedly reduced in the CBDL group, and this effect was significantly prevented by the VSL#3 treatment (Figure 6). The VSL#3 treatment of sham rats affected minimally all these cytokines (Figure 6).

Discussion

The present study indicates that the CBDL-induced endothelial dysfunction involves predominantly a reduced EDH component and, also, to some extent a decreased NO component in the mesenteric artery. The blunted EDH component is associated with a reduced expression of SK Ca and IK Ca levels, and Cx30, Cx37 and Cx40 levels, which are all involved in the EDH response, most likely as a consequence of the activation of the local angiotensin system leading to an increased vascular oxidative stress, and the formation of pro-inflammatory mediators. They further indicate that ingestion of the probiotic VSL#3 formulation effectively prevented the CBDL-induced endothelial dysfunction, at least in part, by targeting the vascular angiotensin system.

Biliary cirrhosis induced by chonic bile duct ligation in rats and evidenced by an increased spleen weight and enhanced bile duct proliferation is associated with an unaffected acetylcholineinduced endothelium-dependent relaxation in mesenteric artery rings. In contrast, when experiments were performed to study selectively either the NO component or the EDH component then a significant reduction of both components was observed in the CBDL group as observed previously in some but not all studies [11], [22], [23]. A severely reduced EDH component despite an unaffected ACh-induced relaxation has also been observed previously in aging-related endothelial dysfunction [24]. Such a difference is most likely explained by the fact that the NO component and the EDH component, although blunted, act in synergy to maintain a normal endothelium-dependent relaxation whereas such a compensating mechanism cannot take place when a single component is evaluated. Alternatively, the present findings due not rule out the possibility that vasoactive prostanoids contribute to preserve the acetylcholine-induced endotheliumdependent relaxation in the CBDL group. However, previous findings have indicated that although prostacyclin evoked relaxations in mesenteric resistance arteries from control rats, the prostanoid induced also contractile responses in arteries from spontaneously hypertensive rats presenting an endothelial dysfunction [25]. Moreover, chronic COX inhibition did not affect the CBDL-induced decrease in mean arterial pressure in CBDL rats [26].

The impaired EDH-mediated relaxation is most likely the consequence of the decreased expression in IK Ca and SK Ca involved in EDH formation, and also of Cx37, Cx40 and Cx43, which form myoendothelial gap junctions transmitting the endothelial hyperpolarization towards the underlying smooth muscle [11]. In addition to the present findings, oxidative stress has been suggested to contribute to the impaired EDH-mediated relaxation [11]. The present findings further extent these previous observations by showing that oxidative stress is generated by several sources including NADPH oxidase, uncoupled eNOS, cytochome P450, COXs and the mitochondrial respiration chain. Moreover, an upregulation of several pro-oxidant enzymes including NADPH oxidase, COX-1, and COX-2 is observed in the arterial wall of CBDL rats. Besides the EDH component, the NO component of the relaxation is also impaired in CBDL rats despite an increased expression of eNOS. This latter response is most likely part of a compensatory mechanism due to the degradation of the excessive formation of NO by superoxide anions as indicated by the increased peroxynitrite level.

The present findings support a key role for the angiotensin system in the CBDL-induced endothelial dysfunction. Indeed, an increased expression of ACE, Ang II and AT1R was observed throughout the arterial wall of CBDL rats. They are also consistent with the fact that losartan, an AT1 receptor antagonist, prevented the CBDL-induced endothelial dysfunction and oxidative stress [11]. A major novel finding of the present study is that ingestion of the probiotic VSL#3 formulation improved the endothelial dysfunction in CBDL rats along with a pronounced improvement of the local angiotensin system and the level of oxidative stress in the arterial wall.

Previous studies have shown that amongst factors involved in the general vasodilatation in cirrhosis is bacterial translocation [3,5,12]. Indeed, an alteration of the gut microflora associated with disruption of the gut mucosal barrier leading to bacterial translocation has been observed in 45-75% of animals with experimental cirrhosis [14,15]. This response, in turn, promotes the subsequent inflammatory response, which will contribute to the hyperdynamic circulatory state in cirrhosis [5,12,16]. Indeed, treatments preventing bacterial translocation using either antiobiotics or pentoxifylline (an inhibitor of TNFa) reduced the excessive vasodilatation in CBDL rats [5,12,16]. However, the long-term use of antibiotics and pentoxifylline is hazardous due to the risk of inducing bacterial resistance with antibiotics and the poor tolerance of pentoxifylline as reported in a pilot study by Tanikella et al. [18]. In contrast, chronic use of probiotics has not been associated with major side effects [19,20]. Furthermore, previous studies have indicated that intake of probiotics such as the VSL#3 formulation leads to major changes in the composition of the gut flora in both experimental animals and in humans. Indeed, intake of VSL#3 in patients with pouchitis was associated with an increased intestinal bacterial diversity and a reduced fungal diversity in comparison with patients treated with a placebo [27]. VSL#3 treatment also prevented the antibiotic-induced decrease in several indigenous bacterial groups as assessed using a standardized human fecal microbiota in a computer-controlled model of large intestine [28]. Moreover, intake of the VSL#3 formulation in rats altered the species richness and diversity of the luminal intestinal microbiota [29]. However, the possibility that the beneficial effect of the VSL#3 treatment on the CBDLinduced endothelial dysfunction involves an improved bacterial translocation still remains to be determined.

The study of Loguercio et al. [20] indicated that administration of VSL#3 to alcoholic liver cirrhosis patients led to an improvement of the liver function and the increased plasma level of oxidative stress and TNF-a. In the present study, a relatively small but significant improvement of spleen weights was observed suggesting that VSL#3 may also have some effects on cirrhosis in CBDL rats, which, however, cannot account solely for the improved endothelial function.

In addition in a rat model of cirrhosis induced by CCl 4, Chiva et al. [30] reported that the addition of Lactobacillus johnsonii to an antioxidant treatment (vitamin C plus glutamate) reduced endotoxemia possibly due to an increased clearance of endotoxins by the monocyte-macrophage system. The VSL#3 treatment also reduced the CBDL-induced pro-inflammatory response as indicated by the improvement of circulating levels of pro-inflammatory cytokines such as IL-1a and TNF-a. Thus, the protective effect of VSL#3 in CBDL rats may involve, besides an improvement of the gut flora composition, possibly also the stimulation of immunological defence mechanisms and perhaps also the recovery of the intestinal motility, which has been shown to be decreased in cirrhosis with bacterial translocation [31].

Another potential protective mechanism of VSL#3 is that upon fermentation, various probiotics (including those contained in VSL#3, [32]) are capable of releasing ACE inhibitory peptides. This has been observed in vitro [32,33] and also in vivo in hypertensive rats [34] and patients [35]. In an in vivo trial, the ingestion of milk fermented with probiotics led to a significant decrease in blood pressure [35]. In the present study, a normalized vascular expression level of ACE, angiotensin II and AT1R along with a reduced expression of NADPH oxidase was observed in CBDL rats treated with VSL#3. Since infusion of angiotensin II leads to an increased vascular expression of NADPH oxidase and oxidative stress [36], it is likely that the VSL#3 treatment may improve oxidative stress by reducing the local angiotensin system subsequent to the normalization of ACE expression. Alternatively, VSL#3 may also normalize ACE activity via the release of ACE inhibitory peptides as suggested by previous studies [32,35].

In conclusion, the present findings indicate that oxidative stress possibly due to bacterial translocation inducing a pro-inflammatory response and the local angiotensin system is involved in the endothelial dysfunction in CBDL rats, and that this effect is improved by the ingestion of the probiotic VSL#3 formulation.

Schematic presentation of the 1 st article results:

Common bile duct ligation in rat, induces biliary proliferation at liver level and increase liver size, sign characterize the presence of biliary cirrhosis. This with splenomegaly represents a part of portal hypertension.

Rats received either control drinking water or the probiotic VSL#3 solution (50 billion bacteria.kg body wt -1 .day -1 ) for 7 weeks. After 3 weeks, rats underwent surgery with either resection of the common bile duct or sham surgery. The reactivity of mesenteric artery rings was assessed in organ chambers, expression of proteins by immunofluorescence and Western blot analysis, oxidative stress using dihydroethidium, and plasma pro-inflammatory cytokine levels by flow cytometry.

Results: Both NO-and EDH-mediated relaxations to acetylcholine were reduced in the CBDL group compared to the sham group, and associated with a reduced expression of Cx37, Cx40, Cx43, IK Ca and SK Ca and an increased expression of endothelial NO synthase (eNOS).

In aortic sections, increased expression of NADPH oxidase subunits, angiotensin converting enzyme, AT1 receptors and angiotensin II, and formation of ROS and peroxynitrite were observed. VSL#3 prevented the deleterious effect of CBDL on EDH-mediated relaxations, vascular expression of connexins, IK Ca , SK Ca and eNOS, oxidative stress, and the angiotensin system. VSL#3 prevented the CBDL-induced increased plasma TNF-, IL-1 and MCP-1 levels, (Figure 24).

PART II

RESULTS

PART II

Polyphenol-rich blackcurrant juice prevents endothelial dysfunction in the mesenteric artery of cirrhotic rats with portal hypertension: Role of oxidative stress and the angiotensin system

Rashid Sherzad a , Idris Khodja Noureddine a , Auger Cyril a , Alhosin Mahmoud a , Boehm Nelly b , Oswald-Mammosser Monique, a,c and Schini-Kerth Valérie B a .

Submitted to journal of Free Radical Biology and Medicine

There are numerous reports showing that polyphenols can play an important role in prevention of degenerative diseases such as cancer and attenuating inflammatory diseases like liver fibrosis. Polyphenols are well known to have antioxidant properties and also abilities to modulate the activity of a wide variety of enzymes and cell receptors. For these reasons we are interested to study the effect of polyphenols-rich blackcurrant in rat model of liver cirrhosis.

Chronic liver diseases with portal hypertension are characterized by a progressive vasodilatation associated with an endothelial dysfunction and vascular oxidative stress, which is especially pronounced in the splanchnic and pulmonary beds. It has been suggested that one of the pathophysiological mechanisms leading to the general vasodilatation is related to bacterial translocation and activation of rennin-angiotensin-aldosterone system.

The aim of the present study was to determine whether the ingestion of a polyphenolrich Blackcurrant juice (PRBJ) improves the endothelial dysfunction and vascular oxidative stress in CBDL rats, and, if so, to determine the underlying mechanism.

Male Wistar rats (8 rats per group) received either control drinking water or water containing a dose of 60 mg/kg of polyphenol-rich Blackcurrant juice for 7 weeks. After 3 weeks, the rats underwent surgery either with ligation and resection of the common bile duct (CBDL rats) or sham surgery (sham rats). Thereafter, the rats were sacrificed after 4 weeks.

Reactivity of mesenteric artery rings was assessed in organ chambers, the expression level of proteins in mesenteric artery and/or aorta sections by immunofluorescence, and the vascular formation of reactive oxygen species (ROS) by dihydroethidium (DHE). Plasma levels of pro-inflammatory cytokines including TNF-, IL-1 , MCP-1 and IL-4 were evaluated by flow cytometry.

Both the nitric oxide (NO)-and the endothelium-dependent hyperpolarization (EDHF)-mediated relaxations to acetylcholine in rings with endothelium were significantly reduced in CBDL rats compared to sham rats. In contrast, relaxations to sodium nitroprusside

Introduction

Advanced chronic liver diseases with portal hypertension are characterized by a generalized vasodilatation with endothelial dysfunction, which is especially observed in the splanchnic and pulmonary beds (Abraldes, Iwakiri et al. 2006). The mechanism contributing to the generalized vasodilatation still remains poorly characterized (Nunes, Lebrec et al. 2001;Zhang, Ling et al. 2003;Ling, Zhang et al. 2004;Sztrymf, Rabiller et al. 2004;[START_REF] Zhang | Intestinal endotoxemia plays a central role in development of hepatopulmonary syndrome in a cirrhotic rat model induced by multiple pathogenic factors[END_REF]. In a rat model of partial portal vein ligation, Albrades et al (Abraldes, Iwakiri et al. 2006) reported that the initial event subsequent to the induction of portal hypertension is an up-regulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the intestinal microcirculation. Indeed, an increased formation of NO has been involved in the endothelial dysfunction in chronic liver diseases in patients [START_REF] Rolla | Exhaled nitric oxide and impaired oxygenation in cirrhotic patients before and after liver transplantation[END_REF]Gomez, Barbera et al. 2006), and also in the chronic bile duct ligated (CBDL) rat model (Nunes, Lebrec et al. 2001;Ling, Zhang et al. 2004;Zhang, Ling et al. 2003). In addition, endothelial dysfunction in chronic liver diseases in patients and also in experimental portal hypertension has also been shown to involve a high level of oxidative stress in the arterial wall (Chen, Mo et al. 1997;Dal-Ros, Oswald-Mammosser et al. 2010).

The increased formation of reactive oxygen species (ROS) has been suggested to be initiated by bacterial translocation and the subsequent development of endotoxaemia, and also to the chronic activation of the systemic and local angiotensin system [START_REF] Yang | Effect of 1-week losartan administration on bile ductligated cirrhotic rats with portal hypertension[END_REF][START_REF] Bode | Activation of the innate immune system and alcoholic liver disease: effects of ethanol per se or enhanced intestinal translocation of bacterial toxins induced by ethanol?[END_REF][START_REF] Helmy | Nitric oxide mediates the reduced vasoconstrictor response to angiotensin II in patients with preascitic cirrhosis[END_REF], which promotes the NADPH oxidase-mediated formation of superoxide anions [START_REF] Sarr | Red wine polyphenols prevent angiotensin IIinduced hypertension and endothelial dysfunction in rats: role of NADPH oxidase[END_REF]Dal-Ros, Oswald-Mammosser et al. 2010;[START_REF] Yu | Oxidative stress with an activation of the renin-angiotensin system in human vascular endothelial cells as a novel mechanism of uric acid-induced endothelial dysfunction[END_REF]

Material and Methods

Ethics statement

This study conforms to the Guide of Care and the Use of laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996)

Animal models

Male Wistar rats (10-12 weeks old) were anaesthetized with an intraperitoneal mixture of ketamine (80 mg.kg body wt -1 ) and xylazine (4 mg.kg body wt -1 ). Biliary cirrhosis was induced by CBDL. After laparotomy, the bile duct was isolated, double ligated and resected between the two ligatures. Sham rats had laparotomy, underwent mobilization of the common bile duct without ligation. After surgery, rats were housed in a thermo-neutral environment, on a 12:12 h photoperiod and were provided food and water ad libitum .

Protocols

Eight CBDL rats and seven sham rats received three weeks before surgery and four weeks thereafter a polyphenol-rich blackcurrant juice (PRBJ, 60 mg GAE.kg body wt -1 daily) in the drinking water, and seven CBDL rats and five sham rats received only drinking water.

Four weeks after surgery, rats were sacrificed. After excision, the mesenteric artery and the aorta were placed in Krebs-Ringer bicarbonate solution for the subsequent determination of vascular reactivity using organ chambers. The liver and spleen were removed and weighted.

Liver samples (taken from the median lobe) from each rat were incubated in Bouin-Holland fixative and embedded in paraffin for subsequent histological analysis. Segments of the main superior mesenteric artery and the thoracic aorta were cleaned, embedded in Tissu-Tek O.C.T.

compound and snap-frozen for subsequent immunofluorescence studies and the determination of the level of oxidative stress. Samples of blood were taken from heart puncture, and thereafter plasma was stored at -20°C for subsequent determination the levels of cytokines.

Assessment of liver cirrhosis and portal hypertension

Sections of liver samples (5 µm thick) were stained with hematoxylin, eosin and Masson's trichrome stain and evaluated by light microscopy. Histologically, liver of chronic bile duct ligation showed evidence of intrahepatic tubular duct hypertrophy with severe fibrosis. Portal hypertension was reflected by an increase in the spleen weight.

Vascular reactivity studies

Vascular reactivity was performed using the main superior mesenteric artery as

Immunofluorescence studies

Frozen arteries were cryosectioned at 14 µm and, then, the sections were air-dried for

Determination of vascular and mitochondrial ROS formation

The redox-sensitive fluorescent dye dihydroethidine (DHE) was used to evaluate the in situ formation of ROS as described previously by Sarr et al. [START_REF] Sarr | Red wine polyphenols prevent angiotensin IIinduced hypertension and endothelial dysfunction in rats: role of NADPH oxidase[END_REF].

Dihydroethidine (2.5 µM, Sigma) was applied onto 25 µm unfixed cryosections of aorta for 30 min at 37°C in a light-protected humidified chamber. To determine the nature and the sources of ROS, sections were incubated with a pharmacological modulator for 30 min at 37°C before addition of DHE. MitoSox, a mitochondrion-specific hydroethidine-derivative fluorescent dye, was used to assess the mitochondrial formation of ROS. Briefly, 14 µm unfixed cryosections of aorta were incubated with MitoSox (5 µM at 37°C for 60 min) in a light-protected humidified chamber. After incubation, sections were washed three times, mounted in Vectashield and cover-slipped. Images were obtained with a Leica SP2 UV DM IRBE laser scanning confocal microscope. Quantification of staining levels was performed using FIJI GPL v2 software.

Determination of plasma cytokine levels

The determination of the plasma level of TNF-, IL-1 , MCP-1 and IL-4 was performed using a commercial rat cytokine Kit Flowcytomix™ (eBioscience SAS, Paris, France), according to the protocol supplied by the manufacturer. The raw data were analysed using "The FlowCytomixPro software".

Products and chemicals

Antibodies were purchased as indicated: anti-rat Cx37 polyclonal antibody (Alpha Diagnostic, San Antonio, USA), anti-K Ca 2.

Statistical analyses

Values were expressed as means±SEM. Statistical evaluation was performed using either Student's t test for paired data or an analysis of variance followed by the Bonferroni post-hoc test to identify significant difference as appropriate with GraphPad Prism (version 5

for Microsoft windows. GraphPad software, Inc, San Diego, CA, USA). Values of P<0.05

were considered to be statistically significant.

Results

Characteristics and histologic findings

As shown in Table 1, four weeks after surgery, CBDL rats had an increased liver and spleen weight and there was a pronounced bile duct proliferation (data not shown). Chronic intake of PRBJ 3 weeks before surgery and 4 weeks thereafter was associated with a slight but significant reduction of the weight of the spleen but not of the weight of the liver in CBDL rats (Table 1). The PRBJ treatment alone affected minimally liver and spleen weight in sham rats (Table 1).

PRBJ treatment prevents the CBDL-induced endothelial dysfunction in the mesenteric artery

ACh caused similar concentration-dependent relaxations in mesenteric artery rings from control and CBDL rats (maximal relaxations amounted to 97.4 ± 1.4% and 94.9 ± 2.4%, respectively, n = 6-8). However, the NO component of the relaxation as assessed in the presence of indomethacin and charybdotoxin plus apamin to prevent the formation of vasoactive prostanoids and EDH, respectively, was slightly but significantly blunted in the CBDL group (Figure 1A). In addition, the EDH-mediated component of the relaxation as assessed in the presence of indomethacin plus N G -nitro-L-arginine (a NOS inhibitor), was markedly reduced in the CBDL group (Figure 1B).

Although the PRBJ treatment did not affect the CBDL-induced impaired NO component, it significantly improved the EDH component (Figures 1A andB). The PRBJ treatment alone did affect neither the NO nor the EDH component in the sham group (Figures 1 A andB).

Both sodium nitroprusside (a NO donor)-and levcromakalim (an ATP-sensitive K + channel opener)-induced endothelium-independent relaxations were similar in all groups (Figures 1C andD). 2010), were observed predominantly at the luminal surface and also to some extent in the vascular smooth muscle of the mesenteric artery in the sham group (Figure 2). The fluorescence signals of SK Ca and Cx37 were significantly reduced in the CBDL group (Figure 2). In contrast to SK Ca and Cx37, the eNOS fluorescence signal observed at the luminal surface of the mesenteric artery was significantly increased in the CBDL group (Figure 2).

PRBJ treatment prevents CBDL-induced up-regulation of eNOS and down

The PRBJ treatment significantly prevented the CBDL-induced down-regulation of both SK Ca and Cx37 expression, and the up-regulation of eNOS (Figure 2). The PRBJ treatment alone had minor effects in the sham group (Figure 2).

PRBJ treatment reduces the CBDL-induced vascular oxidative stress involving several sources in the aorta

In the CBDL group, a markedly increased DHE fluorescence signal, MitoSOX fluorescence signal and nitrotyrosine fluorescence signal were observed throughout the aortic wall (Figure 3A). All these effects were significantly prevented by the PRBJ treatment (Figure 3A). The PRBJ treatment alone had no significant effect in the sham group (Figure 3A). The CBDL-induced vascular oxidative stress was significantly reduced by MnTMPyP (a membrane permeant superoxide dismutase mimetic) and PEG-catalase (a membrane permeant analogue of catalase), and slightly but not significantly by native SOD and catalase (Figure 3B). In addition, the CBDL-induced oxidative stress was also markedly inhibited by apocynin (NADPH oxidase inhibitor and antioxidant), L-NA (non-selective NOS inhibitor), sulfaphenazol (cytochrome P450 inhibitor), indomethacin (cyclooxygenase inhibitor) and by a combination of inhibitors of the mitochondrial respiration chain (KCN, myxothiazol and rotenone) (Figure 3C). These findings suggest that several sources of ROS contribute to the increased vascular level of oxidative stress in the CBDL group including NADPH oxidase, COXs, NOS, cytochrome P450 and the mitochondrial respiration chain.

To obtain further evidence for the role of NADPH oxidase, COX and NOS, their expression level was determined by immunofluorescence staining. An increased expression level of the NADPH oxidase subunits p22phox and p47phox was observed in the aorta, and COX-2 and inducible NOS in the mesenteric artery of the CBDL group compared to the sham group (Figure 4). The PRBJ treatment significantly prevented the CBDL-induced expression of these markers (Figure 4). The PRBJ treatment alone had only minor effects in the sham group (Figure 4).

PRBJ improves the CBDL-induced expression of the angiotensin system in the aorta

Previous findings indicating that losartan (an angiotensin type 1 receptor antagonist)

prevented the CBDL-induced endothelial dysfunction and oxidative stress in the mesenteric artery of rats suggested the involvement of the angiotensin system (Dal-Ros, Oswald-

Mammosser et al. 2010).

To determine the possibility that the PRBJ treatment affects the vascular angiotensin system, the expression level of angiotensin II, AT1 receptors and ACE was assessed by immunofluorescence staining in aortic sections. The fluorescence staining of Ang II, AT1 receptors and ACE was significantly increased in the CBDL group compared to that in the sham group (Figure 5). The PRBJ treatment prevented CDBL-induced upregulation of Ang II, AT1R and ACE whereas it did not affected the signals in the sham group (Figure 5).

PRBJ reduces the CBDL-induced plasma level of pro-inflammatory cytokines

A significant increase in the plasma level of pro-inflammatory cytokines including IL-, MCP-1 and TNF-was observed in the CBDL group and this effect was significantly prevented by the PRBJ treatment (Figure 6). In contrast, the plasma level of IL-4, a potent anti-inflammatory cytokine, was markedly reduced in the CBDL group, no such effect was observed in the PRBJ-treated CBDL group (Figure 6). The PRBJ treatment alone affected only minimally the level of the different cytokines in the sham group (Figure 6).

Discussion

The 2010). Moreover, the high level of oxidative stress in the arterial wall of CBDL rats appears to be the key mediator of the endothelial dysfunction. Indeed, increased levels of oxidative stress have been observed in blood of patients with decompensated liver cirrhosis (Chen, Mo et al. 1997), and also in the arterial wall of experimental models of portal hypertension including CBDL [START_REF] Fernando | N-acetylcysteine prevents development of the hyperdynamic circulation in the portal hypertensive rat[END_REF][START_REF] Vercelino | N-acetylcysteine effects on genotoxic and oxidative stress parameters in cirrhotic rats with hepatopulmonary syndrome[END_REF] A major novel finding of the present study is that chronic ingestion of a polyphenolrich blackcurrant juice, a natural source of antioxidants, prevented the endothelial dysfunction affecting the EDH component in CBDL rats most likely as a consequence of the improvement of the vascular level of oxidative stress and the prevention of the down-regulation of the expression of SK Ca and Cx37. Blackcurrant juices is well known to contain high levels of anthocyanins predominantly delphinidin-3-rutinoside, delphinidin-3-glucoside, cyanidin-3rutinoside, and cyanidin-3-glucoside, which have potent antioxidant activity [START_REF] Maatta | High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: ribes species[END_REF][START_REF] Wu | Lipophilic and hydrophilic antioxidant capacities of common foods in the United States[END_REF][START_REF] Borges | Identification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cranberries[END_REF]). However, PRBJ may also improve CBDL-induced oxidative stress indirectly as shown by the present observations since it prevented the upregulation of several sources of ROS including NADPH oxidase, COX-2, and NOSs. Previous studies have also indicated that intake of several other polyphenol-rich sources such as a red wine extract prevented vascular oxidative stress and endothelial dysfunction in the angiotensin II-induced hypertensive rats (Sarr, Chataigneau et al. 2006) and in old rats (Idris Khodja, Chataigneau et al. 2012), and tea catechin in the Otsuka Long-Evans Tokushima fatty rat model of metabolic syndrome [START_REF] Ihm | Catechin prevents endothelial dysfunction in the prediabetic stage of OLETF rats by reducing vascular NADPH oxidase activity and expression[END_REF].

Similarly Although the mechanism underlying the CBDL-induced oxidative stress remains poorly characterized, several lines of evidence suggest the involvement of an increased translocation of bacteria from the intestinal tract to the blood circulation. Indeed, bacterial translocation has been observed in 45-75% of animals with experimental liver cirrhosis (Runyon, Borzio et al. 1995;[START_REF] Wiest | Bacterial translocation in cirrhotic rats stimulates eNOSderived NO production and impairs mesenteric vascular contractility[END_REF]). Moreover, it has been shown to induce an inflammatory response, which, in turn, contributes to the hyperdynamic circulatory state observed in liver cirrhosis (Rabiller, Nunes et al. 2002;Sztrymf, Rabiller et al. 2004;Liu, Liu et al. 2012) . In addition, exposure of a human enteric epithelial cell line to Escherichia coli has been shown to induce a pro-oxidant response, which facilitates bacteria internalization and translocation [START_REF] Schoultz | Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C[END_REF]. Consistent with those previous findings, the present ones indicate an increased plasma level of several pro-inflammatory cytokines including IL-1 , MCP-1 and TNF-and a reduced level of the anti-inflammatory cytokine IL-4 in the CBDL group. The fact that the PRBJ treatment prevented the CBDL-induced vascular oxidative stress and the pro-inflammatory response suggests that the treatment may reduce bacterial translocation. Such a possibility is also supported by the fact that resveratrol, a natural antioxidant, attenuated bacterial translocation in mesenteric lymph nodes, liver and spleen in rats following an intestinal ischemia/reperfusion injury [START_REF] Ozkan | Resveratrol, a natural antioxidant, attenuates intestinal ischemia/reperfusion injury in rats[END_REF].

Several lines of evidence suggest that the CBDL-induced vascular oxidative stress may also involve, besides bacterial translocation, an increased activation of the angiotensin system. Such a response will lead to an excessive formation of angiotensin II, a potent inducer of vascular oxidative stress by stimulating the activation of NADPH oxidase, a major enzymatic source of superoxide anions, and by increasing its expression [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][START_REF] Sarr | Red wine polyphenols prevent angiotensin IIinduced hypertension and endothelial dysfunction in rats: role of NADPH oxidase[END_REF]. Indeed, portal hypertension in both humans and experimental animals has been associated with an increased plasma renin activity and/or angiotensin II level [START_REF] Yang | Effect of 1-week losartan administration on bile ductligated cirrhotic rats with portal hypertension[END_REF][START_REF] Helmy | Nitric oxide mediates the reduced vasoconstrictor response to angiotensin II in patients with preascitic cirrhosis[END_REF]. Moreover, losartan, an AT1 receptor antagonist, has been shown to reduce portal hypertension in patients [START_REF] De | Portal pressure response to losartan compared with propranolol in patients with cirrhosis[END_REF][START_REF] Wagatsuma | Clinical usefulness of the angiotensin II receptor antagonist losartan in patients with portal hypertensive gastropathy[END_REF], and to improve the hyperdynamic circulation, the endothelial dysfunction and vascular oxidative stress in CBDL rats [START_REF] Yang | Effect of 1-week losartan administration on bile ductligated cirrhotic rats with portal hypertension[END_REF][START_REF] Heller | Vascular, hemodynamic and renal effects of low-dose losartan in rats with secondary biliary cirrhosis[END_REF]Dal-Ros, Oswald-Mammosser et al. 2010). Consistent with an activation of the angiotensin system are the present findings indicating an upregulation of Ang II, AT1R and ACE in the arterial wall of CBDL rats.

Furthermore, they also suggest that the beneficial effect of the PRBJ treatment on the CBDLinduced endothelial dysfunction involves the prevention of the activation of the angiotensin system as indicated by similar expression levels of Ang II, AT1R and ACE in the PRBJ + CBDL group and the sham group. Previous findings have also indicated that resveratrol decreases the expression of AT1R through sirtuin1 activation in vascular smooth muscle cells [START_REF] Miyazaki | SIRT1, a longevity gene, downregulates angiotensin II type 1 receptor expression in vascular smooth muscle cells[END_REF]). Potential mechanisms contributing to limit the activation of the angiotensin system by polyphenols involve their ability to inhibit angiotensin converting enzyme activity [START_REF] Kang | Inhibition of inducible nitricoxide synthase expression by silymarin in lipopolysaccharide-stimulated macrophages[END_REF][START_REF] Actis-Goretta | Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins[END_REF]) but possibly also their antioxidant properties since oxidative stress has been shown to induce AT1R and ACE expression [START_REF] Banday | Angiotensin II-mediated biphasic regulation of proximal tubular Na+/H+ exchanger 3 is impaired during oxidative stress[END_REF][START_REF] Mu | Hydrogen peroxide induces overexpression of angiotensin-converting enzyme in human umbilical vein endothelial cells[END_REF].

In conclusion, the present findings indicate that chronic ingestion of a blackcurrant juice prevents endothelial dysfunction and the inflammatory response in CBDL rats. The beneficial effect involves its ability to prevent vascular oxidative stress, in part, by preventing the activation of the local angiotensin system and possibly also bacterial translocation.

Schematic presentation of the 2nd article results:

Chronic liver diseases with portal hypertension are characterized by a progressive vasodilatation (with endothelial dysfunction) which is especially observed in the splanchnic and pulmonary beds. The latter is referred as to the hepatopulmonary syndrome (HPS). It has been suggested that one of the physiopathologic mechanisms leading to the general vasodilatation and especially to the HPS is related to bacterial translocation which has been shown to improve in CBDL rats by changing the intestine microbiota. Therefore, the present study has evaluated the effect of ingestion of polyphenol-rich blackcurrant juice to prevent the endothelial dysfunction in rats following common bile duct ligation (CBDL), and, if so, to determine the underlying mechanism.

Male Wistar rats (8 rats per group) received either control drinking water or a dose of 60 mg/kg of polyphenol-rich blackcurrant juice (PRBJ) for 7 weeks. After 3 weeks, the rats underwent surgery with either the ligations and resections of the common bile duct (CBDL rats) or sham surgery (sham rats), and were followed for 4 weeks. Reactivity of mesenteric artery rings was assessed in organ chambers. The expression levels of connexins (Cx37) potassium channels (SK Ca ), endothelial NO synthase (eNOS), inducible endothelial NO synthase (iNOS), cyclo-oxygenase-2 (COX 2 ), NADPH oxidase subunits and nitrotyrosines were assessed by immunohistochemistry in mesenteric artery and/or aorta. The vascular formation of reactive oxygen species (ROS) was evaluated using dihydroethidine. Plasma levels of pro-inflammatory cytokines including TNF-, IL-1 , MCP-1 and IL-4 were evaluated by 5plex-FlowCytomix.

Both the NO-and the EDHF-mediated relaxations to acetylcholine were significantly reduced in CBDL rats compared to sham rats, whereas relaxations to sodium nitroprusside (an exogenous donor of NO) and to levcromakalim (an ATP-sensitive K + channel opener) were similar. Impaired EDHF-mediated relaxations were associated with reduced vascular expression of Cx37 and SK Ca and increased expression of eNOS. In aortic sections we found increased NADPH oxidase subunits, iNOS, COX 2 and increased vascular formation of ROS and peroxynitrites. The deleterious effect of CBDL on EDHF-mediated relaxations was significantly prevented by Blackcurrant juice through up-regulation of vascular expression of Cx37 and SK Ca and down-expression of eNOS. Blackcurrant treatment also reduced vascular oxidative stress in aorta. Finally the plasma levels of pro-inflammatory cytokines were improved (Figure 25). Taken altogether, these results indicate that Blackcurrant juice ingestion prevented the blunted mediated relaxation and the increased vascular oxidative stress in the mesenteric artery of CBDL rats suggesting the contribution of endotoxemia and inflammation in the endothelial

PART III

RESULTS

PART III

Effect of Fulvestrant, an estrogen receptor antagonist, on the cirrhotic rat lung

Oswald-Mammosser Monique, Rashid Sherzad K., Boehm Nelly, Agin Arnaud, Geny Bernard, Schini-Kerth Valérie, Charloux Anne.

Submitted for publication

In the literature, hepatopulmonary syndrome (HPS) is a well-defined cause of hypoxemia in patients who have liver disease due to abnormal intrapulmonary vascular dilatation. The pulmonary symptoms are attributed to defect in the blood oxygenation that occurs as a result of dilation of pulmonary small vessels. Previous studies have suggested that an increased estradiol level in CBDL rats might contribute to pulmonary vasodilatation via excessive formation of the potent vasodilator NO (Aller, de Luis et al. 2001;Aller, Moya et al. 2002;Yol, Erikoglu et al. 2005). It is well documented that sex hormones play role in the regulation of vascular tone. Furthermore, in cirrhotic patients an imbalance in sex hormone levels have been reported, and for example, increased levels of progesterone and estradiol have been associated to hyperventilation in cirrhotic patients (Aller, Moya et al. 2002). Thus, our hypothesis was that an estrogen receptor antagonist like fulvestrant could improve the HPS in CBDL rats. This has been studied in article 3, which is submitted for publication.

Since HPS, occurring in 10 to 20 % of cirrhotic patients waiting for liver transplantation is of prognostic value, it is important to have an efficient treatment to improve the outcome of these patients before but also after surgery. Moreover, in some cases, improving this syndrome should lead to delay transplantation.

In our study, we investigated the possibility that fulvestrant, an estrogen receptor blocker could improve the HPS. Fulvestrant was administered, at the same day of surgery, as a single s.c. bolus dose of 10 mg/rat in a volume of 0.2 ml vehicle (Faslodex AstraZeneca).

Sham rats received the same volume of castor oil. Four weeks after surgery, rats were killed.

Blood was collected for hormonal and plasma nitrite determination, arterial blood was collected from the aorta in a heparinized syringe for the determination of carboxyhemoglobin (COHb) levels and haematocrit using an ABL Cooxymeter. Serum was stored at -80°C for the subsequent determination of hormone and nitrite/nitrate levels. Liver, spleen and lung samples were obtained for histological and immunohistological studies.

Plasma nitrite and COHb levels were significantly increased in the CBDL group compared to the sham group, and these effects were not affected by fulvestrant treatment.

In the lung, the endothelial nitric oxide synthase (eNOS), and nitrotyrosine protein expressions were increased in CBDL rats as compared to sham rats, this effect was significantly reduced by fulvestrant. Furthermore, the HO-1 immunofluorescence signal was also significantly increased in lung sections of CBDL rats and this effect was prevented by fulvestrant treatment. In contrast there was an increased recruitment of macrophages in the lungs of CBDL rats compared to sham rats, an effect which was not affected by fulvestrant treatment. Macrophages in the lungs were mostly intravascular in CBDL rats. A significantly increased VEGF (the angiogenic factor involved in the development of HPS) immunostaining signal was observed in the lung sections of CBDL rats predominantly in the alveolar-capillary wall and also in the vessel lumen. These signals were not affected by fulvestrant treatment.

Moreover, the diameter of small non-muscular lung vessels in CBDL lungs was increased, and this parameter was unaffected by fulvestrant.

In conclusion, the use of fulvestrant may not be an option to improve the lung abnormalities in CBDL rats since NO may not be biologically active and since other markers implicated in pulmonary abnormalities are not affected by fulvestrant.

Effect of the estrogen receptor antagonist fulvestrant on the cirrhotic rat lung

Oswald-Mammosser Monique

Abstract

Background

It has been postulated that vasodilatation in the lung observed in cirrhosis and the subsequent hepatopulmonary syndrome, is explained in part by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In our study we assessed whether the estrogen receptor antagonist fulvestrant (F) could improve the lung abnormalities due to cirrhosis.

Methods

Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham (S), S+F. Histological, immunohistochemical and western blot analyses were performed on lung samples.

Results

The endothelial nitric oxide synthase (eNOS), and nitrotyrosine protein expressions were increased in CBDL as compared to S, this effect was significantly reduced by fulvestrant. Surprisingly the level of pVASP (an indirect marker of NO formation and action)

was decreased in CBDL, this was not affected by fulvestrant. In contrast, level of vascular endothelial growth factor, diameter of small lung vessels and number of macrophages in CBDL lungs were increased, these parameters were unaffected by fulvestrant.

Conclusions

Fulvestrant may not be an option to improve the lung abnormalities in cirrhosis since NO may not be biologically active and since key events contributing to the lung abnormalities are not affected by fulvestrant. Chronic liver diseases are characterized by a progressive vasodilatation, which is especially observed in the splanchnic and the pulmonary beds. The vasodilatation is due to portal hypertension associated to either cirrhosis or extrahepatic portal venous obstruction without cirrhosis (Abraldes et al., 2006). In a rat model of partial portal vein ligation, Abraldes et al (Abraldes et al., 2006) reported that the initial event because of portal hypertension is an up-regulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the intestinal microcirculation. The mechanisms involved in the occurrence of the subsequent generalized vasodilatation and hyperdynamic syndrome are incompletely understood, although animal models have led to a number of hypotheses (Ling et al., 2004;Nunes et al., 2001;Sztrymf et al., 2004;Zhang et al., 2003).

Keywords

Several lines of evidence support a role for an increased formation of nitric oxide ( NO) in the occurrence of the general vasodilatation in chronic liver diseases in patients and also in the chronic bile duct ligated (CBDL) rat model [START_REF] Brussino | Effect on dyspnoea and hypoxaemia of inhaled N(G)-nitro-L-arginine methyl ester in hepatopulmonary syndrome[END_REF]Chabot et al., 1996;Gomez et al., 2006;Ling et al., 2004;Nunes et al., 2001;Rolla et al., 1998;[START_REF] Schenk | Methylene blue improves the hepatopulmonary syndrome[END_REF]Zhang et al., 2003). Moreover, from clinical and animal studies it has been suggested that an increased level of estradiol may be implicated in the occurrence of the vasodilatation most likely through subsequently to an increased NO formation (Aller et al., 2001;Aller et al., 2002;Yol et al., 2005). The generalized vasodilatation leads to complications and especially to a hepatopulmonary syndrome (HPS) in approximately 10%-15% of patients awaiting liver transplantation (Rodriguez-Roisin et al., 2004). The HPS, which is characterized by arterial deoxygenation because of intrapulmonary vascular dilatations, is of prognostic value for the survival before and also after liver transplantation (Arguedas et al., 2003;Rodriguez-Roisin et al., 2004). It resolves in most cases with liver transplantation (Arguedas et al., 2003) but having an efficient approach to improve this syndrome is of great clinical importance because this could ameliorate survival and possibly also delay liver transplantation in some cases. The aim of the present study was to investigate the effect of fulvestrant, which is an estrogen receptor antagonist with no agonistic effect, in an animal model of biliary cirrhosis with portal hypertension. Indeed, fulvestrant is expected to prevent the increased estradiol-stimulated NO formation and, thus, improve the abnormalities observed in cirrhotic rat lungs.

Material & Methods

The study was performed in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). The project has been approved by the HUS/CNRS committee (collaboration between the university hospital of Strasbourg and the research department CNRS for research project).

CBDL model

Male Wistar rats (10-12 weeks old) were anesthetized with an intraperitoneal mixture of ketamine (80 mg.kg body wt -1 ) and xylazine (4 mg.kg body wt -1 ). Biliary cirrhosis was induced by CBDL. After laparotomy, the bile duct was isolated, double ligated, and resected between the 2 ligatures. Sham rats had laparotomy and underwent mobilization of the common bile duct without ligation. After surgery, rats were housed in a thermoneutral environment, on a 12:12-hour photoperiod and were provided food and water ad libitum. Rats were divided into four groups: CBDL (n=10), CBDL + F (fulvestrant) (n=10), sham (S) (n=8), S + F (n=9). Fulvestrant was administered, at the same day than surgery, as a single s.c.

bolus dose of 10 mg/rat in a volume of 0.2 ml vehicle (Faslodex AstraZeneca). Sham rats received the same volume of castor oil. Four weeks after surgery, rats were killed. Before the collection of blood for hormonal and plasma nitrite determinations, arterial blood was collected from the aorta in a heparinized syringe for the determination of carboxyhemoglobin (COHb) levels and haematocrit using an ABL Cooxymeter (ABL725, Radiometer,

Copenhagen, Denmark). Serum was stored at -80°C for the subsequent determination of hormone and nitrite/nitrate levels. The liver and the spleen were removed and weighted. Liver samples from each rat were put in Bouin-Holland fixative and embedded in paraffin for histological analysis. After excision, the left lung was snap frozen and the right lung was fixed by infusion of Bouin-Holland fixative at a pressure of 25 cm H 2 O and embedded in paraffin for immunohistochemistry.

Assessment of biliary cirrhosis and portal hypertension

Five-micrometer-thick sections of liver samples were stained with H&E and evaluated by light microscopy. Ten photographs were taken from each liver sample, and the area occupied by the biliary ducts was evaluated from each photograph using the Image-J software (National Institutes of Health, Bethesda, MD; http://rsweb.nih.gov/ij/). The final result for each rat was the mean of the 10 evaluations and was expressed in percentage of the whole area of the photographs. Portal hypertension was reflected by an increased spleen weight.

Immunohistochemical studies and small vessels diameter

Immunolocalization of macrophages in lungs was performed using the mouse anti-rat CD68 monoclonal antibody (AbD Serotec) on 5 µm paraffin sections. Eight photographs were taken from each lung sample and the area occupied by the macrophages was evaluated from each photograph using the Image-J software (National Institutes of Health, Bethesda, MD;

http://rsweb.nih.gov/ij/). On the same sections, we also evaluated the diameter of small, nonmuscular vessels (ten vessels per rat). Only vessels round in shape were retained.

Immunohistochemical staining was performed on lung paraffin 10 µm sections fixed with 4% paraformaldehyde. Fixed sections were incubated with antibodies directed against either inducible heme oxygenase (HO-1) (1/ 500) or VEGF (1/300). Sections were then incubated with the secondary antibody (Alexa 488-conjugated or Alexa 637-conjugated goat anti-rabbit IgG). The preparations were then evaluated using a confocal laser-scanning microscope (Leica SP2 UV DM IRBE) with 20× magnification lens. Quantification of protein levels was performed using Image-J software (National Institutes of Health, Bethesda, MD; http://rsweb.nih.gov/ij/).

Western blot analysis

Frozen lung tissues were homogenized in extraction buffer (composition in mM:

Tris/HCl 20 (pH 7.5; QBiogene), NaCl 150, Na 3 VO 4 1, sodium pyrophosphate 10, NaF 20, okadaic acid 0.01 (Sigma), a tablet of protease inhibitor (Roche) and 1% Triton X-100 (QBiogene)). Total proteins (25 µg) were separated on SDS-polyacrylamide (Sigma) gels and transferred electrophoretically onto polyvinylidine difluoride membranes (Amersham).

Membranes were blocked with blocking buffer containing 5% bovine serum albumin, Trisbuffered saline solution (Biorad) and 0.1% Tween 20 (Sigma) (TBS-T) for 1 hour. For detection of targeted proteins, membranes were incubated with the respective primary antibody (phosphorylated vasodilator-stimulated phosphoprotein, p-VASP, Cell Signaling Technology, 1/1,000; phosphorylated serine/threonine kinase Akt, p-Akt, Cell Signaling Technology, 1/1,000; eNOS, Cell Signaling Technology, 1/1,000; HO-1, Enzo life science, 1/1,000; estradiol receptor-alpha, ER-alpha, Cell Signalling Technology, 1/1,000; inducible NOS, iNOS, BD Transduction Laboratories 1/1,000; nitrotyrosine, Millipore, 1/1,000; VEGF, Santa Cruz Biotechnology INC, 1/2,000). Detection of either -tubulin or -actin protein was used for quantification. After incubation with a primary antibody, membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody and signals were detected using enhanced chemiluminescence (Amersham).

Nitrite determinations

Plasma nitrite levels were determined using the Griess reaction colorimetric method.

Hormonal levels measurements

Progesterone, testosterone and estradiol levels were determined using

Results

Characteristics of CBDL rats

Rats were sacrificed 4 weeks after surgery. CBDL rats showed a significant increase in liver and spleen weight and a clear bile duct proliferation (increased biliary density on the histologic analyses). These effects were not affected by fulvestrant treatment (Table I). Initial and final body weights did not differ between the four groups (Table I).

Hormone levels

Testosterone levels were significantly decreased in the CBDL group compared to the sham group and this effect was not affected by fulvestrant treatment (Table II). Estradiol levels were similar in the control and CBDL group (Table II). Although fulvestrant did not affect estradiol levels in sham it significantly increased estradiol levels in the CBDL group (Table II). Progesterone levels were similar in all groups (Table II).

Plasma parameters

Plasma nitrite and COHb levels were significantly increased in the CBDL group compared to the sham group, and these effects were not affected by fulvestrant treatment (Table III). The hematocrit level was significantly decreased in the CBDL group compared to the sham group; this was not affected by fulvestrant (Table III).

Western blot analysis

The expression level of eNOS, iNOS, nitrotyrosine and VEGF was significantly increased in lungs of the CBDL group compared to the control group (Figures 1 and2).

Fulvestrant treatment prevented the CBDL-induced upregulation of both eNOS and nitrotyrosine expression but did not affect that of iNOS and VEGF (Figures 1 and2). p-VASP, an indirect marker of NO formation and action, was significantly decreased in the CBDL group and this effect was not affected by fulvestrant treatment (Figure 1). The level of pAkt, a marker of the VEGF-induced pro-angiogenic effect, was similar in the four groups (Figure 2). The expression level of ER-was similar in the sham and the CBDL groups (Figure 2). Although fulvestrant did not affect the ER-expression level in sham, it significantly increased that in the CBDL group (Figure 2).

Immunofluorescence and histochemical analyses

There was an increased presence of macrophages in the lungs of CBDL rats compared to sham rats, an effect which was not affected by fulvestrant treatment (Table III).

Macrophages in the lungs were mostly intravascular in CBDL rats (Figure 4). A significantly increased VEGF immunostaining signal was observed in the lung sections of CBDL rats predominantly in the alveolar-capillary wall and also in the vessel lumen (Figure 3). These signals were not affected by fulvestrant treatment (Figure 3). The HO-1 immunofluorescence signal was also significantly increased in lung sections of CBDL rats and this effect was prevented by fulvestrant treatment (Figure 3).

Small blood vessel diameter

The mean diameter of small non-muscular blood vessels was 21.8±5.7 µm in sham and 38.6±9.7 µm in CBDL groups, which were not affected by fulvestrant treatment (Table III).

Discussion

Consistent with previous findings [START_REF] Brussino | Effect on dyspnoea and hypoxaemia of inhaled N(G)-nitro-L-arginine methyl ester in hepatopulmonary syndrome[END_REF]Chabot et al., 1996;Gomez et al., 2006;Ling et al., 2004;Nunes et al., 2001;Rolla et al., 1998;[START_REF] Schenk | Methylene blue improves the hepatopulmonary syndrome[END_REF]Zhang et al., 2003), the induction of CBDL in rats was associated with an upregulation of eNOS, iNOS and HO-1 protein levels in lungs. Treatment with fulvestrant prevented the CBDL-induced expression of eNOS and HO-1, and also reduced that of iNOS but this effect did not reach statistical significance. Surprisingly, p-VASP, which is an indirect marker of the NO-cyclic GMP pathway, was decreased in CBDL rats and this effect was not affected by fulvestrant. In addition, the expression level of VEGF, which has been reported to contribute to lung angiogenesis in cirrhotic rats (Zhang et al., 2009), was also increased in CBDL rats as assessed by both Western blot analysis and immunofluorescence staining, such an effect was not affected by fulvestrant. Furthermore, fulvestrant treatment did affect neither the increased diameter of small non-muscularized blood vessels nor the infiltration of macrophages into lungs of CBDL rats. Altogether, these findings do not support a major role of estrogens in the impaired lung function in cirrhosis.

Liver diseases have been associated with an imbalance of serum estradiol and testosterone levels in men [START_REF] Guechot | Effect of liver transplantation on sex-hormone disorders in male patients with alcohol-induced or post-viral hepatitis advanced liver disease[END_REF], and also as observed in the present study of the testosterone level in CBDL rats. Although estradiol levels were similar in sham and CBDL rats, the values observed in male rats were close to the detection limit of the radioimmunoassay used and, thus, have to be analyzed with caution. Of interest, fulvestrant treatment markedly increased the estradiol level in CBDL rats without affecting that in sham rats. Thus, these findings indicate that the estrogen receptor antagonist revealed an enhanced estradiol level in CBDL rats presumably subsequent to its binding to estrogen receptors thereby preventing their interaction with estradiol.

Previous studies have suggested that an increased estradiol level in CBDL rats may contribute to pulmonary vasodilatation leading ultimately to HPS via an excessive formation of the potent vasodilator NO (Aller et al., 2001;Aller et al., 2002;Yol et al., 2005). The present findings are in agreement with these previous ones and indicate that an increased plasma level of nitrite and also of eNOS, iNOS and nitrotyrosine protein levels were observed in the lungs of CBDL rats. Fulvestrant treatment markedly reduced the stimulatory effect of CBDL on the lung protein expression level of both eNOS and nitrotyrosine and also reduced slightly but not significantly that of iNOS. Thus, these findings support the concept that estradiol may contribute to some extent to the excessive vasodilatation in lungs of cirrhotic rats subsequent to the activation of estradiol receptors. Such a concept is also consistent with the fact that an increased plasma estradiol level and an increased lung ER-alpha expression level are observed in CBDL rats treated with fulvestrant.

An increased presence of macrophages especially in the intravascular field is observed in lungs of CBDL rats and is associated with an increased expression of iNOS (Ling et al., 2004;Nunes et al., 2001;Sztrymf et al., 2005;Sztrymf et al., 2004). Previous studies have indicated that 17 -estradiol increased iNOS expression in peritoneal macrophages in rats [START_REF] You | 17 beta-estradiol increases inducible nitric oxide synthase expression in macrophages[END_REF]. Since fulvestrant treatment did not affect the accumulation of macrophages and reduced only slightly the expression of iNOS in lungs of CBDL rats, it is likely that estrogens contribute only to some extent to the increased expression of iNOS. Besides estrogens, oxidative stress as indicated by the increased expression of nitrotyrosine in the lung of CBDL rats (Dal-Ros et al., 2010) is also likely to contribute to iNOS expression as suggested previously by Kang et al (Kang et al., 2002).

The present findings also indicate an increased expression in HO-1 in the lung of CBDL rats which was significantly reduced by fulvestrant. Both endothelial cells and macrophages are likely to contribute to the increased HO-1 level since they have been shown

previously to express HO-1 [START_REF] Baruscotti | Estradiol stimulates capillary formation by human endothelial progenitor cells: role of estrogen receptor-{alpha}/{beta}, heme oxygenase 1, and tyrosine kinase[END_REF]Zhang et al., 2003). [START_REF] Baruscotti | Estradiol stimulates capillary formation by human endothelial progenitor cells: role of estrogen receptor-{alpha}/{beta}, heme oxygenase 1, and tyrosine kinase[END_REF] have observed that estradiol stimulates HO-1 expression in endothelial progenitor cells. Such an effect may contribute to explain the inhibitory effect of fulvestrant on the CBDL-induced expression of HO-1 in lungs. In addition, Baruscotti et al [START_REF] Baruscotti | Estradiol stimulates capillary formation by human endothelial progenitor cells: role of estrogen receptor-{alpha}/{beta}, heme oxygenase 1, and tyrosine kinase[END_REF] further suggested that the stimulatory effect of estradiol involves an indirect mechanism via an increased expression of VEGF, which, in turn, activates Akt and HO-1. In the present study, the CBDL-induced upregulation of VEGF expression was not affected by fulvestrant indicating that estradiol has only a minor role. In addition, Carter et al (Carter et al., 2002) have observed an NO-dependent upregulation in HO-1 expression in the lungs of CBDL rats possibly via a cyclic GMP-independent mechanism as has been observed

in vascular smooth muscle cells [START_REF] Hartsfield | Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide[END_REF]. Thus, the inhibitory effect of fulvestrant on the CBDL-induced upregulation of HO-1 expression in lungs may possibly be due to a reduced NO formation.

An increased expression level of eNOS, iNOS and HO-1 was observed in lungs suggesting that these mechanisms may contribute to the occurrence of vasodilatation in the CBDL rat lung by the formation of the potent vasodilators NO and CO. Indeed, plasma nitrite and carboxyhemoglobin levels were increased in CBDL rats. However, the level of p-VASP, an indirect marker of NO formation and action, was significantly reduced in the lungs of CBDL rats. These apparently contradictory findings suggest that NO may either act via a cyclic GMP-independent pathway or be biologically inactive. In a previous study (Dal-Ros et al., 2010), we have observed that the NO-mediated component of the relaxation in the CBDL rat mesenteric artery ring is not increased in comparison to sham rats. One possible explanation is that oxidative stress may promote to the degradation of NO by superoxide anions leading to an increased nitrotyrosine expression as observed in the CBDL lung. Thus, the increased formation of NO subsequently to the stimulation of eNOS by estradiol may not be biologically active but rather lead to an increased formation of peroxynitrite, a strong prooxidant. The inhibitory effect of fulvestrant on the CBDL-induced lung peroxynitrite formation most likely reflects the reduced formation of NO. Thus, the present findings taken in conjunction with previous ones [START_REF] Chang | Sorafenib treatment improves hepatopulmonary syndrome in rats with biliary cirrhosis[END_REF]Dal-Ros et al., 2010;[START_REF] Roberts | Genetic risk factors for hepatopulmonary syndrome in patients with advanced liver disease[END_REF]Zhang et al., 2009) suggest that NO may have a minor role in the occurrence of HPS.

An increased expression of VEGF was observed in the lungs of CBDL rats and this effect was not affected by fulvestrant. Similarly, an increased expression of VEGF especially VEGF-A production by monocytes was also observed in the pulmonary vascular bed in CDBL rats (Zhang et al., 2009). These authors have highlighted the role of angiogenesis in the occurrence of the HPS in CBDL rats. Thus, vascular remodeling rather than acute vasodilatation may be the cornerstone of the occurrence of HPS. Such a concept is supported by previous findings indicating that acute inhibition of NO did not lead to increased oxygenation in patients as reported by Gomez et al (Gomez et al., 2006), and the resolution of the HPS after liver transplantation takes often several months [START_REF] Schiffer | Hepatopulmonary syndrome increases the postoperative mortality rate following liver transplantation: a prospective study in 90 patients[END_REF][START_REF] Swanson | Natural history of hepatopulmonary syndrome: Impact of liver transplantation[END_REF]. Therefore, angiogenesis is likely to be an important event contributing to the human disease. Nevertheless, the mechanism leading to angiogenesis remains unclear. An increased activation of the Akt pathway associated to an increased expression of eNOS has been suggested to be one of the mechanisms potentially leading to angiogenesis [START_REF] Dimmeler | Akt takes center stage in angiogenesis signaling[END_REF][START_REF] Papapetropoulos | Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells[END_REF]. However, the contribution of Akt in the present study is not supported by the fact that p-Akt was not increased in the lungs of CBDL rats despite an increased expression of VEGF.

HPS is related to dilated blood vessels in the lung and the subsequent hypoxia to an impaired interaction of oxygen with haemoglobin in red blood cells, which are too far from the alveolar membrane in dilated capillaries and precapillaries. In the present study, the diameter of small non-muscularized vessels was significantly increased in CBDL rats and this effect was not affected by fulvestrant. Moreover, fulvestrant treatment did not affect the infiltration of macrophages in the CBDL rat lung, which have been shown to express iNOS and VEGF and to play a critical role in the occurrence of the HPS (Thenappan et al., 2011).

The results of our study suggest that fulvestrant may not be an option to improve the HPS but there are some limitations. We chose to treat the rats with a bolus of 10 mg of fulvestrant (in castor oil) which has been shown to induce anti-oestrogenic activity for 1 month or more [START_REF] Wakeling | A potent specific pure antiestrogen with clinical potential[END_REF]. Nevertheless we can question if a greater dose of fulvestrant would have had more effects on the lungs. A second limitation is that only male rats were studied, if the results could differ in female rats should be tested in the future.

Conclusions

In conclusion, the use of fulvestrant to prevent the stimulatory effect of estrogens on NO formation may not be an interesting option to improve the HPS since NO is mostly biologically inactive and appears to play only a minor role in the occurrence of the HPS. It is also not supported by the fact that neither the expression of iNOS and VEGF nor the increased infiltration of macrophages, which are key events contributing to the lung abnormalities observed in cirrhosis, are affected by fulvestrant. Schematic presentation of the third article results:

After common bile duct ligation or sham operation, Rats were sacrificed 4 weeks after surgery. CBDL rats showed a significant increase in liver and spleen weight and a clear bile duct proliferation. These effects were not affected by fulvestrant treatment.

Hormone levels

Estradiol levels were similar in the control and CBDL group. Although fulvestrant did not affect estradiol levels in sham it significantly increased estradiol levels in the CBDL group.

Plasma nitrite and COHb levels were significantly increased in the CBDL group compared to the sham group, and these effects were not affected by fulvestrant treatment. The expression level of eNOS, iNOS, nitrotyrosine and VEGF was significantly increased in lungs of the CBDL group compared to the control group. Fulvestrant treatment prevented the CBDL-induced upregulation of both eNOS and nitrotyrosine expression but did not affect that of iNOS and VEGF. The expression level of ER-was similar in the sham and the CBDL groups. Although fulvestrant did not affect the ER-expression level in sham, it significantly increased that in the CBDL group.

There was an increased presence of macrophages in the lungs of CBDL rats compared to sham rats, an effect which was not affected by fulvestrant treatment. Macrophages in the lungs were mostly intravascular in CBDL rats. The HO-1 immunofluorescence signal was also significantly increased in lung sections of CBDL rats and this effect was prevented by fulvestrant treatment.

Finally, the mean diameter of small non-muscular blood vessels was 21.8±5.7 µm in sham and 38.6±9.7 µm in CBDL groups, which were not affected by fulvestrant treatment. A better understanding of the underlying mechanisms is mandatory for the design of new drugs to be used in clinic. Therefore, rodent models are being developed to mimic human liver disease. However, despite all efforts, no model to date can completely recapitulate the corresponding human disorders. Limiting factors are the time frame required in humans to establish a certain liver disease and the fact that rodents possess a distinct immune system compared with humans and have different metabolic rates affecting the homeostasis.

Classical animal models of experimental liver fibrosis

Liver fibrosis and its advanced form, cirrhosis, represent the common final pathway of most types of chronic liver disease. The fact that rats generally respond to fibrotic stimuli with development of a fibrotic reaction that is much more robust than that of other rodent [START_REF] Bissell | Therapy for hepatic fibrosis: revisiting the preclinical models[END_REF] makes it necessary to have a second animal models like mice taking in the advantage of the availability of genetic manipulation [START_REF] Hillebrandt | Genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus Hfib1 on chromosome 15[END_REF].

The available animal models of chemicaly induced liver fibrosis using hepatotoxins are:

Carbon tetrachloride (CCl 4 )

Carbon tetrachloride is one of the most widely used hepatic toxins for experimental induction of liver injury in laboratory animals. The limitations of using this model are: a higher mortality rate and the degree of lesions achieved can vary depending on species, strain, dose, route and frequency of administration. In addition, the pathology is reversible upon its discontinuation (Bosma et al, 1988;Charbonneau et al, 1986).

Thioacetamide

Thioacetamide is not toxic by itself to the liver, but its metabolic intermediates, in particular, thioacetamide-S-oxide. Compared to CCl 4 the fibrotic lesions are more prominent in thioacetamide-induced cirrhosis in rats (Salguero et al, 2008), and they can persist for several weeks after discontinuation of thioacetamide. The limitations of using this model are a relative long time needed to induce significant fibrosis and the increased risk of premature loss of test animals due to development of cholangiocarcinoma and hepatocellular carcinoma.

Dimethyl or diethyl nitrosamine

Dimethyl or diethyl nitrosamine is hydroxylated by CYP2E1 in liver cells yielding in the generation of diazonium ions, the bioactive intermediates. In this model, liver fibrosis and cirrhosis develop stably and progressively, even several weeks or months after withdrawal of the toxins. This model is useful for studying the progression of liver fibrosis to hepatocellular carcinoma (Jenkins et al, 1985;Schiffer et al, 2005).

Common bile duct ligation (CBDL)

Bile duct ligation is the classical experimental model for secondary biliary fibrosis.

The technique has been mentioned previously in chapter four. Rats are especially adapted to the bile duct ligation model because they lack the gall bladder. In addition, CBDL is also widely used in mice. Relatively high mortality rates due to leakage of bile or rupture of biliary cyst is a major drawback when performing CBDL. Importantly, spontaneous resolution of biliary fibrosis is observed in rats following biliojejunal anastomosis (Issa et al, 2001;Popov et al, 2010). Therefore, CBDL has also been used to study reversibility of fibrosis. Using CBDL, we can obtain most pathological consequences of chronic liver diseases seen in humans like portal hypertension and hepatopulmonary syndrome. However, the presence of different factors like the long duration of chronic liver disease progression in human and the much faster metabolic rates of rodents may rule out the possibility of directly translate the results from animal experiments to the humans.

The factors implicated in the development of chronic liver diseases in experimental

animals and humans and its consequences

Role of NO/NOS

Portal hypertension (PH) is a common clinical complication of chronic liver disease that is associated with high risk of morbidity and mortality. PH is classified as either; prehepatic, intra-hepatic or post-hepatic, with intra-hepatic being the most often form caused by cirrhosis irrespective of etiology [START_REF] Buob | Portal hypertension: pathophysiology, diagnosis, and treatment[END_REF]. Many vasoactive substances contribute to the development of PH. Among these, NO is the key mediator that paradoxically regulates the intra-hepatic and systemic/splanchnic circulation.

A hyperdynamic splanchnic circulatory state is a major characteristic of portal hypertension. The mechanisms underlying this phenomenon are not fully understood, but overproduction of endogenous vasodilators and decreased vascular reactivity to vasoconstrictors have been involved [START_REF] Rodriguez-Vilarrupla | Current concepts on the pathophysiology of portal hypertension[END_REF]. There is evidence of eNOS up-regulation and increased NO release by the superior mesenteric artery endothelium that occurs before the development of hyperdynamic splanchnic circulation (Wiest et al, 1999). Furthermore, to support that NO has a role in portal hypertension, certain studies mentioned that overproduction of NO in the splanchnic and systemic circulation contributes to this phenomenon as NOS inhibition effectively improved splanchnic hyperemia [START_REF] Martin | Nitric oxide as a mediator of hemodynamic abnormalities and sodium and water retention in cirrhosis[END_REF][START_REF] Thiesson | Nitric oxide synthase inhibition does not improve renal function in cirrhotic patients with ascites[END_REF]. In contrast, an attenuated splanchnic blood flow was observed in eNOS knock-out mice injected by CCL4. However, this was associated with an increased intra-hepatic vascular resistance, presumably due to the reduced NO formation within the liver [START_REF] Theodorakis | Role of endothelial nitric oxide synthase in the development of portal hypertension in the carbon tetrachloride-induced liver fibrosis model[END_REF]. Taken together, these results indicate an up-regulation of eNOS expression during splanchnic hyperemia associated with relative eNOS deficiency in the liver. In addition, several other studies demonstrated the importance of iNOS in the hyperdynamic circulation of cirrhosis [START_REF] Ferguson | Inducible nitric oxide synthase activity contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites[END_REF][START_REF] Kajita | iNOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats[END_REF][START_REF] Theodorakis | The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice[END_REF][START_REF] Wei | Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct-ligated rats[END_REF]. In cirrhosis, endotoxins, cytokines and bacterial translocation promote iNOS expression and overproduction of NO [START_REF] Ferguson | Inducible nitric oxide synthase activity contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites[END_REF][START_REF] Bauer | Small intestinal bacterial overgrowth in human cirrhosis is associated with systemic endotoxemia[END_REF][START_REF] Guarner | Increased serum nitrite and nitrate levels in patients with cirrhosis: relationship to endotoxemia[END_REF][START_REF] Albillos | Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement[END_REF]. Moreover, this concepts is supported by Ferguson et al, who observed that a selective iNOS inhibitor, N- [3-(aminomethyl) benzyl] acetamidine, caused peripheral vasoconstriction in patients with cirrhosis [START_REF] Ferguson | Inducible nitric oxide synthase activity contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites[END_REF]. It is of outmost importance to note that there exists also an interaction between eNOS and iNOS in the vasculature. For example, in cirrhosis, an increased expression of eNOS is observed in large arteries and this result in systemic hypotension and increased blood flow. Such effects can be blunted by activated iNOS in the small vessels of the splanchnic circulation since iNOS activation resulted in the inhibition of eNOS expression in the small vessels [START_REF] Bhimani | Hepatic and mesenteric nitric oxide synthase expression in a rat model of CCl(4)-induced cirrhosis[END_REF]. However, this was not the case in our study using the mesenteric artery we observed a parallel up-regulation of both eNOS and iNOS in CBDL. Furthermore, previous studies suggest also that (neuronal NOS) nNOS may promote vasodilation of the splanchnic circulation, though its contribution is overall less significant [START_REF] Kwon | Increased neuronal nitric oxide synthase interaction with soluble guanylate cyclase contributes to the splanchnic arterial vasodilation in portal hypertensive rats[END_REF][START_REF] Lores-Arnaiz | Hippocampal mitochondrial dysfunction with decreased mtNOS activity in prehepatic portal hypertensive rats[END_REF].

Oxidative stress

Oxidative stress is a major event occurring during biological and pathological processes. Molecular oxygen (O 2 ) is fundamental for the surviving of all aerobic organisms.

Aerobic energy metabolism relies on oxidative phosphorylation in mitochondria. However during this process, partially reduced and highly reactive O 2 metabolites, such as superoxide anions (O 2 -), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical ( • OH), can be found within the cells. These chemically reactive molecules containing oxygen are called reactive oxygen species. The generation of reactive oxygen species in a biological environment expose most living organisms to the so-called 'oxygen paradox': oxygen is necessary for life and reactive oxygen species have important roles in redox homeostasis and cell signaling but they are also potentially hazardous since reactive oxygen species may easily become a source of cell stress and tissue injury, like in most acute and chronic inflammatory processes and infections.

However, most living organisms have mechanisms and strategies to overcome the excess generation of reactive oxygen species and oxidative stress, as well as to 'make use' of reactive oxygen species under physiological conditions [START_REF] Novo | Redox mechanisms in hepatic chronic wound healing and fibrogenesis[END_REF]. The sources of reactive oxygen species can be of two types, exogenous or endogenous. The exogenous sources include atmospheric pollution, cigarette smoking, ionizing radiation, xenobiotic metabolism and alcohol. The endogenous sources of reactive oxygen species are mainly enzymatic in nature like NADPH oxidase, un-coupled eNOS, COXs, cytochromes P450, mono-oxygenases, xanthine oxidase, 5-lipo-oxygenase and the mitochondrial respiratory chain. NADPH oxidase and uncoupled eNOS are particularly important and well studied in the field of the cardiovascular system. The NADPH oxidase (Figure 27), is an important source of superoxide anions in human and animal blood vessels associated with vascular pathologies (Rajagopalan et al, 1996;[START_REF] Griendling | NAD(P)H oxidase: role in cardiovascular biology and disease[END_REF]. Furthermore, Guzik et al.

highlighted that the production of superoxide anions by NADPH oxidase is correlated with the degree of endothelial dysfunction [START_REF] Guzik | Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors[END_REF]. It is worthy to mention that in every living organism, there are endogenous cellular defense mechanisms including both small molecular weight anti-oxidants (vitamins C and E etc.) and anti-oxidant enzymes (CuZn-and Mn-SOD, glutathione peroxidase, catalase, etc.). In normal physiological conditions, there is a balance response between oxidative stress and the endogenous cellular defense mechanisms. Recent data have shown that these anti-oxidant defense mechanisms, especially catalase, can be compromised under conditions of endothelial dysfunction [START_REF] Sharma | Lung antioxidant enzymes are regulated by development and increased pulmonary blood flow[END_REF]. Thus, the effect of H 2 O 2 on eNOS can be separated into either a physiologic response (if catalase levels are maintained) or a pathologic response (when catalase levels are compromised). Thus, under physiological conditions of acute increases in oxidative stress, eNOS will be activated to produce NO, while under sustained oxidative stress like "inflammation", there can be a lack of the essential co-factor tetrahydrobiopterin (BH 4 ) due its transformation to dihydrobiopterin (BH 2 ) (by reactive oxygen species), eNOS dimerization will be compromised and finally the generation of NO signaling will be attenuated [START_REF] Rafikov | eNOS activation and NO function: structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity[END_REF]. In this pathologic case, the uncoupled eNOS subsequently generates superoxide anions rather than NO and, thus, increases the formation of peroxynitrite, which reduces the bioavailability of NO [START_REF] Kawashima | Dysfunction of endothelial nitric oxide synthase and atherosclerosis[END_REF]). In our model of CBDL, we have shown the implication of reactive oxygen species in the development of the pathology associated with bile duct ligation manifested as augmentation of oxidative stress formation detected by the dihydroethidium redox-sensitive probe in mesenteric and aortic sections. In addition, there is an upregulation of both subunits of NADPH oxidase system the cytosolic p47phox, and membranous p22phox, further suggesting the involvement of NADPH oxidase system in our model. Furthermore, in both studies of VSL#3 and polyphenol-rich blackcurrant juice (PRBJ), we have shown that there are beneficial effects in reducing oxidative stress, peroxynitrite and preventing the upregulation of NADPH oxidase subunits.

Role of renin angiotensin aldosterone system RAAS

It is well recognized that increased activation of the RAAS is being as important event leading to endothelial dysfunction, arterial stiffness and cardiovascular diseases (Yki-Jarvinen and Westerbacka 2007; [START_REF] Milan | Arterial stiffness: from physiology to clinical implications[END_REF][START_REF] Targosz-Korecka | Stiffness memory of EA.hy926 endothelial cells in response to chronic hyperglycemia[END_REF][START_REF] Underwood | The renin angiotensin aldosterone system and insulin resistance in humans[END_REF]. Furthermore, The RAS (renin-angiotensin system) is now recognized as an important regulator of liver fibrosis and portal pressure in cirrhosis [START_REF] Grace | Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options[END_REF].

Hepatic injury (ischemia or inflammation) induces the hepatic expression of components of the RAS, such as ACE and the AT1 receptors, which play an active role in promoting inflammation and deposition of extracellular matrix. In addition, the more recently recognized structural homologue of ACE, ACE2, is also up-regulated [START_REF] Grace | Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options[END_REF]). ACE2 catalyses the conversion of Ang II into Ang (1-7), and there is accumulating evidence that this 'alternative axis' of the RAS has anti-fibrotic, vasodilatory and anti-proliferative effects, thus counter-balancing the effects of Ang II in the liver (Figure 28). Although the intra-hepatic circulation in cirrhosis is hypercontractile in response to Ang II, resulting in increased hepatic resistance; the splanchnic vasculature is hyporesponsive, promoting the development of the hyperdynamic circulation that, characterizes portal hypertension [START_REF] Grace | Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options[END_REF]. The first approach in the management of portal hypertension is to reduce intra hepatic vascular tone induced by the activity of powerful vasocontrictors such as Ang II, endothelin-1 and the sympathetic nervous system, and mediated via contraction of peri-sinusoidal myofibroblasts and perivascular smooth muscle cells. The second approach is to reduce mesenteric and portal blood flow. In addition to the conventional circulating RAAS, the presence of RAAS components have been detected in tissues such as heart, kidney, vasculture, adipose tissue, immune cells, and the brain [START_REF] Hayden | Possible Mechanisms of Local Tissue Renin-Angiotensin System Activation in the Cardiorenal Metabolic Syndrome and Type 2 Diabetes Mellitus[END_REF][START_REF] Nguyen Dinh | A new look at the renin-angiotensin system-focusing on the vascular system[END_REF][START_REF] Kumar | The intracrine renin-angiotensin system[END_REF][START_REF] Underwood | The renin angiotensin aldosterone system and insulin resistance in humans[END_REF][START_REF] Bader | Tissue renin-angiotensin-aldosterone systems: Targets for pharmacological therapy[END_REF]. Recent studies have shown that vascular smooth muscle cells, VSMCs, synthesize angiotensin II intracellularly. In vascular smooth muscle cells, the intracellular Ang II regulates the expression of angiotensinogen and renin, generating a feedback loop. The first reaction of intracellular Ang II synthesis (conversion of angiotensinogen to Ang I) is catalysed by renin and/or cathepsin D, depending on the cell type, and chymase, not ACE, catalyzes the second step (conversion of Ang I to other forms) [START_REF] Kumar | The intracrine renin-angiotensin system[END_REF][START_REF] Bader | Tissue renin-angiotensin-aldosterone systems: Targets for pharmacological therapy[END_REF]. In addition, to the classical Ang II system, the role of nonclassical angiotensin peptides generated by tissue ACE2 comprising Ang (1)(2)(3)(4)(5)(6)(7)(8)(9) and Ang (1-7), which generally antagonize the actions of Ang II are increasingly recognized for their biological activity [START_REF] Nguyen Dinh | A new look at the renin-angiotensin system-focusing on the vascular system[END_REF][START_REF] Hayden | Possible Mechanisms of Local Tissue Renin-Angiotensin System Activation in the Cardiorenal Metabolic Syndrome and Type 2 Diabetes Mellitus[END_REF][START_REF] Bader | Tissue renin-angiotensin-aldosterone systems: Targets for pharmacological therapy[END_REF][START_REF] Kumar | The intracrine renin-angiotensin system[END_REF]. Ang (1)(2)(3)(4)(5)(6)(7) is also converted to Ang (1)(2)(3)(4)(5) by ACE. Ang III, Ang IV, Ang (3)(4)(5)(6)(7) are other peptides formed from Ang II [START_REF] Kumar | The intracrine renin-angiotensin system[END_REF][START_REF] Bader | Tissue renin-angiotensin-aldosterone systems: Targets for pharmacological therapy[END_REF]. The role of these peptides in vascular tissue is not well understood. The role of cross talk between Ang II and aldosterone signaling is increasingly recognized in the development of endothelial dysfunction and arterial stiffness [START_REF] Bender | Mineralocorticoid receptor-mediated vascular insulin resistance: an early contributor to diabetes-related vascular disease?[END_REF][START_REF] Whaley-Connell | Aldosterone: role in the cardiometabolic syndrome and resistant hypertension[END_REF][START_REF] Rautureau | Cross-talk between aldosterone and angiotensin signaling in vascular smooth muscle cells[END_REF][START_REF] Mcgraw | Mineralocorticoid receptors in vascular disease: connecting molecular pathways to clinical implications[END_REF]. Hence, aldosterone activates NADPH oxidase, thereby increasing oxidative stress and decreasing NO bioavailability as one of the mechanism implicated in endothelial dysfunction [START_REF] Aroor | Insulin resistance and heart failure: molecular mechanisms[END_REF][START_REF] Whaley-Connell | Aldosterone: role in the cardiometabolic syndrome and resistant hypertension[END_REF][START_REF] Hwang | Mineralocorticoid receptors modulate vascular endothelial function in human obesity[END_REF].

Role of gut microbiota in liver diseases

The liver is well known as a detoxification center in almost all mammalians gastrointestinal systems. Furthermore, in normal conditions the liver encounters food-derived antigens and bacterial components such as lipopolysaccharide translocated from the gastrointestinal tract into the portal circulation. However, the liver has the unique capacity to bacteria such as E.coli as well as by increasing intestinal stability (i.e. reduced intestinal permeability), and reduced hepatic inflammation [START_REF] Osman | Endotoxin-and D-galactosamine-induced liver injury improved by the administration of Lactobacillus, Bifidobacterium and blueberry[END_REF][START_REF] Davis | Extravasation of doxorubicin from vascular access devices[END_REF][START_REF] Kirpich | Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study[END_REF]. These data support our hypothesis that the beneficial effects of the VSL#3 treatment on endothelial dysfunction in rats with portal hypertension is related to a decreased bacterial translocation.

VSL#3 effect on local angiotensin system and portal hypertension

In our study, the findings indicate that ingestion of the probiotic VSL#3 formulation effectively prevented the CBDL-induced endothelial dysfunction, at least in part, by targeting the vascular angiotensin system. The results showed an increased expression of Ang II, ACE and AT1R in the aortic sections of CBDL rats associated with endothelial dysfunction. While, treating the CBDL rats with VSL#3 was able to prevent the activation of vascular angiotensin system. These results are consistent with the fact that losartan, an AT1 receptor antagonist, NADPH oxidase activation and the activation of the local angiotensin system and vis versa (Rajagopalan et al, 1996). Our findings suggest that VSL#3 treatment prevents bacterial translocation since the plasma concentrations of pro-inflammatory cytokines were reduced in CBDL rats treated with VSL#3, and that this effect was associated with a decreased oxidative stress and down-regulation of NADPH oxidase, and, ultimately, a normalized expression level of the local angiotensin system. Furthermore, some few other studies suggested the production of small peptides from probiotics, which have inhibitory effect on angiotensin converting enzyme. This could be additional mechanism for VSL#3 in our work.

In the recent study done by Gupta et al, treating patients with compensated liver cirrhosis with VSL#3 in adjuvant with propranolol was non-inferior to antibiotics plus propranolol [START_REF] Gupta | Effects of the adjunctive probiotic VSL#3 on portal haemodynamics in patients with cirrhosis and large varices: a randomized trial[END_REF]. They observed that VSL#3 plus propranolol and antibiotics plus propranolol reduced both the hepatic venous pressure gradient and plasma TNF-level in comparison to propranolol alone [START_REF] Gupta | Effects of the adjunctive probiotic VSL#3 on portal haemodynamics in patients with cirrhosis and large varices: a randomized trial[END_REF]. In addition, during the course of the two month treatment, VSL#3 treatment was well tolerated by the treated patients. More recent clinical studies using VSL#3 probiotics showed a beneficial effect in patients with irritable bowel syndrome and childhood constipation (Sisson et al, 2014;Yoon et al, 2014). However, no data are available regarding the safety of using VSL#3 in severely immune-compromised patients. The possibility of VSL#3 probiotic, are translocating in immune-deficient patients needs to be addressed.

Uses of probiotics in clinical studies

In a double blind study probiotics vs. placebo, evaluating the effect of probiotics in improving the symptoms of irritable bowel syndrome, they observed that probiotics improved the symptoms such as abdominal colic, changes in bowel motions and dyspepsia (Sisson et al, 2014). They concluded that probiotics offers a potential new treatment for irritable bowel syndrome (Sisson et al, 2014). In a study evaluating the effect of probiotics on childhood constipation (a randomized controlled double blind clinical trial done using probiotics vs.

Placebo), it was observed that probiotics have a positive role in increasing the frequency and improving the consistency of bowel motion at the end of the 4 th week of treatment (Yoon et al, 2014). In all these clinical studies, probiotics are well tolerated and no notable side effects were noticed, confirming the safety of using probiotics in humans. Finally, we can conclude that VSL#3 gives promising therapy for use in various inflammatory diseases in humans.

The protective effects of polyphenol-rich blackcurrant juice (PRBJ) in CBDLinduced endothelial dysfunction

In our work of polyphenol-rich blackcurrant juice (PRBJ) on CBDL rats, we have observed that chronic ingestion of PRBJ prevents endothelial dysfunction. This effect involving an improvement of EDH-mediated relaxation in the mesenteric artery associated with an improvement of factors implicated in the EDH-mediated pathway, the endothelial gap junction protein the connexin Cx37 and the expression of calcium-activated potassium channels, SK Ca and the oxidative stress. These further support an important role of polyphenols in preventing cardiovascular diseases, inflammatory diseases and cancer.

Polyphenols are well known not only to have antioxidant properties due to their structure but also to have the ability to modulate the activity of different kinds of enzymes and cell receptors. The fact that polyphenols are abundant in our diet makes it necessary to determine the nature and distribution of different kinds of polyphenols, so that we can identify which sources and key compounds are likely to provide an optimal protection especially in the cardiovascular system.

PRBJ and the CBDL-induced vascular oxidative stress

There is a lot of evidence supporting the anti-oxidant effect of polyphenols in vascular cells and, in particular, in smooth muscle cells. The anti-oxidant effect of polyphenols involves predominantly their ability to inhibit the expression and the activity of pro-oxidant enzymes such as NADPH oxidase and xanthine oxidase, and to increase that of anti-oxidant enzymes such as catalase [START_REF] Lin | Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)epigallocatechin-3-gallate, and propyl gallate[END_REF][START_REF] Ying | Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin IIinduced hyperpermeability in endothelial cells[END_REF]. Our observations in the PRBJ study, provides informations regarding the effect of polyphenols in the prevention of the vascular upregulation of NADPH oxidase subunits p22phox and p47phox and of eNOS expression in an animal model of portal hypertension. Furthermore, this effect is associated with the reduction of vascular reactive oxygen species formation and ultimately, improvement of endothelial function. These observations are comparable with those of Sarr et al., (2006) who studies an animal model of hypertension induced by Ang II, where the chronic oral ingestion of red wine polyphenols prevented the development of hypertension and the associated endothelial dysfunction via reduction of vascular oxidative stress. The other interesting findings of the PRBJ study, is the anti-inflammatory properties as indicated by the fact that PRBJ in reduced the plasma concentration of pro-inflammatory cytokines including IL-1, TNF-and MCP-1. It is known that the CBDL model is an aggressive inflammatory condition associated with systemic inflammatory response. The ability of PRBJ to reduce CBDL-induced inflammatory marker, supports their evaluation in human with other diseases.

Our observations are comparable with those of Ergün et al [START_REF] Ergun | Enteral resveratrol supplementation attenuates intestinal epithelial inducible nitric oxide synthase activity and mucosal damage in experimental necrotizing enterocolitis[END_REF] who highlighted the role of an increased iNOS in experimental necrotizing enterocolitis and the benefit of resveratrol treatment on inflammation and mucosal integrity. Some other studies observed the ability of polyphenols to reduce bacterial translocation in rat models. A decreased in bacterial translocation has been observed in mesenteric lymph nodes, liver and spleen with resveratrol in rats submitted to an intestinal ischemia/reperfusion injury [START_REF] Ozkan | Resveratrol, a natural antioxidant, attenuates intestinal ischemia/reperfusion injury in rats[END_REF]. All together, these support the need to determine the active polyphenolic molecules and the characterization of the underlying molecular mechanisms.

Other biological effects of Blackcurrant

In addition to the antioxidant effect of the blackcurrant juice that we have observed in our 2 nd study, other studies have observed anti-inflammatory and immune-modulatory effects of blackcurrant. It is known that in living tissues, the inflammatory response involves a cascade of eicosanoids, signalling molecules made from oxidation of essential fatty acids.

One of the precursors of essential fatty acids is -linolenic acid which is abundant in blackcurrant seed oil. Moreover, eicosanoids derived from -linolenic acid, exerts antiinflammatory responses (Barre, 2001). Furthermore, studies involving an administration of a blackcurrant extract on a human monocytic cell line derived from an acute monocytic leukemia patients, revealed a suppressive effect against TNF-and its downstream molecules, especially NF-kB and IL-6 in response to lipopolysaccharide (Lyall et al, 2009).

The PRBJ and bioavailability of polyphenols

Although it is important to know the biological function of natural polyphenols and the underlying mechanism, the bioavailability of these compounds is also of importance.

However, previous studies have indicated that anthocyanins and procyanidins have a low absorption and bioavailability [START_REF] Tsang | The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats[END_REF][START_REF] Basu | Berries: emerging impact on cardiovascular health[END_REF][START_REF] Shoji | Apple procyanidin oligomers absorption in rats after oral administration: analysis of procyanidins in plasma using the porter method and high-performance liquid chromatography/tandem mass spectrometry[END_REF]. Despite their relatively poor apparent bioavailability, numerous studies have reported that intake of polyphenols-rich products has a beneficial effect on the endothelial function in experimental animals and humans [START_REF] Schini-Kerth | Nutritional improvement of the endothelial control of vascular tone by polyphenols: role of NO and EDHF[END_REF]. In some feeding studies with human subjects and animal models, typically approximately <0.1% of the quantity ingested, has been detected in urine within 24 h of consumption of catechins from ready-to-drink tea [START_REF] Del Rio | Berry flavonoids and phenolics: bioavailability and evidence of protective effects[END_REF]. However, the structure of the anthocyanin aglycon and the attached glucoside moiety seem to have a strong effect on their absorption, metabolism and excretion [START_REF] Manach | Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies[END_REF]. Despite, the fact that most of the polyphenols undergo hydrolysis by intestinal enzymes or the colonic microflora before absorption [START_REF] Day | Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity[END_REF][START_REF] Nemeth | Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans[END_REF] intact glycosides are the major circulating forms of anthocyanins [START_REF] Manach | Polyphenols: food sources and bioavailability[END_REF] possibly due to the instability of the aglycone form, anthocyanidins, and to the existence of a specific mechanism of absorption or metabolism. Furthermore, some recent studies have shown that, in rat and mice, anthocyanins are absorbed from the stomach [START_REF] Talavera | Anthocyanins are efficiently absorbed from the stomach in anesthetized rats[END_REF][START_REF] Passamonti | Fast access of some grape pigments to the brain[END_REF][START_REF] Matuschek | The jejunum is the main site of absorption for anthocyanins in mice[END_REF]. Other studies reported that, cyanidinbased anthocyanins can undergo cleavage of the sugar moiety followed by ring fission of the released cyanidin, which produces phenolic acids such as 3,4-dihydroxybenzoic acid (protocatechuic acid) by the microflora in the colon [START_REF] Aura | In vitro metabolism of anthocyanins by human gut microflora[END_REF][START_REF] Vitaglione | Protocatechuic acid is the major human metabolite of cyanidin-glucosides[END_REF].

The possibility that these phenolic acids are absorbed and contribute to different biological function (anti-oxidant and anti-inflammatory) remains to be determined. Moreover, some authors have shown that metabolites of polyphenols affect growth of certain intestinal bacteria colonies. Hui Cheng et al observed in their study, that different strains of intestinal bacteria had varying degrees of growth inhibition to tea phenolic and metabolites. Growth of certain pathogenic bacteria such as Clostridium perfringens, Clostridium difficile and Bacteriodes spp was significantly suppressed by tea phenolics and their derivatives, while commensal anaerobes like Bifidobacterium spp. and probiotics such as Lactobacillus sp. were less severely affected (Lee et al, 2006).

Does the estrogen-mediated up-regulation of pulmonary eNOS is the sole events in hepatopulmonary syndrome

Estrogens have a regulatory effect on eNOS activity through genomic (transcriptional) and non-genomic (non-transcriptional) pathway. Experimental work in human endothelial cells showed that, estrogens enhance eNOS protein and mRNA abundances in the absence of changes in the stability of eNOS mRNA [START_REF] Chambliss | Estrogen modulation of endothelial nitric oxide synthase[END_REF][START_REF] Morales | Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model[END_REF]. In a model of vascular injury, estrogens affect vascular injury by promoting angiogenesis [START_REF] Morales | Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model[END_REF]. Estradiol-enhanced endothelial cell activation is important in neo-vascularization and suggests a promoting influence of estrogens on angiogenesis [START_REF] Morales | Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model[END_REF]. It is well known from previous studies that pulmonary dilatation in cirrhotic patients or animals is associated at least in part by an upregulation of pulmonary eNOS (Nunes et al, 2001;Gomez et al, 2006). Moreover, several clinical and animal studies have suggested that an increased level of estradiol may be implicated in the occurrence of the vasodilatation most likely subsequently to an increased NO formation (Aller et al, 2001;Aller et al, 2002;Yol et al, 2005). The generalized vasodilatation leads to complications and especially to a hepatopulmonary syndrome (HPS) in approximately 10%-15% of patients awaiting liver transplantation (Rodriguez-Roisin et al, 2004). The HPS, which is characterized by arterial deoxygenation because of intrapulmonary vascular dilatations, is of prognostic value for the survival before and also after liver transplantation (Rodriguez-Roisin et al, 2004;Arguedas et al, 2003). Estrogens have been shown to contribute positively in the evolution of different pathologies like pulmonary hypertension and trauma hemorrhage-induced lung injury [START_REF] Resta | Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression[END_REF][START_REF] Kan | Estrogen ameliorates trauma-hemorrhage-induced lung injury via endothelial nitric oxide synthase-dependent activation of protein kinase G[END_REF] and certain developmental processes [START_REF] Macritchie | Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium[END_REF]. In the fulvestrant study, we have observed an attenuation of CBDL-induced pulmonary eNOS expression by fulvestrant as assessed using Western blot and immunofluorescence. Unfortunately, the lung pathology persisted as indicated by pulmonary capillary dilatation. These observations indicate that estrogens do not have a fundamental role and that other marker and factors contribute to the development of hepatopulmonary syndrome.

Conclusions and perspective

It is well known from previous works that probiotics are tolerable and have low Our observations on polyphenols in general and on Blackcurrant juice, provide evidences suggesting that natural products might be of interest for improving liver diseases. In accordance with that, there are numerous reports that support an important role of polyphenols in preventing cardiovascular diseases, inflammatory diseases and cancer. In addition to the antioxidant properties of polyphenols, their ability to modulate the activity of different kinds of enzymes and cell receptors are also of interest. The fact that polyphenols are abundant in our diet makes it necessary to determine the nature and distribution of different kinds of polyphenols, so that we can identify which sources and key compounds are likely to provide the greatest protection for endothelial cells. Further works need to detect the exact active molecule of polyphenols and their metabolites plus more details about their bioavailability, before we can proceed to introduce them in human research.

Our results on fulvestrant, suggest that fulvestrant may not be an option to improve the hepatopulmonary syndrome. However, there are some limitations. We chose to treat the rats with a bolus of 10 mg of fulvestrant (in castor oil) which has been shown to induce antioestrogenic activity for 1 month or more. Nevertheless we can question if a greater dose of fulvestrant would have had more effects on the lungs. A second limitation is that only male rats were studied, if the results could differ in female rats should be tested in the future.

Overall further work needs to dentify the pathophysiological process of portal hypertension and its complications like hepatopulmonary syndrome. Furthermore, it will be interesting to test both VSL#3 and PRBJ to study other organs affected by portal hypertension like lung and kidneys. Our studies support that both VSL#3 and polyphenols are of potential interest for therapeutic use in liver diseases.

AAFIGURESFigure 1 :

 1 FIGURES

Figure 2 :

 2 Figure 2: NO generation from L-Arginine and its functional properties.

Figure 3 :

 3 Figure 3: NO synthesis pathway through eNOS phosphorylation in calcium dependent and independent pathway.

Figure 4 :

 4 Figure 4: Hypothesis describing the nature of EDHF pathway.

Figure 5 :

 5 Figure 5: Arachidonic acid metabolism pathway.

Figure 6 :

 6 Figure 6: The pathways of endothelial-derived relaxation.

Figure 7 :

 7 Figure 7: The renin angiotensin system.

Figure 8 :

 8 Figure 8: Pathways of endothelial derived contraction.

Figure 9 :

 9 Figure 9: Overview of how portal hypertension developed.

Figure 10 :

 10 Figure 10: Overview of factors contributing to PH initiation and it is worsening.

Figure 11 :

 11 Figure 11: Production of CO by the enzymatic activity of Heme-oxygenase.

Figure 12 :

 12 Figure 12: Potential mechanisms and target therapeutic options in hepatopulmonary syndrome (HPS) and HCS.

Figure 13 :

 13 Figure 13: Role of angiogenesis in the pathology of HPS.

Figure 14 :

 14 Figure 14: Strauctures of some polyphenols.

Figure 15 :

 15 Figure 15: Schematic of pharmacokinetically relevant flavonol features pertaining to the composition in foods, abdorption, metabolism, distribution and excretion.

Figure 16 :

 16 Figure 16: Various photographs of blackcurrants (Ribes nigrum) showing a commercial blackcurrant plantation (A), shrub with fruits (B and C), and isolated fruits (D).

Figure 17 :

 17 Figure 17: Chemical structures of the major anthocyans present in blackcurrants.

Figure 18 :

 18 Figure 18: Chemical structures of the major phytoconstituents present in blackcurrants.

Figure 19 :Figure 20 :

 1920 Figure 19: Histological study of liver section using heamatoxylin and eosin staining, sample taken at the end of study; A, sham; B, CBDL Figure 20: CBDL operation; consist of identification of the common bile duct and then put double ligature and cut between the ligature with scissor.

Figure 21 :

 21 Figure 21: Study of the vascular reactivity.

Figure 22 :

 22 Figure 22: Bacterial translocation and the hyperdynamic circulatory state in cirrhosis.

Figure 23 :

 23 Figure 23: Beneficial effects of commensal and probiotic bacteria.

Figure 24 :

 24 Figure 24: Schema presenting the effects of CBDL in rats and the therapeutic preventive effects of VSL#3 in the mesenteric arteries and both liver and spleen size.

Figure 25 :Figure 26 :

 2526 Figure 25: Schema presenting the effects of CBDL in rats and the therapeutic preventive effects of PRBJ in the mesenteric arteries and /or aorta and both liver and spleen size

Figure 27 :

 27 Figure 27: Structure of NADPH oxidase.

Figure 28 :

 28 Figure 28: Schematic representation of different biological events depending on the pathway that angiotensinogen pass through.

Figure 29 :

 29 Figure 29: Role of TLR in liver pathology. Treg: T regulatory cell.

2 (

 2 triade : défaut d'oxygénation artériel dû à des dilatations vasculaires pulmonaires dans un contexte de maladie hépatique. Il est important d'en faire le diagnostic car c'est un facteur pronostique indépendant de survie qui conditionne aussi la mortalité post-opératoire au cours de la greffe hépatique. Pour le moment seule la transplantation hépatique permet une régression ou une disparition du SHP mais un traitement médical efficace est nécessaire pour deux raisons. D'une part étant donné qu'il n'existe pas de corrélation étroite entre la survenue d'une hypoxémie et la sévérité de l'atteinte hépatique, il y a des cas où l'hypoxémie profonde nécessite la transplantation hépatique alors que d'un point de vue hépatique elle n'est pas encore justifiée. Améliorer l'hypoxémie pourrait donc surseoir à la greffe. D'autre part la survenue d'un SHP sévère entraîne un risque opératoire important et un traitement médical atténuant la complication pulmonaire devrait pouvoir faciliter la prise en charge pré-et postopératoire de ces patients. Les mécanismes conduisant à la vasodilatation due à l'hypertension portale ne sont pas complètement élucidés. La molécule qui a été incriminé le plus souvent est le NO. C'est un vasodilatateur bien connu et dont on a montré la surproduction dans un modèle de rat de cirrhose biliaire accompagnée d'un SHP. En expérimentation animale un traitement chronique par le N G -nitro-L-arginine methyl ester (L-NAME), inhibiteur non spécifique des NO synthases atténuait ou empêchait la survenue du SHP. Néanmoins des essais cliniques de traitement par inhalation de L-NAME donnent des résultats contradictoires. Ceci peut s'expliquer par le fait que d'autres facteurs jouent un rôle. Il en est ainsi du système-rénineangiotensine-aldostérone probablement par le biais d'un stress oxydant. Ce dernier a aussi été lié à la translocation bactérienne qui est présente chez une proportion importante de patients présentant une cirrhose évoluée. Enfin d'autres facteurs ont été incriminés : le monoxide de carbone qui est un vasodilatateur produit par la hème-oxygénase et il a aussi été suggéré que les estrogènes (taux élevés chez certains patients) pouvaient entraîner une dilatation vasculaire pulmonaire. Le but des différents travaux était donc d'estimer l'action de probiotiques agissant sur la translocation bactérienne, de polyphénols agissant sur la composante stress oxydant et d'un antagoniste des récepteurs de l'estrogène dans un modèle de rat ayant une cirrhose biliaire. L'analyse des effets a été essentiellement réalisée dans la circulation splanchnique et dans une moindre mesure (étude fulvestrant) au niveau pulmonaire. Premier chapitre Un rappel général sur les cellules endothéliales et notamment leur action sur le tonus vasculaire par le biais des différents facteurs vasoconstricteurs et vasodilatateurs produits par l'endothélium est fait dans un premier chapitre. Ainsi ont été revus comme vasodilatateurs le monoxyde d'azote (NO), le facteur hyperpolarisant dérivé de l'endothelium (endotheliumderived-hyperpolarization-factor, EDHF), et la prostacycline (PGI2). Parmi les facteurs vasoconstricteurs ont été présentés : le thromboxane A 2 (TXA 2 ), et la prostaglandine H PGH 2 ), les espèces réactives de l'oxygène (ROS), l'endotheline 1 (ET1) and l'angiotensine II. Deuxième chapitre Le deuxième chapitre est dédié à la dysfonction endothéliale dans l'hypertension portale. D'après des études antérieures réalisées sur un modèle de ligature partielle de la veine porte chez le rat, les premières anomalies survenant au niveau de la microcirculation intestinale est une augmentation de l'expression du facteur de croissance vasculaire (vascular-endothelialgrowth factor, VEGF) et de la NO synthase endothéliale (eNOS). Survient par la suite une vasodilatation généralisée avec syndrome hyperdynamique imputable à des mécanismes encore non complètement élucidés. Plusieurs facteurs sont très probablement impliqués : NO, le monoxyde de carbone (CO, possiblement en relation étroite avec le système NO), VEGF et peut-être la prostacycline (peu étudiée à vrai dire). Il est aussi insisté dans ce chapitre sur le rôle très probable de la translocation bactérienne (BT) par le biais de cytokines et des ROS en partie étroitement liés à l'activation du système rénine-angiotensine (SRA). Enfin, ces dernières années a été souligné le rôle très probable d'une angiogénèse anormale qui survient tant au niveau de la circulation splanchnique qu'au niveau pulmonaire. Troisième chapitre Les résultats observés au cours d'études antérieures justifient les choix faits en termes d'expérimentations réalisées pour cette thèse : action sur la translocation bactérienne par l'utilisation de probiotiques, action sur le stress oxydant en évaluant un traitement par polyphénols. La troisième étude a permis d'estimer la possibilité d'agir sur la composante NO par le biais de l'administration de fulvestrant. Ce dernier est un antagoniste des récepteurs de l'estrogène dont le taux est souvent augmenté dans la cirrhose et qui stimule l'expression de eNOS. Le modèle de cirrhose biliaire a été bien validé et consiste à ligaturer la voie biliaire du rat sous anesthésie générale. Ceci induit une cirrhose biliaire avec hypertension portale, un état circulatoire hyperkinétique et un syndrome hépato-pulmonaire. Les rats ont été étudiés 4 semaines après l'intervention. La figure ci-dessous montre à gauche un foie chez un rat normal et à droite le foie d'un rat présentant une cirrhose biliaire. Dans ce dernier cas il existe une nette prolifération des canaux biliaires. Foie normal Cirhhose biliaire Dans les études concernant l'action des probiotiques et polyphénols ont été essentiellement étudiés les effets au niveau de l'artère mésentérique et de l'aorte : réactivité vasculaire dans des cuves à organe isolé en présence d'indométacine pour prévenir la formation de prostanoïdes vasoactifs. La composante NO de la relaxation est étudiée en présence de charybdotoxine et d'apamine (inhibiteurs de la réponse EDHF-dépendante). La composante EDHF (endothelial derived hyperpolarizing factor) est étudiée en présence de N G -nitro-L-arginine, inhibiteur de la réponse NOdépendante. formation d'espèces réactives de l'oxygène (ROS) déterminée à l'aide d'une sonde fluorescente, la dihydroéthidine et détermination des différentes sources de ROS quantifications de l'expression de différents composants de la réactivité vasculaire et du système rénine-angiotensine-aldostérone (SRAA) par immunofluorescence et en western blot dosages plasmatiques de cytokines inflammatoires Quant à l'action du fulvestrant (antagoniste des récepteurs de l'estrogène) il a été étudié au niveau du poumon : étude des macrophages intravasculaires pulmonaires et des petits vaisseaux (immunohistochimie sur des coupes du poumon) western blot sur tissu pulmonaire pour la quantification de l'expression de eNOS, iNOS, nitrotyrosine, VEGF et des marqueurs de l'action de NO (p-VASP) et de VEGF (p-Akt), récepteurs-des estrogènes dosages plasmatiques de nitrites, HbCO et du taux d'hormones. Etude de la réactivité vasculaire La première étude a montré qu'un traitement par le probiotique VSL#3 améliorait la composante EDHF-dépendante de la relaxation au niveau mésentérique. Ceci était accompagné de la diminution de production des ROS et des expressions facteurs du SRA ainsi qu'une diminution des cytokines pro-inflammatoires dans le plasma. Le stress oxydant dû à la translocation bactérienne et conduisant à la production de cytokines proinflammatoires et à une stimulation du SRA local est probablement un facteur important dans la survenue de la dysfonction endothéliale chez le rat cirrhotique. L'administration de probiotiques a amélioré ces anomalies. Le probiotique VSL#3 améliore la fonction EDHF-dépendante de la relaxation de l'artère mésentérique chez le rat cirrhotique ; A) composante NO en présence d'indométacine (10 µM) et d'apamine plus charybdotoxine (100 nM chacun), B) composante EDH en présence d'indométacine et de N G -nitro-L-arginine (300 µM) C) réponse au nitroprussiate de sodium (donneur exogène de NO), D) réponse au levcromakalim (ouvreur de canaux potassiques ) ; CBDL : rats cirrhotiques ; *P<0.05 CBDL vs sham, et # P<0.05 CBDL+VSL#3 vs CBDL. Immmuno-fluorescence au niveau de l'artère mésentérique : VSL#3 diminue l'expression de eNOS et augmente celle des canaux potassiques SK Ca et IK Ca (A) et des connexines (B) impliquées dans la réponse EDHF-dépendante. Panneau du haut : exemple d'immunofluorescence, panneau du bas : données cumulées pour 4-5 rats ; *P<0.05 CBDL vs sham, et # P<0.05 CBDL+VSL#3 vs CBDL. Le traitement par VSL#3 diminue le stress oxydant et l'expression de eNOS dans la paroi de l'aorte des rats cirrhotiques (A) ; rôle d'anions superoxydes intracellulaires et du superoxyde d'hydrogène, anneaux aortiques exposés au MnTMPyP (membrane permeant superoxide dismutase mimetic), à la superoxyde dismutase (SOD), PEG-catalase (membrane permeant catalase) et à la catalase. (B) ; sources cellulaires des ROS, anneaux aortiques exposés à l'antioxydant et inhibiteur de la NADPH oxydase apocynine, au L-NA inhibiteur de la NOS, au sulfaphenazol inhibiteur du cytochrome P450, à l'indométacine inhibiteur de la cyclooxygénase et à la combinaison KCN, myxothiazol et roténone (KCN+MY+Rot), inhibitrice de la respiration mitochondriale, 30 minutes avant le marquage DHE (C) ; A) *P<0.05 CBDL vs sham, et # P<0.05 CBDL+VSL#3 vs CBDL; B et C) *P<0.05 pour CBDL avec et sans inhibiteurs. Le traitement par VSL#3 prévient la surexpression des sous-unités p22phox et p47phox de la NADPH oxydase et de COX-2 mais pas de COX-1 dans la paroi aortique. Panneau de gauche : exemple d'immunofluorescence, panneau de droite : données cumulées pour 4-5 rats ; *P<0.05 CBDL vs sham, et # P<0.05 CBDL+VSL# 3 vs CBDL. Action du VSL#3 sur les composantes du système RAS (Ang II : angiotensine II, AT1R : récepteur 1 de l'angiotensine, ACE : enzyme de conversion de l'angiotensine) au niveau de la paroi aortique; Panneau de gauche : exemple d'immunofluorescence, panneau de droite : données cumulées pour 4 rats différents; *P<0.05 CBDL vs sham, et # P<0.05 CBDL+VSL#3 vs CBDL. Diminution des cytokines pro-inflammatoires IL-1 , MCP-1 et TNF-et augmentation de la cytokine anti-inflammatoire IL-4 par le VSL#3 ; *P<0.05 CBDL vs sham, et # P< 0.05 CBDL+VSL#3 vs CBDL. La deuxième étude a montré que l'administration de polyphénols (jus de cassis riche en polyphénols, PRBJ) améliorait la dysfonction endothéliale chez le rat cirrhotique : amélioration de la relaxation EDHF-dépendante, diminution de l'expression de eNOS, des ROS, du SRA et des cytokines pro-inflammatoires. Le stress oxydant, dont une part peut être due à la translocation bactérienne mais d'autres facteurs sont également en jeu, est probablement un facteur clé dans la dysfonction endothéliale chez le rat cirrhotique. Le rôle du SRA lié au stress oxydant est à nouveau mis en exergue dans cette étude ce qui confirme une de nos études antérieures où un traitement par losartan (antagoniste des récepteurs 1 de l'angiotensine) améliorait la dysfonction endothéliale chez le rat présentant une cirrhose biliaire. Le traitement par PRBJ améliore la fonction EDHF mésentérique chez le rat cirrhotique µM) et d'apamine plus charybdotoxine (100 nM chacun d'indométacine et de N G -nitro (donneur exogène de NO), D) répons CBDL : rats cirrhotiques ; *P<0.05 CBDL vs sham, et Le traitement par PRBJ améliore la fonction EDHF-dépendante de la relaxation de l'artère mésentérique chez le rat cirrhotique ; A) composante NO en présence d'indométacine (10 µM) et d'apamine plus charybdotoxine (100 nM chacun), B) composante EDHF en présence nitro-L-arginine (300 µM) C) réponse au nitroprussiate de s (donneur exogène de NO), D) réponse au levcromakalim (ouvreur de canaux potassiques ) <0.05 CBDL vs sham, et # P<0.05 CBDL+PRBJ vs CBDL dépendante de la relaxation de l'artère composante NO en présence d'indométacine (10 ), B) composante EDHF en présence arginine (300 µM) C) réponse au nitroprussiate de sodium e au levcromakalim (ouvreur de canaux potassiques ) ; <0.05 CBDL+PRBJ vs CBDL Immmuno-fluorescence au niveau de l'artère mésentérique eNOS et augmente celle des canaux potassiques SK la réponse EDHF-dépendante. fluorescence au niveau de l'artère mésentérique : PRBJ diminue l'expression de et augmente celle des canaux potassiques SK Ca et de la connexine 37 impliqués dans dépendante. Panneau du haut : exemple d'immunofluorescence, pan : PRBJ diminue l'expression de et de la connexine 37 impliqués dans Panneau du haut : exemple d'immunofluorescence, panneau du bas : données cumulées pour 4 vs CBDL bas : données cumulées pour 4-5 rats ; *P<0.05 for CBDL vs sham, et # P<0.05 CBDL+P <0.05 CBDL+PRBJ Le traitement par PRBJ diminue le stress oxydant et l'aorte des rats cirrhotiques (A) d'hydrogène, anneaux aortiques exposés au dismutase mimetic), à la superoxyde dismutase (SOD) catalase) et à la catalase (B) Le traitement par PRBJ diminue le stress oxydant et l'expression de eNOS dans la paroi de l'aorte des rats cirrhotiques (A) ; rôle d'anions superoxydes intracellulaires et du d'hydrogène, anneaux aortiques exposés au MnTMPyP (membrane permeant superoxide dismutase mimetic), à la superoxyde dismutase (SOD), PEG-catalase (membrane permeant (B) ; sources cellulaires des ROS, anneaux aortiques ex l'expression de eNOS dans la paroi de ; rôle d'anions superoxydes intracellulaires et du superoxyde MnTMPyP (membrane permeant superoxide catalase (membrane permeant ; sources cellulaires des ROS, anneaux aortiques exposés à l'antioxydant et inhibiteur de la NADPH oxydase apocynine, au L-NA inhibiteur de la NOS, au sulfaphenazol inhibiteur du cytochrome P450, à l'indométacine inhibiteur de la cyclooxygénase et à la combinaison KCN, myxothiazol et roténone (KCN+MY+Rot), inhibiteur de la respiration mitochondriale, avant le marquage DHE (C). Panneaux du haut : exemple d'immunofluorescence, panneaux du bas : données cumulées pour 4 rats A) *P<0.05 CBDL vs sham, et # P<0.05 CBDL+VSL#3 vs CBDL; B and C) *P<0.05 for CBDL avec et sans inhibiteurs. Le traitement par PRBJ prévient la surexpression des sous NADPH oxydase, de COX-2 et de iNOS dans la paroi aortique. Panneau de ga d'immunofluorescence, panneau de droite CBDL vs sham, et # P<0.05 CBDL+PRBJ vs CBDL. prévient la surexpression des sous-unités p22phox et p47phox de la 2 et de iNOS dans la paroi aortique. Panneau de ga d'immunofluorescence, panneau de droite : données cumulées pour 4 rats différents 0.05 CBDL+PRBJ vs CBDL. p22phox et p47phox de la 2 et de iNOS dans la paroi aortique. Panneau de gauche : exemple : données cumulées pour 4 rats différents ;*P<0.05 Action du PRBJ sur les composantes du système RAS ( récepteur 1 de l'angiotensine, ACE paroi aortique; Panneau du haut : exemple d'immunofluorescence, pan Action du PRBJ sur les composantes du système RAS (Ang II : angiotensine II, AT1R récepteur 1 de l'angiotensine, ACE : enzyme de conversion de l'angiotensine) au niveau Panneau du haut : exemple d'immunofluorescence, panneau du bas : données : angiotensine II, AT1R : : enzyme de conversion de l'angiotensine) au niveau de la neau du bas : données cumulées pour 4 rats différents CBDL. pour 4 rats différents ; *P<0.05 CBDL vs sham, et # P<0.05 CBDL+PRBJ vs 0.05 CBDL+PRBJ vs Diminution des cytokines pro-inflammatoires IL-1 , MCP-1 et TNF-et augmentation de la cytokine anti-inflammatoire IL-4 par le PRBJ ; *P<0.05 CBDL vs sham, et # P< 0.05 CBDL+PRBJ vs CBDL. Dans la troisième étude, un traitement par fulvestrant (antagoniste des récepteurs de l'estrogène) diminuait l'expression de eNOS et des nitrotyrosines au niveau du poumon. Néanmoins le taux de p-VASP qui est un marqueur de l'action de NO était diminué dans le poumon du rat cirrhotique et le fulvestrant n'avait aucun effet sur ce marqueur. L'hypothèse est que malgré l'augmentation de l'expression de eNOS, le NO produit est capté par les ROS ce qui empêcherait son activité. Ceci pourrait d'ailleurs aussi expliquer la diminution de la composante NO de la relaxation dans les 2 études précédentes. Le fulvestrant n'a aucun effet non plus sur l'expression de VEGF ni sur la quantification des macrophages dans les vaisseaux pulmonaires, ni sur le diamètre des petits vaisseaux pulmonaires. En conclusion, soit le fuvestrant n'est pas efficace, soit l'hyperoestrogénie relative chez nos rats cirrhotiques mâles n'a pas de rôle à jouer dans le survenue du SHP. Résultats des western blots pour eNOS (NO synthase inductiblel), nitrotyrosine, p-VASP (phosphorylated vasodilator dans les poumons. Le fulvestrant diminue l'expression de eNOS et de l p-VASP qui est un marqueur de l'action de NO, est diminué cirrhotique et le fulvestrant n'a aucun effet sur c * : P<0.05 pour CBDL vs sham rats; ** : P<0.01 pour vs CBDL+F Résultats des western blots pour eNOS (NO synthase endothéliale), iNOS VASP (phosphorylated vasodilator-stimulated phosphoprotein) Le fulvestrant diminue l'expression de eNOS et de la nitrotyrosine ; de manière surprenante, qui est un marqueur de l'action de NO, est diminué dans le poumon du rat cirrhotique et le fulvestrant n'a aucun effet sur ce marqueur * : P<0.05 pour CBDL vs sham rats; ** : P<0.01 pour CDL vs sham; # : P<0.01 pour CBDL endothéliale), iNOS (NO synthase stimulated phosphoprotein) ; de manière surprenante, dans le poumon du rat CDL vs sham; # : P<0.01 pour CBDL Résultats des western blots pour VEGF -alpha des estrogènes), p-Akt (phosphorylated serine/threonine kinase Akt) da poumons. Le fulvestrant augmente l'expression de ER Akt.* : P<0.05 pour CBDL vs sham; ** : P<0.01 pour CBDL vs tern blots pour VEGF (vascular endothelial growth factor), ER Akt (phosphorylated serine/threonine kinase Akt) da Le fulvestrant augmente l'expression de ER-mais n'a aucun effet sur VEGF ni p P<0.05 pour CBDL vs sham; ** : P<0.01 pour CBDL vs CBDL+F (vascular endothelial growth factor), ER-(récepteur Akt (phosphorylated serine/threonine kinase Akt) dans les mais n'a aucun effet sur VEGF ni p-CBDL+F Marquage en immunofluorescence pour VEGF (vascular hème-oxygénase-1 dans les poumons. Les deux facteurs sont élevés mai diminuée avec le traitement par fulvestrant (possib stimuler HO-1, par le fulvestrant). ** : P<0.05 pour CBDL vs sham; # : P<0.05 pour CBDL Marquage en immunofluorescence pour VEGF (vascular endothelial growth factor) et la dans les poumons. Les deux facteurs sont élevés mai diminuée avec le traitement par fulvestrant (possible effet de la diminution de NO, qui peut 1, par le fulvestrant). ** : P<0.05 pour CBDL vs sham; # : P<0.05 pour CBDL vs CBDL+F endothelial growth factor) et la dans les poumons. Les deux facteurs sont élevés mais seule HO-1 est le effet de la diminution de NO, qui peut Immunohistochimie d'échantillons pulmonaires. On ob intravasculaires (étoiles) et une augmentation du d poumon de rats cirrhotiques (panneaux du bas, 2 rat normaux (panneaux du haut). Conclusions L'ensemble de nos études soulignent le rôle importa stimulation du SRA dans la survenue de la dysfoncti cirrhose biliaire. L'action sur des facteurs pouvan Immunohistochimie d'échantillons pulmonaires. On observe de nombreux macrophages intravasculaires (étoiles) et une augmentation du diamètre de petits vaisseaux (flèches) dans le poumon de rats cirrhotiques (panneaux du bas, 2 rats différents) en comparaison avec des rats L'ensemble de nos études soulignent le rôle important du stress oxydant qui est lié à la stimulation du SRA dans la survenue de la dysfonction endothéliale chez le rat présentant une cirrhose biliaire. L'action sur des facteurs pouvant induire le stress ox serve de nombreux macrophages iamètre de petits vaisseaux (flèches) dans le comparaison avec des rats nt du stress oxydant qui est lié à la on endothéliale chez le rat présentant une t induire le stress oxydant comme la translocation bactérienne et le traitement par des antioxydants (polyphénols) a clairement montré des actions bénéfiques chez nos rats cirrhotiques. Le rôle du NO, en tous cas en tant que vasodilatateur pur, n'est peut-être pas primordial ce qui peut expliquer les résultats décevants en clinique publiés par certains auteurs et ce qui peut également expliquer en partie les résultats de notre troisième étude.

Figure 1 .

 1 Figure 1. The three layers of normal artery

Figure 2 .Figure 3 .

 23 Figure 2. NO generation from L-Arginine and its functional properties. (Ghalayini 2004)

Figure 4 .

 4 Figure 4. Hypothesis describing the nature of EDHF pathway (Grgic et al, 2009).

Figure 5 . 1 . 2 . 5 .

 5125 Figure 5. Arachidonic acid metabolism pathway. (Tang and Vanhoutte, 2009)

Figure 6 .

 6 Figure 6. The pathways of endothelial-derived relaxation. AA, arachidonic acid; AC, adenylyl cyclase; ATP, Adenosine triphosphate ; Big ET-1, precursor of endothelin-1; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate ; eNOS, endothelium NO synthase; ECE, endothelin converting enzyme; ET-1, endothelin-1, ET-B1 endothelin receptor; IP 1 , prostacyclin receptor; GTP, guanosine triphosphate; NO , nitric oxide; PI3K, phosphatidylinositol 3kinse; Src, Src family. Dash line represents inhibition.

Figure 7 .

 7 Figure 7. The renin-angiotensin system. (Grace et al, 2013) Ang I , angiotensin I; ACE, angiotensin converting enzyme; Ang II, angiotensin II; ACE2, angiotensin converting enzyme type 2; AT1R, angiotensin receptor type 1; AT2R, angiotensin receptor type 2; Ang (1-7) , angiotensin (1-7).

Figure 8 .

 8 Figure 8. Pathways of endothelial derived contraction.

Figure 9 .

 9 Figure 9. Overview of how portal hypertension develops. (Iwakiri 2012)

Figure 10 .

 10 Figure 10. Overview of factors contributing to PH initiation and it is worsening. (Iwakiri 2012).

Figure 11 .

 11 Figure 11. Production of CO by the enzymatic activity of Heme-oxygenase. Araujo et al, 2012)

Figure 12

 12 Figure 12. Potential mechanisms and target therapeutic options in hepatopulmonary syndrome (HPS) and hyperdynamic circulatory state. (Palma and Fallon 2006)

Figure 13 .

 13 Figure 13. Role of angiogenesis in the pathology of HPS. (Rodriguez-Roisin and Krowka 2008)

Flavonoids; flavonoids are

  further divided into many subclasses: anthocyanins, flavan-3-ols (as monomers forming catechins and as polymers forming proanthocyanidins (condensed tannins), flavones, flavanones, flavonols, isoflavones, neoflavonoids and chalcones. They consist of a C6-C3-C6 backbone (Figure14).Anthocyanidins have a basic structure, known as flavylium cation, which contains positive charge in the C-ring, and by this positive charge it is distinguished from other flavonoids. In nature, highly unstable anthocyanidins are protected from degradation by glycosylation (anthocyanidins with sugar group(s) are called anthocyanins) generally with a number of sugars, in particular glucose, sophorose, rutinose, galactose, arabinose and xylose at different positions of the C-ring. Anthocyanins are water soluble pigments, providing the distinctive and vibrant palette of colors (red, blue and purple) found in fruits, vegetables, flowers, and other plant tissues or products.

Figure 14 .

 14 Figure 14. Structures of some polyphenols (Manach et al, 2004).

Figure 15 .

 15 Figure 15. Schematic of pharmacokinetically relevant flavonol features pertaining to the composition in foods, absorption, metabolism, distribution and excretion. This can be applied to other flavonoids with exception some flavonoids are present in the form of glycosides, ester, or polymers that cannot be absorbed in their native form. Most of the glycosides probably resist acid hydrolysis in the stomach and thus arrive intact in the intestine (Heiss et al, 2010).

  States. Modern laboratories have demonstrated the potent anti-inflammatory, antioxidant and antimicrobial effects of blackcurrant constituents on a myriad of disease states. The properties of the blackcurrants are conferred from its biochemical constituents, some of which include anthocyanins (specifically delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside), flavonols, phenolic acids and polyunsaturated fatty acids(Gopalan et al, 2012, Figure 16).

Figure 16 .

 16 Figure 16. Various photographs of blackcurrants (Ribes nigrum) showing a commercial blackcurrant plantation (A), shrub with fruits (B and C), and isolated fruits (D) (Gopalan et al, 2012).

(

  anthocyanidins) are salt derivatives of the 2-phenylchromenylium (flavylium) cation. Its positive charge differentiates anthocyanidins from other flavonoids. Anthocyanins are sugar derivatives of anthocyanidins. Four major anthocyanins have been identified in the blackcurrant: delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside,

Figure 17 .Figure 18 .

 1718 Figure 17. Chemical structures of the major anthocyanins present in blackcurrants. (Gopalan et al, 2012)

2 -

 2 On other hand a previous study byDal-Ros et al. (Dal-Ros et al, 2010) concluded that impaired EDHF-mediated relaxation in CBDL is partly if not totally explained by an excessive vascular level of oxidative stress. And the enzymatic sources of this vascular oxidative stress are due to for example; NADPH oxidase, cytochrome P450, un-coupled eNOS etc. Therefore, the effect of a polyphenol-rich blackcurrant juice (PRBJ), a potent antioxidant product on vascular endothelial function in CBDL rat was tested.

Figure 19 .

 19 Figure 19. Histological study of liver section using heamatoxylin and eosin staining, sample taken at the end of study; A, sham: normal liver architecture; B, CBDL: disturbed liver architecture

Figure 20 .

 20 Figure 20. CBDL operation; consist of identification of the common bile duct and then put double ligature and cut between the ligature with scissor.

  Rings were suspended in organ baths containing oxygenated (95% O 2 , 5% CO 2 ) Krebs bicarbonate solution (mM: NaCl 119, KCl 4.7, KH 2 PO 4 1.18, MgSO 4 1.18, CaCl 2 1.25, NaHCO 325 and d-glucose 11, pH 7.4, 37°C) for the determination of changes in isometric tension. The rings were stretched step by step until an optimal resting tension of 1 g was reached and then allowed to equilibrate for at least 60 min. After the equilibration period, the rings were exposed to Krebs bicarbonate solution containing a high concentration of potassium (80 mM) until reproducible contractile responses were obtained. After washing with Krebs bicarbonate solution, the rings were precontracted with phenylephrine (PE, 1 µM) to about 80% of the maximal contraction obtained by high K + solution before the addition of acetylcholine (ACh, 1 µM) to check the presence of a functional endothelium. After washout and a further 30-min equilibration period, rings were again contracted with PE before the application of increasing concentrations of either ACh, sodium nitroprusside (an exogenous NO donor) or levcromakalim (an ATP-sensitive K + channel opener) to construct concentration-response curves. Sodium nitroprusside-and levcromakalim-induced relaxations were examined in endothelium denuded rings of mesenteric artery. In some experiments, rings were exposed to an inhibitor for 30 min before contraction with PE. The NO-mediated component of relaxation was determined in the presence of indomethacin (10 µM) and charybdotoxin (CTX, 100 nM) plus apamin (APA, 100 nM) to inhibit the participation of prostanoids and EDHF, respectively. The EDHF-mediated component of relaxation was determined in the presence of indomethacin (10 µM) and N • -nitro-L-arginine (L-NA, 300 µM) to inhibit the formation of prostanoids and NO, respectively. Relaxations are expressed as percentage of the contraction induced by PE (Figure21).

Figure 21 .

 21 Figure 21. Study of the vascular reactivity.

I Article 1 3 1 :

 131 Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system Sherzad K. Rashid a , Noureddine Idris-Khodja a , Cyril Auger a , Mahmoud Alhosin a , Nelly Boehm b , Monique Oswald-Mammosser c and Valérie B. Schini-Kerth a PLOS ONE, 2014; 10.1371/journal.pone.0097458 PART II Article 2 Polyphenol-rich blackcurrant juice prevents endothelial dysfunction in the mesenteric artery of cirrhotic rats with portal hypertension: Role of oxidative stress and the angiotensin system Rashid Sherzad a , Idris Khodja Noureddine a , Auger Cyril a , Alhosin Mahmoud a , Boehm Nelly b , Oswald-Mammosser Monique, a,c and Schini-Kerth Valérie B a .Submitted to journal of Free Radical Biology and MedicinePART IIIArticleEffect of Fulvestrant, an estrogen receptor antagonist, on the cirrhotic rat lungOswald-Mammosser Monique, Rashid Sherzad K., Boehm Nelly, Agin Arnaud, Geny Bernard, Schini-Kerth Valérie, Charloux Anne. Probiotic VSL#3 as a candidate to prevent endothelial dysfunction in cirrhotic rats with PH.The intestinal microbiota consists of more than 500 bacterial species that inhabit the human intestinal tract. In the healthy individuals, the composition of bacterial densities change in different parts of the intestine for example the lower part of ileum has higher bacterial concentration than the upper part of ileum[START_REF] Madsen | Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice[END_REF]. The intestinal microbiota has been shown to play important immunomodulatory and homeostatic roles[START_REF] Tlaskalova-Hogenova | Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases[END_REF]. The intestinal microbiota and the mucosal lining are closely related components of the intestinal epithelial barrier. Clinical observations and animal experiments have suggested that intestinal bacteria can trigger ongoing mucosal inflammation in some susceptible individuals[START_REF] Madsen | Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice[END_REF]. It is known that in patients and animal with cirrhosis, gram-negative bacterial translocation plays a pivotal role with the development of spontaneous bacterial infections and the worsening of the hyperdynamic circulatory state further deteriorating the endothelial function(Wiest and Groszmann 1999;[START_REF] Albillos | Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement[END_REF]. Bacterial translocation in patient with advanced cirrhosis, is favored by gram-negative bacterial overgrowth, increased intestinal permeability, and impaired immunity in addition to the presence of ascites (Albillos and de la Hera 2002;Wiest and Garcia-Tsao 2005). In addition to splanchnic vasodilatation, it has been shown that systemic vasodilatation (as evidenced by lower mean arterial pressure) is greater in animals with bacterial translocation. These abnormalities correlate with increased levels of endotoxin, TNF-, and nitric oxide[START_REF] Wiest | Bacterial translocation up-regulates GTPcyclohydrolase I in mesenteric vasculature of cirrhotic rats[END_REF] (Figure22).

Figure 22 .

 22 Figure 22. Bacterial translocation and the hyperdynamic circulatory state in cirrhosis.[START_REF] Fernandez | Bacterial infections in cirrhosis: epidemiological changes with invasive procedures and norfloxacin prophylaxis[END_REF]

  bacterial translocation is one of the factors thought to lead to endothelial dysfunction in cirrhosis we have assessed the effects of VSL#3 in CBDL rats. The results of our study are reported in the article entitled "Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system" published in Plos One:May 15, 2014; 10.1371/journal.pone.0097458.

Figure 23 :

 23 Figure 23: Beneficial effects of commensal and probiotic bacteria. (Petrof 2009) Schematic of the many vital roles of bacteria in the intestine, commensal/ probiotic bacteria are purple, epithelial cells are in light blue, apical and basolateral sides of epithelial cells are also indicated. Intestinal bacteria serve an important role by 1) inhibiting pathogen growth through production of antimicrobials; 2) enhancing tight junction barrier function; 3) priming dendritic cell (drawn in black) and immune system; 4) assisting in digestion and breakdown of micronutrients from otherwise undigestable material (indicated in light green), synthesis of short chain fatty acids (SCFAs); 5) synthesizing key vitamins such as vitamin K and B vitamins. Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system Sherzad K. Rashid a , Noureddine Idris-Khodja a , Cyril Auger a , Mahmoud Alhosin a , Nelly Boehm b , Monique Oswald-Mammosser c and Valérie B. Schini-Kerth a PLOS ONE, 2014; 10.1371/journal.pone.0097458

Figure 1 .

 1 Figure 1. Ingestion of VSL#3 improves the CBDL-induced blunted EDH but not NO-mediated relaxations in mesenteric artery rings. Concentration-relaxation curves to acetylcholine in mesenteric artery rings with endothelium in sham, CBDL, sham+VSL#3 and CBDL+VSL#3 rats. A) The NO component of the relaxation was assessed in the presence of indomethacin (10 mM) and apamin plus charybdotoxin (100 nM each), and B) the EDH component in the presence of indomethacin and N G -nitro-L-arginine (300 mM). C) Relaxations to sodium nitroprusside (an exogenous donor of NO) and D) levcromakalim (an ATP-sensitive K + channel opener) in mesenteric artery rings without endothelium are also shown. Results are shown as mean6SEM of 5-7 different rats; *P,0.05 CBDL vs sham, and # P,0.05 CBDL+VSL#3 vs CBDL. doi:10.1371/journal.pone.0097458.g001

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. The VSL#3 treatment prevents the CBDL-induced A) up-regulation eNOS and down-regulation of SK ca and IK ca , and B) down-regulation of connexins (Cx37, Cx40 and Cx43) in the mesenteric artery. Upper panels show representative immunofluorescence staining, and lower panels corresponding cumulative data. C) Expression of eNOS and SK Ca protein levels as assessed by Western blot analysis. Results are shown as mean6SEM of 4 different rats for A and B, and 3 for C. *P,0.05 for CBDL vs sham, and # P,0.05 CBDL+VSL#3 vs CBDL. doi:10.1371/journal.pone.0097458.g002

Figure 5 .

 5 Figure 5. The VSL#3 treatment prevents the CBDL-induced upregulation of several components of the local angiotensin system in the aorta. The expression level of angiotensin II (Ang II), AT1 receptors (AT1R) and angiotensin-converting enzyme (ACE) was determined by immunofluorescence staining. Upper panels show representative immunofluorescence staining; Lower panels corresponding cumulative data. Results are shown as mean6SEM of 4 different rats. *P,0.05 CBDL vs sham, and # P,0.05 CBDL+VSL#3 vs CBDL. doi:10.1371/journal.pone.0097458.g005

Figure 6 .

 6 Figure 6. The VSL#3 treatment prevents the CBDL-induced increased formation of pro-inflammatory cytokines including IL-1a, MCP-1 and TNF-a, and decreased formation of the anti-inflammatory cytokine IL-4 in plasma. Results are shown as mean6SEM of 6 different rats. *P,0.05 CBDL vs sham, and # P,0.05 CBDL+VSL#3 vs CBDL. doi:10.1371/journal.pone.0097458.g006

Figure 24 .

 24 Figure 24. Schema presenting the effects of CBDL in rats and t of VSL#3 in the mesenteric arteries and both liver hyperpolarizing factor, eNOS: endothelial NO syntha NO: nitric oxide, AT-1: angiotensin receptor type, AA: arachidonic acid, and SK Ca : calcium dependent potassium channels of intermedi effects of CBDL, increase and decrease,

(

  NO donor) and levcromakalim (an ATP-sensitive K + channel opener) in rings without endothelium were minimally affected. Impaired endothelium-dependent relaxations were associated with a reduced vascular expression of Cx37 and SK Ca and an increased expression of eNOS (endothelial NO synthase), cyclooxygenase-2 and inducible NOS. In aortic sections of CBDL rats, an increased expression of NADPH oxidase subunits, angiotensin II, angiotensin-converting enzyme (ACE) and angiotensin type 1 receptors (AT1) are observed as well as an increased vascular formation of ROS and peroxynitrites. The endothelial dysfunction in CBDL rats was significantly prevented by PRBJ, and this effect was associated with the normalization of the vascular expression of Cx37 and SK Ca, and eNOS. PRBJ treatment also reduced vascular oxidative stress in the aorta, and the increased plasma level of pro-inflammatory cytokines including TNF-, IL-1 , MCP-1 and IL-4 in CBDL rats. In conclusion, these results indicate that PRBJ ingestion prevented the blunted NO-and EDHF-mediated endothelium-dependent relaxation in the mesenteric artery of CBDL rats most likely by preventing the excessive oxidative stress in the arterial wall and attenuating the activation of local angiotensin system. 7213, Faculty of Pharmacy, Université de Strasbourg, Illkirch. b Institut d'Histologie et INSERM U1119, Université de Strasbourg, Faculté de Médecine, Strasbourg. c Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologie Thoracique, Hôpitaux Universitaires de Strasbourg, Université de Strasbourg, Faculté de Médecine, Strasbourg. .oswald@chru-strasbourg.frAbstract:Chronic liver diseases with portal hypertension are characterized by a progressive vasodilatation associated with an endothelial dysfunction and vascular oxidative stress, and have been suggested to involve bacterial translocation and the angiotensin system. Since blackcurrant berries are a rich natural source of antioxidants, the aim of the present study was to determine the possibility that ingestion of a polyphenol-rich blackcurrant juice (PRBJ) prevents endothelial dysfunction in a rat model of cirrhosis induced by chronic bile duct ligation (CBDL), and, if so, to determine the underlying mechanism.Male Wistar rats received either control drinking water or water containing 60 mg/kg gallic acid equivalents (GAE) of PRBJ for 7 weeks. After 3 weeks, rats underwent surgery with CBDL or sham surgery.Both the acetylcholine-induced nitric oxide (NO)-and the endothelium-dependent hyperpolarization (EDH)-mediated relaxations in mesenteric artery rings are significantly reduced in CBDL rats compared to sham rats, and associated with a reduced vascular expression of connexion Cx37 and small conductance calcium-dependent K + channel (SK Ca ), and an increased expression of endothelial NO synthase (eNOS). An increased formation of reactive oxygen species (ROS), and expression level of nitrotyrosine, p22phox and p47phox NADPH oxidase subunits, cyclooxygenase-2, inducible NOS, angiotensin II, angiotensinconverting enzyme (ACE) and angiotensin type 1 receptors (AT1) are observed in aortic sections in the CBDL group. Chronic intake of PRBJ prevented the CBDL-induced impaired

  . Moreover, oxidative stress has been shown to contribute to endothelial dysfunction in CBDL rats by affecting the endothelium-dependent hyperpolarization component (Dal-Ros, Oswald-Mammosser et al. 2010), and possibly also by promoting an inflammatory response subsequent to the expression of pro-inflammatory cytokines such as IL-1 and TNF-(Bode and Bode 2005; Zhang, Han de et al. 2007; Yu, Sanchez-Lozada et al. 2010). The aim of the present study was to evaluate the possibility that chronic intake of a natural rich source of antioxidants such as the polyphenol-rich backcurrant juice prevents endothelial dysfunction in an animal model of biliary cirrhosis with portal hypertension, and, if so, to characterize the underlying mechanism.

  described previously(Dal-Ros, Oswald-Mammosser et al. 2010). Briefly, the main superior mesenteric artery was cleaned of connective tissue, cut into rings (2-3 mm in length) and suspended in organ baths containing oxygenated Krebs bicarbonate solution for the determination of changes in isometric tension. After equilibration and test of the presence of a functional endothelium, rings were contracted with phenylephrine (PE, 1 µM) before the construction of concentration-relaxation curve in response to acetylcholine (ACh) in endothelium-intact rings, and to either sodium nitroprusside or levcromakalim in endothelium-denuded rings. In some experiments, rings were exposed to an inhibitor for 30 min before addition of PE. Relaxations are expressed as percentage of the reversal of the contraction induced by PE.

  -regulation of SK Ca and Cx37 in the mesenteric artery Pronounced fluorescence signals for SK Ca, the small conductance calcium-activated K + channel involved in EDH (Dal-Ros, Oswald-Mammosser et al. 2010), and Cx37 involved in myoendothelial gap junction transmitting the hyperpolarization from endothelial cells to the underlying smooth muscle cell to cause relaxation (Dal-Ros, Oswald-Mammosser et al.

  present findings indicate that the CBDL-induced endothelial dysfunction affecting predominantly the EDH component in the mesenteric artery is associated with an increased vascular level of oxidative stress subsequent to the upregulation of the local angiotensin system and an increased level of circulating pro-inflammatory mediators. They further indicate that chronic ingestion of a blackcurrant juice, a polyphenol-rich natural source of antioxidants, prevented the CBDL-induced endothelial dysfunction, vascular oxidative stress and the pro-inflammatory response by improving the local angiotensin system. Chronic bile duct ligation in rats is an experimental model of cirrhosis, which is characterized by an increased spleen weight reflecting portal hypertension, associated with an increased proliferation of biliary ducts. Consistent with previous observations (Dal-Ros, Oswald-Mammosser et al. 2010), CBDL leads to an endothelial dysfunction affecting predominantly the EDH component and also as shown in the present study to a slightly but significantly reduced NO component. The blunted EDH component is explained, at least in part, by a reduced expression of SK Ca and Cx37 levels, and also as shown previously of IK Ca , Cx40 and Cx43, which all contribute to the initiation and transmission of the hyperpolarization from endothelial cells to the underlying smooth muscle cells (Dal-Ros, Oswald-Mammosser et al.

  ; Dal-Ros, Oswald-Mammosser et al. 2010). Moreover, apocynin, an antioxidant and NADPH oxidase inhibitor, prevented the CBDL-induced impaired EDH-mediated component of the relaxation, vascular oxidative stress and the decreased expression of calcium-activated potassium channels and connexins (Dal-Ros, Oswald-Mammosser et al. 2010). A determinant role for oxidative stress is also supported by the fact that the antioxidant N-acetylcysteine prevented the development of the hyperdynamic circulation in portal hypertensive rats associated with a reduced urinary level of F 2 -isoprostanes, a marker of oxidative stress (Fernando, Marley et al. 1998). The present findings further extent these previous observations (Dal-Ros, Oswald-Mammosser et al. 2010) by indicating that CBDL-induced vascular oxidative stress involves both the intracellular formation of superoxide anions and hydrogen peroxide. Furthermore, the pharmacological characterization of the sources of ROS has indicated the involvement of several sources including NADPH oxidase, uncoupled NOS, cytochrome P450, COXs and the mitochondrial respiration chain. Moreover, CBDL is associated with the upregulation of several sources of ROS including p22phox and p47phox NADPH oxidase subunits, COX-2 and both endothelial and inducible NOS in the arterial wall. Since apocynin has been shown to prevent the CBDL-induced upregulation of NADPH oxidase and eNOS, oxidative stress appears to be an early determinant stimulatory event to promote the upregulation of prooxidant enzymes, and, thus, to sustain oxidative stress (Dal-Ros, Oswald-Mammosser et al. 2010).

  to the EDH component, the NO component of the relaxation is impaired although only to some extent in the CBDL group despite an upregulation of eNOS expression in the arterial wall. This latter response is most likely part of a compensatory mechanism due to the degradation of the excessive formation of NO by superoxide anions leading to the formation of peroxynitrite. The PRBJ treatment improved the CBDL-induced upregulation of eNOS expression and vascular oxidative stress, which, however, did not result in an improvement of the NO-mediated relaxation in contrast to the enhanced EDH component. Such a difference is most likely explained by the fact that the EDH component is more sensitive to oxidative stress than the NO component as observed previously in vascular aging and angiotensin II-induced endothelial dysfunction and hypertension (Dal-Ros, Bronner et al. 2009; Idris Khodja, Chataigneau et al. 2012).

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. The PRBJ ingestion prevents the CBDL-induced up-regulation eNOS and down-regulation of both SK ca and connexin Cx37 in the mesenteric artery. Upper panels show representative immunofluorescence staining, and lower panels corresponding cumulative data. Results are shown as mean±SEM of 4 to 5 different rats. * P<0.05 for CBDL vs sham, and # P<0.05 for CBDL+PRBJ vs CBDL.

Figure 3 .

 3 Figure 3. The PRBJ treatment prevents the CBDL-induced oxidative stress in the aortic wall. (A) In situ determination of the level of oxidative stress as assessed using dihydroethidine (DHE), MitoSox, and peroxynitrite (nitrotyrosine) in the aorta. Top: representative fluorescence staining; bottom: corresponding cumulative data for 4 to 5 rats/group. (B) Aortic sections from CBDL rats were exposed either to B) MnTMPyP (membrane-permeant superoxide dismutase mimetic), superoxide dismutase (SOD), PEG-catalase (membrane-permeant catalase) or catalase, and (C) apocynin (Apo, NADPH oxidase inhibitor and antioxidant), L-NA (NO synthase inhibitor), sulfaphenazol (Sulfa, cytochrome P450 inhibitor), indomethacin (Indo, cyclooxygenase inhibitor) or the combination KCN, myxothiazol (Myxo) and rotenone (Rot, inhibitors of the mitochondrial respiration chain) for 30 min before DHE staining. Top: representative ethidium staining; bottom: corresponding cumulative data for 4 rats. Results are shown as mean±SEM. * P<0.05 for CBDL vs sham, and # P<0.05 for CBDL+PRBJ vs CBDL for A; *P<0.05 for CBDL with inhibitors vs CBDL in B and C.

Figure 4 .

 4 Figure 4. The PRBJ treatment prevents the CBDL-induced up-regulation of the NAPDH oxidase subunits p22phox and p47phox in aortic sections, and COX-2 and inducible NOS (iNOS) in mesenteric artery sections. Left panels: Representative immunofluorescence staining; Right panels: corresponding cumulative data. Results are shown as mean±SEM of 4 different rats. * P<0.05 for CBDL vs sham, and # P<0.05 for CBDL+PRBJ vs CBDL.

Figure 5 .

 5 Figure 5. The PRBJ treatment prevents the CBDL-induced up-regulation of several components of the local angiotensin system in the aorta. The expression level of angiotensin II (Ang II), angiotensin II type 1 receptors (AT1R) and angiotensin-converting enzyme (ACE) was determined by immunofluorescence staining. Upper panels: Representative immunofluorescence staining; lower panels: corresponding cumulative data. Results are shown as mean±SEM of 4 different rats. * P<0.05 for CBDL vs sham, and # P<0.05 for CBDL+PRBJ vs CBDL.

Figure 6 .

 6 Figure 6. The PRBJ treatment prevents the CBDL-induced increase levels of pro-inflammatory cytokines including IL-1 , MCP-1 and TNF-, and the decreased level the anti-inflammatory cytokine IL-4 in plasma. Results are shown as mean±SEM of 4 different rats. * P<0.05 for CBDL vs sham, and # P< 0.05 for CBDL+PRBJ vs CBDL.

  Figure 3

  Taken altogether, these results indicate that Black EDHF-mediated relaxation and the increased vascul of CBDL rats suggesting the contribution of endotox dysfunction.

Figure 25 .

 25 Figure 25. Schema presenting the effects of CBDL in of PRBJ in the mesenteric arteries and /or aorta and bot endothelium derived hyperpolarizing factor, eNOS: e species, K + : potassium, NO: nitric oxide, AT COX: cycloogynase, IK Ca and SK conductance, : effects of CBDL increase and decrease.

Figure 25 .

 25 Figure 25. Schema presenting the effects of CBDL in rats and the therapeutic preventive effects BJ in the mesenteric arteries and /or aorta and both liver and spleen size. endothelium derived hyperpolarizing factor, eNOS: endothelial NO synthase, ROS: reactive oxygen : potassium, NO: nitric oxide, AT-1: angiotensin receptor type, AA: arachidonic acid, and SK Ca : calcium dependent potassium channels of intermedi effects of CBDL, increase and decrease, : effects of CBDL+PRBJ,

  Abbreviations:

Fig. 1 Fig. 2 Fig. 3 1 (Fig. 4

 12314 Fig. 1 Western blot analyses for eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nitrotyrosine, p-VASP (phosphorylated vasodilator-stimulated phosphoprotein) in the lungs

Figure 4

 4 Figure 4

Figure 26 .

 26 Figure 26. Schema presenting the effects of CBDL in preventive effects of fulvestrant synthase, ROS: reactive oxygen species, HO estrogen alpha receptor, VEGF: vascular endothelial species, ONOO -: peroxynitrite , increase and decrease.

Figure 26 . 1 .

 261 Figure 26. Schema presenting the effects of CBDL in rats' lung and the therapeutic preventive effects of fulvestrant: eNOS: endothelial NO synthase, iNOS: inducibl synthase, ROS: reactive oxygen species, HO-1: hemooxygenase-1, NO: nitric oxide, ER estrogen alpha receptor, VEGF: vascular endothelial growth factor, ROS: reactive oxygen peroxynitrite : effects of CBDL, : effects of CBDL+ Fulvestrant

Figure 27 .

 27 Figure 27. Structure of NADPH oxidase (Novo and Parola 2008).

Figure 28 .

 28 Figure 28. Schematic representation of different biological events depending on the pathway that angiotensinogen passes through (Grace et al, 2012).

  induce immune tolerance. The Toll-like receptors (TLR) are responsible for immunetolerance, which comprises a highly conserved family of receptors that recognize specific pathogen-associated molecular patterns (PAMPs), which play a key role in innate immunity by triggering inflammatory responses to the main ligands of TLR. On the other, a breakdown in TLR tolerance results in persistent inflammation and contributes to the development of chronic liver diseases[START_REF] Miyake | Role of gut microbiota in liver diseases[END_REF]. The breakdown of TLR tolerance has been shown to occur in different inflammatory conditions affecting the intestinal integrity like inflammatory colitis, obstructive jaundice and ischemia reperfusion injury. However, previous observations have indicated that bacterial translocation occurs in both normal and diseased livers[START_REF] Singh | Status of bacterial colonization, Toll-like receptor expression and nuclear factor-kappa B activation in normal and diseased human livers[END_REF]. It is the different TLR types and the pro-inflammatory cytokines released by monocytes like, IL-1 and IL-6 in response to bacterial components such as LPS, flagellin and others, that affect liver prognosis regarding inflammation and fibrosis (Figure29, Miyake and Yamamoto 2013).

Figure 29 .

 29 Figure 29. Role of TLR in liver pathology. Treg = T regulatory cell (Miyake and Yamamoto 2013).

  prevented the CBDL-induced endothelial dysfunction and oxidative stress in previous work done in our lab(Dal-Ros et al, 2010). However, we were not able to precise the exact underling mechanism through which VSL#3 affects the vascular angiotensin system. It is likely that bacterial translocation is a key event triggering a cascade of immune and inflammatory responses. The activation of local and systemic inflammatory responses manifested by increased plasma concentrations of pro-inflammatory cytokines such as IL-1 , MCP-1 and TNF-leads to increased oxidative stress through different mechanisms among them involving the NADPH oxidase system. Previous studies have shown a link between

  toxicity. Our observations on VSL#3 study in accordance with what have been published, support potential benefit of VSL#3 in chronic inflammatory diseases including chronic liver diseases. Perspective work on VSL#3 need to address the following, VSL#3-induced changes on the intestinal microbial composition are well established, the identification of the specific bacteria affected in the vast microbial community still remains unknown. The role of the VSL#3-induced changes in the intestinal microbiota and of specific bacteria in the improvement of the endothelial function in the CBDL rat model as observed in the first study remains unknown. We need to address this rather difficult challenge in a subsequent study in collaboration with microbiologists. The possibility whether or not ACE inhibitory peptides are generated by the VSL#3 treatment in the CBDL rat model has not yet been tested, it well be of high interest to test that in the future studis.

Table

. Endothelium-derived vasoactive factors. (Calles-Escandon and Cipolla, 2001) 1.2.1. Endothelium-derived vasorelaxing factors 1.2.2. Nitric oxide (NO)

Table 3 . Physiological and pathophysiological stimuli shown to regulate eNOS expression and their mode of regulation: (increased : , decreased: ) (Searles 2006).

 3

CHPTER II ENDOTHELIAL DYSFUNCTION AND PORTAL HYPERTENSION Chapter two 2. Relationship between endothelial dysfunction and portal hypertension. 2.1. Introduction

	Chronic liver diseases are characterized by a progressive vasodilatation, which is
	especially observed in the splanchnic and pulmonary beds. The vasodilatation is due to portal
	hypertension (PH) associated to either cirrhosis or extra-hepatic portal venous obstruction
	without cirrhosis (Abraldes et al, 2006).
	Portal hypertension is a common clinical consequence of chronic liver disease which
	is associated with high morbidity and mortality rate (Hu et al, 2013). Portal hypertension is
	classified as either pre-hepatic, intra-hepatic or post-hepatic, with intra-hepatic PH being the
	form most frequently induced by cirrhosis, irrespective to the etiology
	Gq-protein; IP 3 , inisitol triphosphate; PIP 2 , phosphatidylinositol 4,5-biphophonate; PGH 2,
	prostaglandin H 2 ; ROS, reactive oxygen species ; TP, thromboxane prostanoid receptor; TXA 2 ,
	thromboxane A 2. Dash line represents an inhibitory effect.

AA, arachidonic acid; ACE, angiotensin converting enzyme; Ang I, angiotensin I; Ang II, angiotensin II; ATR, angiotensin II receptor type I; Big ET-1, precursor of endothelin-1; ET-A , endothelin receptor; ET-B2 , endothelin receptor; ECE, endothelin converting enzyme; ET-1, endothelin-1; Gq,

  . The present protocol was approved by the local Ethics Committee (Comite Ŕe ´gional d'Ethique en Matie `re d'Expe ´rimentation Animale, approval AL/01/09/09/05).

Table 1 .

 1 Effect of VSL#3 ingestion on body, liver and spleen weight in both sham and CBDL rats.

		Initial body weight (g)	Final body weight (g)	Liver (% total weight)	Spleen (% total weight)
	Sham	365619.5	505630.2	3.2460.06	0.2060.02
	Sham+VSL#3	368614.3	506626.4	3.3360.18	0.2360.02
	CBDL	369616.3	485630.5	6.6960.63 *	0.6960.05 *
	CBDL+VSL#3	365615.7	507647.6	6.0260.55	0.5660.09 #
	Values are shown as mean 6 SEM of 6 rats. * P,0.05 CBDL vs sham, and # P,0.05 for CBDL+VSL#3 vs CBDL.
	doi:10.1371/journal.pone.0097458.t001

  The samples were analyzed using a confocal laser-scanning microscope (Leica SP2 UV DM IRBE) with 20 × magnification lens. Quantification of fluorescence signals was performed using Image J software (http://rsweb.nih.gov/ij/).

	15 min and stored at -80°C until use. Sections were first fixed with paraformaldehyde at 4%,
	washed and treated with 10% milk or 5% goat serum in PBS containing 0.1% Triton X-100
	for 1 h at room temperature to block non-specific binding. Sections were then incubated
	overnight at 4°C with an antibody directed against either eNOS (1/100), inducible NOS
	(1/200), connexin C37 (Cx37, 1/200), which is part of the myoendothelial gap junctions
	transmitting hyperpolarization to the smooth muscle cells, small conductance calcium-
	dependent K + channels (SK Ca , 1/200), angiotensin II (Ang II, 1/500), angiotensin II type 1
	receptor (AT1R, 1/400), angiotensin-converting enzyme (ACE, 1/200), nitrotyrosine (1/200),
	p47phox (1/200), p22phox (1/200) or cyclooxygenase-2 (COX-2, 1/200). Sections were then
	washed with PBS, incubated with the secondary antibody (Alexa 488-conjugated goat anti-

rabbit IgG or Alexa 637-conjugated goat anti-rabbit, 1/300) in the same buffer for 2 h at room temperature in the dark, and washed before being mounted in Vectashield (Vector Laboratories, Inc. Burlingame, CA 94010) and cover-slipped. For negative controls, primary antibodies were omitted.

  ACE antibody (Abbiotec, San Diego, CA, USA), rabbit anti-angiotensin II (Peninsula laboratories, San Carlos, CA, USA), and Alexa fluor-488 or -637 labelled goat anti-rabbit IgG (Invitrogen, Molecular Probes). All chemicals were from Sigma-Aldrich except apamin (APA) and charybdotoxin (CTX) from Latoxan (Valence, France), and MnTMPyP from

	Alexis Biochemicals Corporation (San Diego, CA, USA). Blackcurrant juice concentrate
	(66.2 °Brix) was kindly provided by Eckes-Granini (Nieder-Olm, Germany). Final
	blackcurrant juice was reconstituted by dilution to 11.6 °Brix in distilled water (3.3 g/l
	polyphenols expressed as GAE measured by the Folin-Ciocalteu method).
	3 (small conductance Ca 2+ -activated K + channel 3,
	SK Ca , Alomone Labs, Jerusalem, Israel), antibody directed against either p22phox, p47phox

NADPH oxidase subunits or AT1 receptors (Santa Cruz Biotechnology, Santa Cruz, USA), purified mouse anti-eNOS, anti-iNOS, and COX-2 monoclonal antibody (BD Transduction Laboratories TM , San Jose, USA), anti-nitrotyrosine (Millipore, Upstate Cell Signaling, USA),

Table 1 .

 1 Effect of chronic PRBJ ingestion on body, liver and spleen weight in both sham and CBDL rats.

		Initial	body	Final	body	Liver	Spleen
		weight		weight	(%	total	(% total weight)
		(g)		(g)	weight)
	Sham	365±19.5		505±30.2	3.24±0.06	0.20±0.02
	Sham+PRBJ 365±23.7		538±29.8	3.14±0.13	0.19±0.01
	CBDL	369±16.3		485±30.5	6.69±0.63*	0.69±0.05*
	CBDL+PRBJ 367±13.8		500±31.2	5.91±0.8	0.56±0.12

#

Values are shown as mean±SEM of 5 rats. * P<0.05 for CBDL vs sham, and # P<0.05 for CBDL+PRBJ vs CBDL.

  1 , Rashid Sherzad 2 , Boehm Nelly 3 , Agin Arnaud 4 , Geny Bernard 1 , Schini-Kerth Valérie 2 , Charloux Anne 1 1 Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologie Thoracique, Hôpitaux Universitaires de Strasbourg et EA 3072, Université de Strasbourg, Faculté de Médecine, Strasbourg, France, mail : monique.oswald@chru-strasbourg.fr (Oswald-Explorations Fonctionnelles, Pôle de Pathologie Thoracique et EA 3072, Hôpitaux Universitaires de Strasbourg, 1, Place de l'Hôpital, BP 426, 67091 Strasbourg Cedex, France Phone : (33) 369551574 Fax : (33) 369551 826 Mail : monique.oswald@chru-strasbourg.fr

	Mammosser	Monique) ;	bernard.geny@chru-strasbourg.fr	(Geny	Bernard) ;
	anne.charloux@chru-strasbourg.fr (Charloux Anne)

2 UMR CNRS 7213, Laboratoire de Biophotonique et de Pharmacologie, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France, mail : valerie.schini-kerth@pharma.ustrasbg.fr (Schini-Kerth Valérie); sherzadnephrology@gmail.com (Rashid Sherzad) 3 Institut d'Histologie et INSERM U1119, Université de Strasbourg, Faculté de Médecine, Strasbourg, France ; mail : nelly.boehm@medecine.u-strasbg.fr 4 Laboratoire d'Hormonologie et Icube, CNRS, FMTS, Université de Strasbourg, Faculté de Médecine, Strasbourg, France ; mail : arnaud.agin@chru-strasbourg.fr Corresponding author: Monique Oswald-Mammosser, Service de Physiologie et d'

Table I :

 I Characteristics of CBDL rats and sham rats, results are given as mean±SEM or median

	(range)
		Initial body	Final	body	Biliary density	Liver weight	Spleen weight
		weight	weight		(% of total area)	(% vs total weight)	(% vs total weight)
		(g)	(g)
	Sham	340.5±3.8	414.4±5.8	0.36 (0.06-1.01)	3.53 (3.14-4.39)	0.25 (0.21-0.32)
	Sham+F 333.6±8.2	404.3±11.5	0.32 (0.12-0.64)	3.58 (3.10-3.69)	0.22 (0.17-0.26)
	CBDL	336.5±6.1	398.1±7.9	20.12 (10.42-43.56)*	7.22 (6.31-8.41)*	0.67 (0.48-0.81)*
	CBDL+F 344.5±5.4	398.9±8.7	19.08 (3.86-39.76) *	7.33 (4.20-8.87) *	0.63 (0.33-0.86) *
	CBLD: chronic bile duct ligated rats, F: rats treated with fulvestrant
	* P<0.001 CBDL vs sham

Table II :

 II Plasma hormonal levels in sham and CBDL rats, results are given as mean±SEM or median

	(range)
		Estradiol	Testosterone	Progesterone
		(ng.L -1 )	(ng.mL -1 )	(nmol.L -1 )
	Sham	4.0 (4.0-4.0) (n=6)	2.64±0.67 (n=7)	4.73 (2.52-6.25) (n=7)
	Sham+F	4.0 (4.0-4.0) (n=5)	2.05±0.38 (n=8)	2.55 (1.06-11.24) (n=8)
	CBDL	4.1 (4.0-15.5) (n=8)	1.00±0.21 (n=10)**	2.76 (1.68-25.39) (n=10)
	CBDL+F	11.6 (6.0-17.9) (n=7)*	1.04±0.48 (n=6) §	2.94 (2.15-17.29) (n=8)
	CBDL: chronic bile duct ligated rats, F: fulvestrant
	* P=0.008 for CBDL vs CBDL+F; ** P=0.017 and § P=0.15 for sham vs CBDL rats

Table III :

 III Infiltration of lung macrophages, small blood vessel diameter and plasma markers in sham and CBDL, results are given as mean±SEM or median (range)

		Nitrite	Lung	Small vessel	COHb	Hematocrit (%)
		(µmol.L -1 )	macrophages (%)	diameter	(%)
				(µm)
	Sham	22.7 (15.7-29.5)	3.4±0.8	21.8±5.7	0.63±0.05	49.6±0.9
		(n=8)	(n=5)	(n=5)	(n=8)	(n=8)
	Sham+F	21.0 (18.7-25.7)	2.3±0.1	22.7±6.2	0.56±0.05	50.6±0.8
		(n=5)	(n=5)	(n=5)	(n=9)	(n=9)
	CBDL	83.7 (51.3-171.9) *	15.2±1.8**	38.6±9.7*	1.21±0.08*	45.5±1.2 #
		(n=10)	(n=9)	(n=8)	(n=8)	(n=8)
	CBDL+F 71.1 (24.7-153.9) *	14.2±0.9*	39.8±9.7*	1.23±0.06*	46.3±1.8 §
		(n=8)	(n=9)	(n=9)	(n=8)	(n=8)
	CBDL: chronic bile duct ligated rats, F: rats treated with fulvestrant, COHb: carboxyhemoglobin
	* P<0.001; ** P=0.007; # P=0.01; § P=0.04 for CBDL vs sham rats

& & & 6 & & ' & & & % ' & 6 & %

PLOS ONE | www.plosone.org

May 2014 | Volume 9 | Issue 5 | e97458

The study was supported by an unrestricted research grant from Actial Farmaceutica Lda (Funchal, Madeira). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. an unrestricted funding support from a commercial source (Actial Farmaceutica Lda),

Author Contributions

Conceived and designed the experiments: MOM VBSK. Performed the experiments: SKR NIK CA MA. Analyzed the data: SKR NIK CA. Contributed reagents/materials/analysis tools: NB. Wrote the paper: SKR MOM VBSK.