Hemodynamic Changes in Splanchnic Blood Vessels in Portal Hypertension

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1 THE ANATOMICAL RECORD 291: (2008) Hemodynamic Changes in Splanchnic Blood Vessels in Portal Hypertension ISABELLE COLLE,* ANJA M. GEERTS, CHRISTOPHE VAN STEENKISTE, AND HANS VAN VLIERBERGHE Department of Hepatology and Gastroenterology, Ghent University Hospital, Ghent, Belgium ABSTRACT Portal hypertension (PHT) is associated with a hyperdynamic state characterized by a high cardiac output, increased total blood volume, and a decreased splanchnic vascular resistance. This splanchnic vasodilation is a result of an important increase in local and systemic vasodilators (nitric oxide, carbon monoxide, prostacyclin, endocannabinoids, and so on), the presence of a splanchnic vascular hyporesponsiveness toward vasoconstrictors, and the development of mesenteric angiogenesis. All these mechanisms will be discussed in this review. To decompress the portal circulation in PHT, portosystemic collaterals will develop. The presence of these portosystemic shunts are responsible for major complications of PHT, namely bleeding from gastrointestinal varices, encephalopathy, and sepsis. Until recently, it was accepted that the formation of collaterals was due to opening of preexisting vascular channels, however, recent data suggest also the role of vascular remodeling and angiogenesis. These points are also discussed in detail. Anat Rec, 291: , Ó 2008 Wiley-Liss, Inc. Key words: portal hypertension; splanchnic; angiogenesis; hemodynamic Portal hypertension (PHT) is the major hemodynamic complication of a variety of diseases that obstruct portal blood flow. Portal pressure gradient (DP) is the result of portal vascular resistance (R) and portal venous inflow (Q) in analogy with Ohm s law: DP = R Q. In normal circumstances the portal pressure gradient varies between 2 and 6 mmhg. Two major theories were put forward to explain portal hypertension. The backward theory assumes that the resistance to portal flow results in portal hypertension (Benoit et al., 1985). Portal vascular resistance consists of the sum of the serial resistance in portal vein and intrahepatic vascular bed and of the parallel resistance of the collaterals. The cause of enhanced vascular resistance can be localized pre-, intra-, and posthepatic. Prehepatic portal hypertension is mostly due to a portal vein thrombosis and is associated with a quite normal liver function. Posthepatically portal hypertension can be caused, for example, by thrombosis of the hepatic veins (Budd Chiari syndrome), occlusion of the caval vein (web), and constrictive pericarditis. The intrahepatic form of portal hypertension can be subdivided into presinusoidal (hepatic schistosomiasis, granulomatosis, i.e., primary biliary cirrhosis and idiopathic portal hypertension), sinusoidal (cirrhosis and fibrosis) and postsinusoidal (veno-occlusive disease). The second theory, the forward theory, proposed an increase in portal inflow as the most important factor leading to portal hypertension (Vorobioff et al., 1984). Portal venous inflow includes the flow of the total portal venous system and the portosystemic collaterals. During the development of portal hypertension, as a result of a hyperdynamic circulation caused by enhanced plasma volume together with a decrease in the splanchnic arteriolar vascular resistance and an increase in cardiac output, an increased blood flow in portal tributaries develops maintaining portal hypertension. *Correspondence to: Isabelle Colle, Department of Hepatology and Gastroenterology, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium. Fax: isabelle.colle@ugent.be Received 14 May 2007; Accepted 19 December 2007 DOI /ar Published online in Wiley InterScience ( com). Ó 2008 WILEY-LISS, INC.

2 700 COLLE ET AL. Presently, the consensus is that both the rise in resistance and the enhanced portal inflow play an important role in the development of portal hypertension. In this review, we will only discuss the hemodynamic changes in the splanchnic vascular bed and the development of portal systemic collaterals, associated with portal hypertension. MECHANISM OF SPLANCHNIC HYPERDYNAMIC CIRCULATION Portal hypertension is associated with a hyperdynamic circulation characterized by increased cardiac output and total blood volume in response to decreased systemic vascular resistance (Moreau et al., 1988; Groszmann, 1993, 1994; Bosch and Garcia-Pagan, 2000; Fig. 1). This diminished systemic vascular resistance is largely the result of a decrease in splanchnic arterial resistance owing to splanchnic vasodilation (Bosch and Garcia- Pagan, 2000). At least three mechanisms have been proposed to contribute to this splanchnic vasodilation in association with portal hypertension: (1) Increased local production of vasodilators; (2) Increased concentrations of systemic circulatory vasodilators and; (3) Decreased vascular response to vasoconstrictors (Groszmann and Abraldes, 2005). Recently, a fourth mechanism of a decrease in splanchnic arterial resistance has been proposed, namely mesenteric neoangiogenesis (Fernandez et al., 2004, 2005; Geerts et al., 2006a). Figure 1 shows the different mechanisms playing a role in portal hypertension and hyperdynamic circulation. Increased Production of Local Vasodilators Early local events. In the early stages of PHT, Abraldes et al. demonstrated in rats with minimal portal hypertension (partial portal vein ligation PPVL over a 16-gauge needle) an early increase (within 24 hr) in vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (enos) expression in the jejunal mucosal microcirculation (Abraldes et al., 2006). This study confirms previous data in severe PHT, that up-regulation of enos precedes the development of splanchnic arterial vasodilation and portal systemic shunts (Abraldes et al., 2006; Wiest et al., 1999a). The up-regulation of VEGF in the intestinal microcirculation accounts in large part for the initial enos activation (Abraldes et al., 2006). After venous pressure elevation, the redistribution of flow within the bowel from the mucosa to the muscularis may cause a certain degree of hypoxia in the mucosa that is sufficient to stimulate VEGF production (Granger et al., 1979, 1989; Davis and Gore, 1985). Because mucosal arterioles account for 25% of the total mesenteric vascular resistance, NO activation at this level, can be the main site for the transduction of the increased portal pressure into splanchnic vasodilation and being the initial step in the development of the hyperdynamic circulation. A second event that occurs in rats with more severe PHT after partial portal vein ligation (PPVL over a 20- gauge needle) is a myogenic response in the superior mesenteric artery (SMA) as early as 10 hr after induction of PHT (Tsai et al., 2003). This SMA vasoconstriction triggers enos catalytic activity and thus NO hyperproduction (Tsai et al., 2003). In contrast, a milder increase in portal pressure did not result in reflex SMA vasoconstriction and enos was not up-regulated at the SMA (Tsai et al., 2003). Iwakiri et al. showed that enos activity was increased before enos expression, suggesting activation of enos at the posttranslational level (Iwakiri et al., 2002a). This increased enos activity is mediated by an Akt-dependent enos phosphorylation at the Serine 1177 level (Iwakiri et al., 2002a). These phenomena may be the early molecular signals that induce the cascade of events leading to the splanchnic hyperdynamic circulation. Later local events. In PHT, once the hyperdynamic circulation with high cardiac output is established, in vivo aortic levels of enos mrna and enos protein are increased, in response to shear stress (Pateron et al., 2000; Tazi et al., 2002). In both studies, treatment with propranolol significantly decreased in vivo aortic enos mrna and protein levels (Pateron et al., 2000; Tazi et al., 2002). Furthermore, in PHT rats there is an increased production of NO in response to shear stress in the superior mesenteric vascular bed (Hori et al., 1998; Wiest et al., 1999a). In cirrhotic rat aortas, endothelial small calcium-dependent potassium channels (SKCa) are overexpressed and overactive in response to shear stress, stimulating the calcium/calmodulin/enos pathway (Barriere et al., 2001). Moreover, shear stress in portal hypertensive aortas results in activation of heat shock protein (Hsp)90, stimulating enos catalytic activity. Hsp90 also contributes to the control of the tone of the mesenteric vascular bed (Shah et al., 1999; Tazi et al., 2002). Intestinal bacterial translocation is common in cirrhosis and is followed by the production of tumor necrosis factor alpha (TNFa) by mononuclear cells. Treatment with norfloxacin (an antibiotic that selectively decontaminates the intestine) in cirrhotic patients and rats (Albillos et al., 2003; Tazi et al., 2005) decreases TNFa production and the hyperdynamic syndrome. Furthermore, cirrhotic rats with bacterial translocation have a more pronounced systemic and splanchnic NO overproduction than those without (Wiest et al., 1999b). Both TNFa and endotoxins may activate inducible NOS (inos) and enos. On one hand, cirrhotic rats have increased levels of aortic inos protein which decrease after treatment with norfloxacin (Tazi et al., 2002, 2005). However, no enhanced levels of inos are found in the splanchnic vasculature of cirrhotic rats with bacterial translocation (Wiest et al., 1999b). Thus probably has inos a minor role in the development of PHT. On the other hand, TNFa in the gastric mucosa of portal hypertensive rats, activates Akt to phosphorylate enos at the Ser 1177 level leading to enos activation (Kawanaka et al., 2002). Moreover, bacterial translocation stimulates the endothelial gene expression of GTPcyclohydrolase I, a key enzyme necessary for the production of tetrahydrobiopterin (BH4; Wiest et al., 2003). The BH4 production is a rate-limiting cofactor in the NO synthesis by enos in the mesenteric arterial bed (Wever et al., 1997; Wiest et al., 2003). Recently, it has been found that neuronal NOS (nnos) protein expression, present in neurons and vas-

3 SPLANCHNIC HEMODYNAMIC CHANGES IN PORTAL HYPERTENSION 701 Fig. 1. Mechanisms of splanchnic vasodilation and hyperdynamic circulation. cular smooth muscle cells, is up-regulated in aorta (Xu et al., 2000) and in mesenteric artery (Jurzik et al., 2005; Kwon et al., 2007) of rats with cirrhosis. Neuronal NOS could also play a role in the development/ maintenance of the hyperkinetic circulation (Xu et al., 2000). Increased Concentrations of Systemic Circulatory Vasodilators Recent investigations indicate that vasodilators synthesized in the splanchnic circulation [nitric oxide (NO), carbon monoxide (CO), glucagon, prostacyclin (PGI 2 ),

4 702 COLLE ET AL. vasoactive intestinal peptide (VIP), adenosine, bile salts, platelet activating factor, substance P, calcitonin generelated peptide, adrenomedullin, atrial natriuretic peptide, endogenous cannabinoids, and others] might be responsible for the hyperdynamic syndrome (Moreau and Lebrec, 1995). At the beginning of the 1990s, Vallance and Moncada suggested the implication of NO, a biologically active gas, in the development of splanchnic vasodilation and the multiple organ malfunctions that characterize the hyperkinetic syndrome (Vallance and Moncada, 1991). We discussed the local splanchnic production of NO in the previous sections, while we discuss here the systemic effects of NO and other vasodilators present in the systemic circulation. Nitric Oxide (NO). Most recent studies focused their attention on the potential role of NO in the pathogenesis of splanchnic vasodilation (Vallance and Moncada, 1991; Hartleb et al., 1997; Martin et al., 1998; Liu and Lee, 1999). Nitric oxide is a very potent vasodilator and is synthesized by NO synthase (NOS) from the amino acid L-arginine (Lowenstein et al., 1994). Three different isoforms of NOS are known: neuronal (nnos), endothelial (enos), and inducible NOS (inos; Moncada et al., 1997). Tonic production of NO by enos is believed to play a major role in the maintenance of an active state of vasodilation in the arterial circulation. A vast number of studies in human and experimental cirrhosis (Niederberger et al., 1995) suggest that NO synthesis is increased and plays an important role in the pathogenesis of arterial splanchnic vasodilation. In patients with ascites, the concentration of NO in peripheral veins is higher than in controls and NO levels in the portal vein are higher than in the peripheral vein, suggesting that NO production is particularly increased in the splanchnic circulation (Battista et al., 1997). Serum nitrite and nitrate, metabolites of NO, and the concentration of NO in exhaled air are also increased in patients with cirrhosis and ascites (Guarner et al., 1993; Matsumoto et al., 1995; Sogni et al., 1995). Increased levels of NO may also play a role in the development of hepatopulmonary syndrome (Fallon et al., 1997). Finally, the infusion of a NOS inhibitor in a peripheral artery of cirrhotic patients with ascites partially restores the impaired reactivity to vasoconstrictors (Albillos et al., 1995; Campillo et al., 1995). Among the three isoforms, enos activation is the major source of NO overproduction in the splanchnic circulation associated with PHT. Multiple factors such as shear stress, inflammatory cytokines (Iwakiri et al., 2002b; Tazi et al., 2003, 2005), and vascular endothelial growth factor (VEGF; Abraldes et al., 2006; Fernandez et al., 2005) stimulate enos-dependent NO production in PHT leading to hyperdynamic circulation. Inducible NOS is produced in macrophages and vascular smooth muscle cells after stimulation by endotoxins and inflammatory cytokines. However, inos is only detected in aorta of cirrhotic rats (Moreau et al., 2002; Tazi et al., 2005), but not in the splanchnic vasculature of experimental animals with PHT and cirrhosis (Cahill et al., 1995; Martin et al., 1996; Morales-Ruiz et al., 1996; Wiest et al., 1999b). From recent data, there is also more evidence that nnos present in neurons and vascular smooth muscle cells, can play an important role in PHT. Neuronal NOS protein expression is up-regulated in cirrhotic rat aorta (Xu et al., 2000) and in the mesenteric artery (Jurzik et al., 2005; Kwon et al., 2007). Treatment with a specific nnos inhibitor decreases the nnos and cgmp levels in the aorta and normalizes the hyperdynamic circulation in cirrhotic rats, suggesting that nnos also plays a role in the maintenance and/or development of the hyperkinetic mesenteric circulation (Xu et al., 2000). Inhibition of NO production only partially inhibits and prevents the development of portosystemic shunts and the hyperdynamic circulation (Lee et al., 1993; Garcia- Pagan et al., 1994; Pilette et al., 1996). This hypothesis is supported by the study of Iwakiri et al., showing that the hyperkinetic syndrome after portal vein ligation still occurs in mice lacking enos (enos2/2) and also develops in double enos/inos knockout mice (Iwakiri et al., 2002b). Prostacyclin (PGI 2 ). Prostacyclin (PGI 2 ) is a product of the metabolism of arachidonic acid by cyclooxygenase (COX) and causes smooth muscle relaxation by stimulation of cyclic adenosine monophosphate (camp). COX-1 is the constitutive form and COX-2 is the inducible form of cyclooxygenase. Whole body production and portal venous levels of PGI 2 are increased in portal hypertensive animals and cirrhotic patients and may play a role in the splanchnic hyperemia, collateral circulation, and portal hypertensive gastropathy (Sitzmann et al., 1994; Ohta et al., 1995). Studies using inhibitors of COX and prostacyclin suggest a pathogenetic role for PGI 2 in the hyperdynamic circulation associated with PHT (Munoz et al., 1999; Tsugawa et al., 1999). Carbon monoxide (CO). Carbon monoxide (CO) is an endogenously produced gas molecule that in a manner similar to NO, activates soluble guanylate cyclase leading to the generation of cgmp, which in turn mediates various physiological functions such as smooth muscle cell relaxation (Wang et al., 1997). CO is produced from the breakdown of heme to biliverdin and free iron by means of the heme-oxygenase (HO) enzyme. Like NOS, HO has three isoforms: inducible HO (HO-1) and two constitutive forms (HO-2 and HO-3; Elbirt and Bonkovsky, 1999). Fernandez et al. found that HO-1 protein levels and activity is significantly increased in the mesentery, spleen, intestine, and liver of portal hypertensive rats (Fernandez and Bonkovsky, 1999; Fernandez et al., 2001). A progressively increased expression of HO-1 is found in mesenteric arteries, aorta and cardiac ventricles from rats with secondary biliary cirrhosis (Liu et al., 2001; Chen et al., 2004). Administration of zinc protoporphyrin, a selective inhibitor of HO, normalizes aortic HO activity, partially restores vascular reactivity to vasoconstrictors and ameliorates the hyperdynamic circulation associated with PHT (Fernandez et al., 2001; Chen et al., 2004). Carbon monoxide also plays a role in the pulmonary vasodilation leading to hepatopulmonary syndrome, with increased levels of carboxyhemoglobin in patients with cirrhosis (Arguedas et al., 2003; Zhang et al., 2003; De Las et al., 2003). Heme oxygenase also mediates hyporeactivity to phenylephrine in the mesenteric vessels of cirrhotic rats with ascites (Bolognesi et al., 2005).

5 SPLANCHNIC HEMODYNAMIC CHANGES IN PORTAL HYPERTENSION Together with NO, carbon monoxide plays a role in splanchnic and pulmonary vasodilation, and both can be seen as portal hypertensive molecules. Whether HO-1 plays a role as protective antioxidant enzyme remains to be elucidated (Moreau, 2001). The precise mechanism whereby HO-1 gene expression is induced is still unknown. However several physical and chemical factors that are present during portal hypertension, including cytokines, endotoxins, and shear stress, can activate HO-1 transcription (Elbirt and Bonkovsky, 1999; Bosch and Garcia-Pagan, 2000). 703 Moreover, it has been shown that endothelial smallconductance Ca 11 -dependent K 1 (SK 1 Ca) channels are overexpressed in cirrhotic aortas (Barriere et al., 2001a). In cirrhosis, selective SK 1 Ca channel blockade by apamin results in a decreased enos hyperactivity and NOdependent smooth muscle relaxation (Barriere et al., 2001). Thus, in cirrhotic arterial walls, activation of K 1 channels located in the plasma membrane of endothelial and smooth muscle cells, induces membrane hyperpolarization, which may contribute to systemic and splanchnic arterial vasodilation. Endocannabinoids. Anandamide is an endogenous lipid ligand that through binding with its cannabinoid CB1 receptor leads to hypotension. Monocytes from cirrhotic patients and rats contain increased levels of anandamide (Batkai et al., 2001; Ros et al., 2002). Thereby, mesenteric vascular CB-1 receptors are maximally activated in cirrhosis (Batkai et al., 2001; Ros et al., 2002). Administration of a CB1 receptor antagonist SR A increases mean arterial pressure (Batkai et al., 2001; Ros et al., 2002), peripheral resistance (Ros et al., 2002), and vascular tone in the mesenteric artery (Domenicali et al., 2005) and decreases portal hypertension and mesenteric blood flow in cirrhotic rats (Batkai et al., 2001). Activation of CB-1 receptors may lead on one hand to enos stimulation and production of NO and on the other hand to potassium channel activation, both leading to vasodilation (Batkai et al., 2001; Ros et al., 2002; Howlett et al., 2002). Endothelium-derived hyperpolarizing factor (EDHF). In normal arterial walls exposed to NOS/cyclooxygenase (COX)-inhibitors, acetylcholine or shear stress induce the release of an endothelium-derived relaxing factor (EDRF; Cohen and Vanhoutte, 1995). This NOS/COX independent EDRF has been called endothelium-derived hyperpolarizing factor or EDHF, because it induces hyperpolarization and arterial vascular smooth muscle cell relaxation (Cohen and Vanhoutte, 1995). The exact nature of EDHF is controversial; however, the main molecules considered to explain EDHFmediated vasodilation are monovalent cation potassium, arachidonic acid metabolites, components of gap junctions, and hydrogen peroxidase (Iwakiri and Groszmann, 2006). EDHF is more prominent in smaller arteries and arterioles than in larger arteries, and its role becomes more important in the absence of NO (Iwakiri and Groszmann, 2006). Thus, the presence of EDHF can explain why hyperdynamic circulation still develops in portal hypertensive enos/inos knockout mice (Iwakiri et al., 2002b). In cirrhotic rats, EDHF is released by the superior mesenteric artery but not in the aorta (Barriere et al., 2000). In this cirrhotic mesenteric artery, EDHF-induced smooth muscle cell relaxation is abolished by a combination of apamin and charybdotoxin and decreased by barium or ouabain (Barriere et al., 2000). Thus, in the superior mesenteric artery from cirrhotic rats, EDHF may be ak 1 ion released by endothelial apamin- and charybdotoxin sensitive K 1 channels, K 1 then activating barium sensitive K 1 channels and Na 1 /K 1 ATPase in the smooth muscle cells leading to hyperpolarization and relaxation of the vascular wall (Barriere et al., 2000). Hydrogen sulfate (H 2 S). Recently it has been suggested that H 2 S is a potent endogenous vasodilator (derived from L-cysteine) in mesenteric arteries and aorta (Hosoki et al., 1997; Zhao and Wang, 2002; Cheng et al., 2004). This H 2 S-mediated vasodilation occurs by means of opening of K 1 ATP channels and thus independently of the cgmp pathway (Zhao et al., 2001). The role of H 2 S is postulated in the vascular abnormalities seen in cirrhosis; however, more studies are needed to confirm this hypothesis (Ebrahimkhani et al., 2005). Other systemic vasodilators. In portal hypertension, despite NOS and COX inhibition, arterial vasodilation, and vascular hyporeactivity are not totally suppressed, suggesting the presence of NOS/COX independent vasodilators (Moreau and Lebrec, 1995). Increased plasma levels of natriuretic peptides, glucagon, adrenomedulin, calcitonin gene-related peptide, substance P, and vasoactive intestinal peptide have been described in cirrhosis (Moreau and Lebrec, 1995, 2005). Probably, other vasodilators will be discovered in the future. Vascular Hyporesponsiveness to Vasoconstrictors In cirrhosis and portal hypertension, the presence of splanchnic vasodilation in the face of highly elevated levels of circulating vasoconstrictors (angiotensin II, norepinephrin, endothelin, vasopressin, and so on), can be explained by vascular hyporesponsiveness. This splanchnic resistance to vasoconstrictor agents (Lee et al., 1992; Sieber et al., 1993) explains why the hyperdynamic circulation increases with progression of the disease despite the stimulation of renin-angiotensin, sympathetic nervous system, and vasopressin release. In contrast, these systems induce vasoconstriction in other organs, such as the brain and kidneys in patients with ascites (Fernandez-Seara et al., 1989; Guevara et al., 1998; Maroto et al., 1993; Maroto et al., 1994). A large number of studies have described hyporesponsiveness in cirrhosis and portal hypertension to different vasoconstrictors (methoxamine, potassiumchloride, phenylephrine, terlipressin, vasopressin, endothelin-1, angiotensin-ii, norepinephrine; Michielsen et al., 1995a; Atucha et al., 1996; Sogni et al., 1996, 1997; Heinemann et al., 1997; Schepke et al., 2001; Chu et al., 2000; Barriere et al., 2001; Colle et al., 2004b). Arterial hyporeactivity to vasoconstrictors may also differ from one vascular bed to another: in cirrhotic rats vascular hyporesponsiveness occurs in the superior mesenteric artery and in the aorta, but is normal in carotid artery (Pateron et al., 1999).

6 704 COLLE ET AL. The increased concentrations of local and systemic vasodilators (NO, HO, and adrenomedullin) as described above, are probably the cause of this hyporeactivity (Kojima et al., 2004; Bolognesi et al., 2005; Erario et al., 2005). However, the molecular mechanisms of this vascular hyporesponsiveness are not well understood and are multiple (Bomzon and Huang, 2001; Moreau, 2001). Vascular hyporeactivity to most relevant endogenous vasoconstrictors in the hepatic artery of cirrhotic patients is not caused by a down regulation of a1-adrenoreceptors a, b, and c; angiotensin II receptor I; vasopressin V1a receptor, and endothelin A and B receptors. These vasoconstrictor receptors are even up-regulated in the hepatic artery (Neef et al., 2003). Under normal conditions, splanchnic arterioles are partially constricted and have the capacity to either further constrict or dilate. The basal contractile state (tone) of arteriolar smooth muscle cells reflects the balance of multiple influences that either cause relaxation or constriction of the vascular smooth muscle cell. Important vasoconstrictors influencing splanchnic arterioles include some circulating agents (e.g., angiotensin II), myogenic factors, certain endothelium-derived substances (e.g., endothelin), and some neurotransmitters (e.g., norepinephrine). All vasoconstrictor receptors belong to the superfamily of guanine nucleotide-binding protein (G-protein) -coupled receptors (GPCR). Stimulation of GPCR on the vascular smooth muscle cell activates G proteins and consequently phospholipase C (PLC) -b. PLC hydrolyzes phosphatidylinositol 4,5-biphosphate into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses in the cytosol and DAG remains in the plasma membrane activating protein kinase C (PKC). Both products cause an increase in intracellular calcium in the vascular smooth muscle cell. The released calcium initiates a cascade of intracellular events, resulting in cross bridging of actin and myosin, leading to contraction (Bomzon and Huang, 2001; Cahill et al., 2001). Figure 2 shows schematically the mechanism of vasoconstriction. Different possible mechanisms contribute to this hyporeactivity to vasoconstrictors. In normal circumstances NO stimulates the production of cgmp which activates cgmp dependent serine-threonine protein kinase called PKG (Lincoln and Cornwell, 1993). PKG inhibits the GPCR signaling pathway used by vasoconstrictors at different levels: (1) PKG increases GTPase activity terminating vasoconstrictor signalling (Tang et al., 2003); (2) PKG may phosphorylate GPCR and thus uncouple the receptor and G-proteins (Lincoln and Cornwell, 1993; Moreau and Lebrec, 2005). In portal hypertension, there is a decreased ex vivo production of IP3 and DAG in response to vasoconstrictors using GPCR in portal hypertensive arteries (Moreau and Lebrec, 1995). Also ex vivo enzymatic activities of PKC-a and PKC-d are decreased in cirrhotic vascular smooth muscle cells (Tazi et al., 2000). Moreover, in vivo PKC-a protein levels are decreased in portal hypertensive aortas (Moreau and Lebrec, 1995; Bomzon and Huang, 2001). In endothelium-denuded hepatic arteries from cirrhotic patients, ex vivo exposure to different vasoconstrictors shows hyporeactivity to some but not to others (Heller et al., 1999). These results suggest that both effects of vasodilators on the vasoconstrictive system, but also that abnormalities within the smooth muscle and endothelial cells may be responsible for this vascular hyporesponsiveness to vasoconstrictors. This has been extensively reviewed in two articles of Cahill et al. and Bomzon et al. (Bomzon and Huang, 2001; Cahill et al., 2001). Recently, the presence of neuropeptide Y1 (NPY) in the superior mesenteric artery in PHT was observed. NPY becomes increasingly important and increased release of NPY may represent a compensatory mechanism to counterbalance arterial vasodilation by restoring the efficacy of endogenous cathecholamines, especially in states of high levels of alfa1-adrenergic activity (Wiest et al., 2006, 2007). Vascular Responsiveness to Vasodilators While for most vasoconstrictors there is an impaired vascular response, conflicting results concerning the response (hypo-, hyper-, and normal response) of the splanchnic vascular bed to different vasodilators have been reported (Tables 1 and 2). By using endotheliumdependent (acetylcholine) and -independent agents [pinacidil, potassium ATP (KATP) channel opener; deta- NONOate and sodiumnitroprusside as NO donors], the integrity of the endothelium and the vascular smooth muscle cell can be evaluated. Figure 3 shows a simplified scheme of different action mechanisms of vasoactive agents. Our group found also an in vivo hyporeactivity of the mesenteric vascular bed for acetylcholine, pinacidil, detanonoate and sildenafil, which persisted after NOS and COX inhibition (Colle et al., 2004a,b). Hyperresponse to vasodilators can contribute to further splanchnic vasodilation, while hyporesponse to vasodilators can be seen as a defence against further aggravation of the hyperdynamic circulation. Increased Splanchnic Angiogenesis Introduction to angiogenesis. During embryogenesis and organogenesis blood vessel formation occurs by aggregation of de novo forming angioblasts or endothelial progenitor cells into a primitive vascular plexus (vasculogenesis), which then undergoes a complex remodeling process, in which growth, migration and sprouting lead to the development of a functional circulatory system (angiogenesis; Carmeliet, 2000). During adulthood in normal situations, angiogenesis occurs only in the cycling ovary and in the placenta during pregnancy. In pathological situations in response to hypoxia and/or inflammation, angiogenesis is reactivated during wound healing and repair. However, this stimulus can become excessive, and the balance between stimulators and inhibitors turns to a pathological angiogenic switch (best known condition is malignant tumor formation) (Carmeliet, 2000, 2003). The vascular endothelial growth factor (VEGF) family and their VEGF receptors (VEGFRs) are considered as the most important factors involved in angiogenesis and receive thereby most of the attention in the current literature (Carmeliet, 2003; Ferrara et al., 2003). VEGF-A has been recognized as the major relatively specific growth factor for endothelial cells and exhibits two major biological activities: first is the capacity to stimulate vascular endothelial cell proliferation, and second is

7 SPLANCHNIC HEMODYNAMIC CHANGES IN PORTAL HYPERTENSION 705 Fig. 2. Vasoconstriction: Schematic presentation of the interactions between endothelial and smooth muscle cell. the ability to increase vascular permeability (Carmeliet, 2000, 2003). In addition to VEGF, placental growth factor (PlGF), originally discovered in human placenta in 1991, plays an important role in pathological circumstances. Loss of PlGF impairs angiogenesis in the ischemic retina, limb, heart, wounded skin, and in cancer, whereas administration of recombinant PlGF (rplgf) promotes collateral

8 706 COLLE ET AL. TABLE 1. Different reactivities in response to acetylcholine Model Methods Author Reference Hyporeactivity CBDL Aortic and SMA rings Barriere 2001 PPVL Aortic rings Atucha 1996 CCl 4 PPVL Aortic rings Karatapanis 1994 PPVL Aortic rings Michielsen 1995a CBDL Aortic rings Rastegar 2001 CBDL Mesenteric superior artery Colle 2004a PPVL Hyperreactivity Human Forearm Albillos 1995 PPVL Preconstricted Aortic rings Gadano 1999 PPVL Preconstricted isolated perfused SMA Heinemann 1996 Normal reactivity CBDL Pulmonary artery Chabot 1996 PPVL Preconstricted renal artery Garcia-Estan 1996 CCl 4 Isolated perfused SMA Mathie 1996 PPVL 6m Isolated Aortic rings Michielsen 1995b CBDL Preconstricted Aortic rings Ortiz 1996 PPVL Preconstricted SMA rings Sogni 1996 CBDL Preconstricted Aortic rings Sogni 1997 SMA 5 superior mesenteric artery; CBDL 5 common bile duct ligation; PPVL 5 partial portal vein ligation; CCl 4 5 carbon tetrachloride. TABLE 2. Different reactivities in response to other vasodilators than acetylcholine Model Method Substance Author Reference Hyporeactivity CBDL Gastric blood flow in vivo Nitroprusside Geraldo 1996 CCl 4 Isolated perfused SMA Nitroprusside Mathie 1996 CBDL Hemodynamics Nitroprusside Safka 1997 CBDL Hemodynamics Prostacyclin Safka 1997 CBDL Hemodynamics Prostacyclin Oberti 1993 PPVL CBDL Hemodynamics Aprikalim Safka 1997 CBDL Hemodynamics Diltiazem Safka 1997 Human Hepatic artery Isoproterenol Heller 1999 PPVL Mesenteric vein Salbutamol Martinez-Cuesta 1996 CBDL Hemodynamics and isolated SMA VIP Lee 1996 CBDL Superior mesenteric artery Pinacidil Colle 2004a PPVL DetaNONOate CBDL SMA Sildenafil Colle 2004b Hyperreactivity PPVL Preconstricted aortic rings NaF Hou 1997 PPVL Preconstricted isolated perfused SMA SIN1 Heinemann 1996 Human Portal vein Isoproterenol Heller 1999 Normal reactivity PPVL Perfused mesenteric bed Nitroprusside Atucha 1996 CBDL Human Forearm Nitroprusside Albillos 1995 CBDL Pulmonary artery Nitroprusside Chabot 1996 PPVL Preconstricted renal artery Nitroprusside Garcia-Estan 1996 CBDL Aortic rings Nitroprusside Rastegar 2001 CBDL Hemodynamics Nicardipine Safka 1997 CBDL Hemodynamics Verapamil Safka 1997 PPVL Preconstricted isolated perfused SMA Foskolin Heinemann 1996 PPVL Aortic rings Glycerilnitrate Karatapanis 1994 SMA 5 superior mesenteric artery; CBDL 5 common bile duct ligation; PPVL 5 partial portal vein ligation; CCl 4 5 carbon tetrachloride; VIP 5 vasoactive intestinal peptide; NaF 5 sodium fluoride; SIN morpholino-sydnonimine (NO donor). vessel growth in models of myocardial and limb ischemia (Carmeliet et al., 2001; Carmeliet, 2003; Autiero et al., 2003). Three receptor tyrosine kinases, binding the VEGF family members have thus far been identified. VEGF receptor-1 (or Flt-1, Fms-like tyrosine kinase-1) binds VEGF-A and PlGF; VEGF receptor-2 (or Flk-1, fetal liver kinase-1) binds VEGF-A and VEGF-C and VEGF receptor-3 (or Flt-4) binds VEGF-C. The major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF-A is VEGFR-2 (Carmeliet et al., 2001, 2003; Autiero et al., 2003). Role of VEGF in splanchnic hyperdynamic circulation. Fukumura et al. demonstrated that VEGF induces NO production by means of activation of enos protein expression and activity (Fukumura et al., 2001). Recently, Abraldes et al. showed that VEGF up-regula-

9 SPLANCHNIC HEMODYNAMIC CHANGES IN PORTAL HYPERTENSION tion in the intestinal mucosal microcirculation accounts largely for the initial enos up-regulation in mild PHT, which precedes the development of vasodilation and the development of portosystemic shunting in mild PHT (Abraldes et al., 2006). Moreover, our group also found increased levels of VEGF in mesenteric tissue of rats with PHT and cirrhosis (Geerts et al., 2006a). This was associated with an increased vascular permeability, only detected in cirrhotic rats but not in pure portal hypertensive rats (Geerts et al., 2006a). 707 Role of angiogenesis in splanchnic hyperdynamic circulation. Nevertheless, these functional alterations (especially vasodilation) described above can not fully explain the observed sustained splanchnic vasodilation. In addition to these functional changes, probably also structural vascular changes are implicated in portal hypertension. Previous studies provided evidence for increased angiogenesis and VEGF production in the splanchnic territory of portal hypertensive rats and cirrhotic patients (Perez-Ruiz et al., 1999; Sumanovski et al., 1999; Cejudo-Martin et al., 2001; Sieber et al., 2001). Recently, our group was able to show an in vivo increased angiogenesis in the mesenteric microcirculation of rats with PHT with and without cirrhosis (Fig. 4)(Geerts et al., 2006a). This increased mesenteric angiogenesis was associated with an increased VEGF and enos protein expression (Geerts et al., 2006a). We could also demonstrate that neo-angiogenesis is present in the mesentery of portal hypertensive mice, and is associated with an up-regulation of VEGF and PlGF protein levels in the mesentery (Geerts et al., 2006a,b)(both oral presentations). PlGF knockout (PlGF2/2) portal hypertensive mice do not develop neo-angiogenesis in the mesentery and have lower portal venous pressures compared with the control portal hypertensive mice (Geerts et al., 2006a,b). These findings confirm the assumption that chronic portal hypertension induces structural, as well as the well-described functional vascular changes. Sieber demonstrated that this increased mesenteric angiogenesis could be reversed by chronically inhibiting NO formation (Sieber et al., 2001). Recently, Fernandez et al. showed that blocking of the VEGF-receptor 2 signaling pathway in portal vein-stenosed mice and rats (using anti-vegfr-2 monoclonal antibodies or using VEGFR-2 autophosphorylation inhibition, both for 5 7 days after surgery) resulted in a significant decrease in the number of mesenteric blood vessels (shown by splanchnic protein levels of CD31) and VEGFR2 protein expression (Fernandez et al., 2004, 2005). These results were accompanied by an increase in splanchnic arteriolar and portal venous resistance resulting in a decreased portal venous inflow, however, portal pressure remained high (Fernandez et al., 2004, 2005). These studies further contribute to the hypothesis that a decrease in VEGF- and PlGF-dependent angiogenesis could reduce vascular density, splanchnic blood flow and thus portal venous inflow in partial portal veinligated animals (Fernandez et al., 2004, 2005; Geerts et al., 2006b). The mechanisms by which VEGF protein expression in portal hypertension is increased are probably multifactorial. Indeed, several factors relevant to the pathogenesis of portal hypertension, such as hypoxia, cytokines, and mechanical stress, have been shown to promote VEGF expression in various cell types and tissues (Carmeliet, 2000, 2003). We can assume that increased portal pressure (even if it is minimal) is the initial factor that triggers VEGF and enos expression in the portal system (Abraldes et al., 2006), which is followed by increased blood flow further exaggerating VEGF overexpression, resulting in enhanced angiogenesis. Recently, a role for NAD(P)H oxidase (a major source of reactive oxygen species) and for the enzyme heme-oxygenase-1 (HO) was suggested to contribute to the angiogenic stimulus in PHT (Abraldes et al., 2006; Angermayr et al., 2006, 2007). Chronic HO inhibition significantly decreased VEGF protein expression in the mesentery of portal hypertensive rats, suggesting that HO enzymatic activity is an important stimulus for VEGF production in portal hypertension (Angermayr et al., 2006). Also, hypoxia inducible factor (HIF) plays probably an initiating role in the activation of VEGF. Role of angiogenesis in other organs associated with PHT. Besides mesenteric angiogenesis there is evidence for up-regulation of angiogenic factors in other splanchnic organs. In patients and in animal models there is an increased expression of VEGF in portal hypertensive gastric mucosa and can be involved in the development of portal hypertensive gastropathy (Tsugawa et al., 2000, 2001). Moreover, Yin et al. demonstrated higher levels of VEGF in the esophagus of portal hypertensive rats (Yin et al., 2005). At this moment, this domain needs further investigation. COLLATERAL CIRCULATION The development of portal hypertension is associated with changes in both the venous and arterial splanchnic circulation. In the venous circulation, portosystemic collaterals are formed which cause shunting of blood from the portal to the systemic circulation. The development of portosystemic shunts, as a compensatory mechanism to decompress the portal circulation and pressure, is responsible for major complications such as encephalopathy, sepsis and bleeding from gastrointestinal varices. At the arterial side, there is an important vasodilation increasing portal venous inflow. By this mechanism, portal pressure remains high, despite the formation of an extensive network of collaterals. Until recently, it was thought that the development of collateral circulation was due to the opening of preexisting vascular channels in response to increased portal pressure, a physiological process which includes NO-mediated vasodilation activated by shear stress and VEGF. Accordingly, all therapeutic strategies are aimed to decrease portal blood inflow and thus pressure. Portosystemic shunting was inhibited by NOS inhibitors in portal vein stenosed rats (Mosca et al., 1992; Lee et al., 1993; Chan et al., 1999). Nonselective b-blockers not only reduce cardiac output but also constrict the collateral circulation (azygos blood flow; Cales et al., 1985a,b) leading to a decreased portal pressure. The administration of propranolol also decreases shear stress and consequently attenuates enos production and systemic arterial vasodilation (Tazi et al., 2002).

10 708 COLLE ET AL. Vasodilation: Schematic presentation of the interactions between endothelial and smooth mus- Fig. 3. cle cell.

11 SPLANCHNIC HEMODYNAMIC CHANGES IN PORTAL HYPERTENSION 709 However, not only vasodilation of pre-existing vessels may be involved in collateral formation, but also vascular remodelling as a long term adaptive response to allow chronic increased blood flow and pressure. In blood vessels of cirrhotic rats, there is a decreased wall thickness, which is reversed by NOS inhibition (Fernandez- Varo et al., 2003). Finally, recent evidence shows the predominant role for angiogenic processes in collateral vessel formation (Fernandez et al., 2004, 2005). Angiogenesis is mostly dependent on VEGF, the major growth factor for blood vessels. VEGF acts through NO-dependent mechanisms as NO is an important downstream mediator of VEGF that facilitates vasodilation and endothelial cell proliferation and migration (Carmeliet, 2000; Fukumura et al., 2001). Additionally, NO also stimulates the release of endothelial progenitor cells from the bone marrow, thereby contributing to vasculogenesis, necessary for the synthesis of de novo vessels (Carmeliet, 2000; Urbich and Dimmeler, 2004). Experimental inhibition of NO formation appears to antagonize the angiogenic response and to reduce flow and shunting through existing portal-systemic collateral vessels (Sumanovski et al., 1999; Sieber et al., 2001). Mechanical forces, most notably shear stress, but also endotoxemia both stimulate NO generation and are thus important in collateral vessel formation. The implication of VEGF/VEGFR-2 pathway in angiogenesis and the formation of collateral circulation was supported by two studies of Fernandez et al. (2004, 2005). The administration of a monoclonal antibody against VEGF-receptor 2 and an inhibitor of VEGF receptor-2 activation, both resulted in a 50% decrease in the formation of portal-systemic collateral vessels in portal hypertensive animal models (Fernandez et al., 2004, 2005). Both studies suggest that VEGFR2-mediated angiogenesis plays an important role in the development of portosystemic shunts and hyperdynamic splanchnic circulation. Importantly, in both studies the portal pressure remained high in portal hypertensive animals despite reduction in mesenteric blood flow. The increase in arterial mesenteric and portal venous resistance, caused possibly by diminishing the number of splanchnic blood vessels and collaterals can be the cause of this phenomenon. So, decreased mesenteric blood flow and increased splanchnic vascular resistance results in almost no change in portal pressure. Fig. 4. Intravital microscopy images of the microvascular density in the mesentery of Sham-operated rats (control rats), portal hypertensive rats (induced by partial portal vein ligation, PPVL), and cirrhotic rats (induced by common bile duct ligation, CBDL). In Sham rats, a normal vasculature is present. In portal hypertensive and cirrhotic rats, the vascular network is irregular and dense arranged. Areas of intense capillary proliferation are seen. CONCLUSIONS In conclusion, portal hypertension is a result of an increased portal blood inflow and increased portal resistance. The increased portal blood flow is due to a process of splanchnic vasodilation and mesenteric neo-angiogenesis. The splanchnic vasodilation is due to (1) Increased splanchnic arterial enos-mediated NO production; (2) Increased inos and nnos-mediated NO production and release of other systemic vasodilators such as endocannabinoids, CO, prostaglandins, glucagons, and others; (3) Hyporesponsiveness of the splanchnic vascular bed toward vasoconstrictors; and finally (4) Mesenteric angiogenesis mediated by vascular endothelial growth factor and placental growth factor. Portosystemic collateral formation related to portal hypertension is a result of the dilation of pre-existing blood vessels, vascular remodeling, and also neo-angiogenesis. Endothelial NOS and VEGF appear to be two important players in these events. Inhibition of splanchnic angiogenesis can be a novel approach to prevent the complications of portal hypertension such as formation of a collateral circulation

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