**The Molecules: Abnormal Vasculatures in the Splanchnic and Systemic Circulation in Portal Hypertension**

Yasuko Iwakiri *Yale University School of Medicine, USA* 

#### **1. Introduction**

Portal hypertension, defined as an increase in pressure within the portal vein, is a detrimental complication in liver diseases. The increased intrahepatic resistance as a consequence of cirrhosis is the primary cause of portal hypertension (Figure 1). Once it is developed, portal hypertension influences extrahepatic vascular beds in the splanchnic and systemic circulation. Two major consequences of portal hypertension in this regard are excessive arterial vasodilation/hypocontractility and the formation of portosystemic collateral vessels. Both excessive arterial vasodilation and portosystemic collateral vessel formation help to increase the blood flow through the portal vein and worsen portal hypertension. This facilitates the development of the abnormal hemodynamic condition, called the hyperdynamic circulatory syndrome, and ultimately leads to variceal bleeding and ascites (Bosch 2000; Bosch 2007; Groszmann 1993; Iwakiri 2011; Iwakiri & Groszmann 2006).

Fig. 1. Overview of portal hypertension.

This chapter summarizes current knowledge of molecules and factors that play critical roles in the development and maintenance of excessive arterial vasodilation and portosystemic collateral vessels in the splanchnic and systemic circulation in cirrhosis and portal

The Molecules: Abnormal Vasculatures in the

**2.1.1.1 Heat shock protein 90 (Hsp90)** 

interactions and posttranslational modifications.

superior mesenteric artery in portal hypertensive rats.

**2.1.1.2 Tetrahydrobiopterin (BH4)** 

excessive arterial vasodilation. **2.1.1.3 Akt/protein kinase B** 

portal hypertensive rats.

necrosis factor alpha.

Splanchnic and Systemic Circulation in Portal Hypertension 3

activity in the superior mesenteric artery (i.e., an artery of the splanchnic circulation) of

This figure shows a general idea of eNOS regulation, not limited to portal hypertension. Caveolin-1 inhibits eNOS activity, while eNOS is activated through interactions with heat shock protein 90 (Hsp90), tetrahydrobiopterin (BH4), guanosine triphosphate (GPT) and calcium calmodulin (CaM). Additionally, eNOS is phosphorylated and activated by Akt, also known as protein kinase B. VEGF; vascular endothelial growth factor, TNF; tumor

Fig. 2. Endothelial nitric oxide synthase (eNOS) is regulated by complex protein-protein

A molecular chaperone, Hsp90, acts as a mediator of a signaling cascade leading to eNOS activation (Garcia-Cardena, et al. 1998). In the superior mesenteric artery isolated from portal hypertensive rats, an Hsp90 inhibitor, geldanamycin (GA), partially attenuated excessive vasodilation (Shah, et al. 1999). This observation suggests that Hsp90, at least in part, plays a role in elevated activation of eNOS, which causes overproduction of NO in the

eNOS requires BH4 for its activity (Cosentino & Katusic 1995; Mayer & Werner 1995). Cirrhosis increases circulating endotoxin, which elevates activity of guanosine triphosphate (GPT)-cyclohydrolase I, an enzyme that generates BH4. One study shows that increased levels of BH4, as a result of cirrhosis, enhance eNOS activity in the superior mesenteric artery (Wiest, et al. 2003). Thus, an increase in BH4 production in the superior mesenteric artery of cirrhotic rats is thought to be one of the mechanisms by which eNOS contributes to

Akt, a serine/threonine kinase, can directly phosphorylate eNOS on Serine1177 (human) or Serine1179 (bovine) and activates eNOS, leading to NO production (Dimmeler, et al. 1999; Fulton, et al. 1999). We have shown that portal hypertension increases eNOS

hypertension. The chapter concludes with a brief discussion about the future directions of this area of study.

#### **2. Key molecules and factors – Excessive arterial vasodilation/hypocontractility**

This section addresses molecules and factors that are involved in the development and maintenance of excessive arterial vasodilation/hypocontractility in cirrhosis and portal hypertension.

#### **2.1 Key molecules**

The molecules discussed here include nitric oxide (NO), carbon monoxide (CO), prostacyclin (PGI2), endocannabinoids, Endothelium-derived hyperpolarizing factor (EDHF), adrenomedullin, tumor necrotic factor alpha (TNF), bradykinin and urotensin II. In addition to these vasodilatory molecules, decreased response to vasoconstrictors, such as neuropeptide Y, also contributes to hypocontractility of mesenteric arterial beds (i.e., arteries of the splanchnic circulation).

#### **2.1.1 Nitric oxide**

Nitric oxide (NO) is the most potent vasodilatory molecule in vessels and contributes to excessive arterial vasodilation in the splanchnic and systemic circulation in portal hypertension perhaps to the most significant degree. NO, synthesized by endothelial NO synthase (eNOS) in the endothelium, defuses into smooth muscle cells and activates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP) (Arnold, et al. 1977; Furchgott & Zawadzki 1980; Ignarro, et al. 1987), facilitating vessel relaxation.

In portal hypertension, elevated eNOS activity causes overproduction of NO and the resultant excessive arterial vasodilation in the splanchnic and systemic circulation. As for the other two NOS isoforms, neuronal NOS (nNOS) and inducible NOS (iNOS), a couple of studies suggest that nNOS, which resides in the nerve terminus and smooth muscle cells of the vasculature, also contributes to excessive arterial vasodilation in portal hypertension, although its effect is small (Jurzik, et al. 2005; Kwon 2004). In contrast to eNOS and nNOS, which are constitutively expressed, iNOS is generally expressed in the presence of endotoxin and inflammatory cytokines and generates a large amount of NO. Interestingly, however, despite the presence of bacterial translocation and endotoxin in cirrhosis, iNOS has not been detected in arteries of the splanchnic and systemic circulation in cirrhosis and portal hypertension (Fernandez, et al. 1995; Heinemann & Stauber 1995; Iwakiri, et al. 2002; Morales-Ruiz, et al. 1996; Sogni, et al. 1997; Weigert, et al. 1995; Wiest, et al. 1999). This paradox remains to be elucidated. Accordingly, eNOS would be the most important among the three isoforms of NOS for excessive vasodilation observed in arteries of the splanchnic and systemic circulation in portal hypertension (Iwakiri 2011; Iwakiri & Groszmann 2006; Wiest & Groszmann 1999).

eNOS is regulated by complex protein-protein interactions, posttranslational modifications and cofactors (Sessa 2004). A summary of mechanisms that activate eNOS is shown in Figure 2. Below presented are several proteins that have been reported to increase eNOS activity in the superior mesenteric artery (i.e., an artery of the splanchnic circulation) of portal hypertensive rats.

#### **2.1.1.1 Heat shock protein 90 (Hsp90)**

2 Portal Hypertension – Causes and Complications

hypertension. The chapter concludes with a brief discussion about the future directions of

This section addresses molecules and factors that are involved in the development and maintenance of excessive arterial vasodilation/hypocontractility in cirrhosis and portal

The molecules discussed here include nitric oxide (NO), carbon monoxide (CO), prostacyclin (PGI2), endocannabinoids, Endothelium-derived hyperpolarizing factor (EDHF), adrenomedullin, tumor necrotic factor alpha (TNF), bradykinin and urotensin II. In addition to these vasodilatory molecules, decreased response to vasoconstrictors, such as neuropeptide Y, also contributes to hypocontractility of mesenteric arterial beds (i.e., arteries

Nitric oxide (NO) is the most potent vasodilatory molecule in vessels and contributes to excessive arterial vasodilation in the splanchnic and systemic circulation in portal hypertension perhaps to the most significant degree. NO, synthesized by endothelial NO synthase (eNOS) in the endothelium, defuses into smooth muscle cells and activates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP) (Arnold, et al.

In portal hypertension, elevated eNOS activity causes overproduction of NO and the resultant excessive arterial vasodilation in the splanchnic and systemic circulation. As for the other two NOS isoforms, neuronal NOS (nNOS) and inducible NOS (iNOS), a couple of studies suggest that nNOS, which resides in the nerve terminus and smooth muscle cells of the vasculature, also contributes to excessive arterial vasodilation in portal hypertension, although its effect is small (Jurzik, et al. 2005; Kwon 2004). In contrast to eNOS and nNOS, which are constitutively expressed, iNOS is generally expressed in the presence of endotoxin and inflammatory cytokines and generates a large amount of NO. Interestingly, however, despite the presence of bacterial translocation and endotoxin in cirrhosis, iNOS has not been detected in arteries of the splanchnic and systemic circulation in cirrhosis and portal hypertension (Fernandez, et al. 1995; Heinemann & Stauber 1995; Iwakiri, et al. 2002; Morales-Ruiz, et al. 1996; Sogni, et al. 1997; Weigert, et al. 1995; Wiest, et al. 1999). This paradox remains to be elucidated. Accordingly, eNOS would be the most important among the three isoforms of NOS for excessive vasodilation observed in arteries of the splanchnic and systemic circulation in portal hypertension (Iwakiri 2011; Iwakiri & Groszmann 2006;

eNOS is regulated by complex protein-protein interactions, posttranslational modifications and cofactors (Sessa 2004). A summary of mechanisms that activate eNOS is shown in Figure 2. Below presented are several proteins that have been reported to increase eNOS

1977; Furchgott & Zawadzki 1980; Ignarro, et al. 1987), facilitating vessel relaxation.

**2. Key molecules and factors – Excessive arterial** 

this area of study.

hypertension.

**2.1 Key molecules** 

**2.1.1 Nitric oxide** 

of the splanchnic circulation).

Wiest & Groszmann 1999).

**vasodilation/hypocontractility** 

This figure shows a general idea of eNOS regulation, not limited to portal hypertension. Caveolin-1 inhibits eNOS activity, while eNOS is activated through interactions with heat shock protein 90 (Hsp90), tetrahydrobiopterin (BH4), guanosine triphosphate (GPT) and calcium calmodulin (CaM). Additionally, eNOS is phosphorylated and activated by Akt, also known as protein kinase B. VEGF; vascular endothelial growth factor, TNF; tumor necrosis factor alpha.

Fig. 2. Endothelial nitric oxide synthase (eNOS) is regulated by complex protein-protein interactions and posttranslational modifications.

A molecular chaperone, Hsp90, acts as a mediator of a signaling cascade leading to eNOS activation (Garcia-Cardena, et al. 1998). In the superior mesenteric artery isolated from portal hypertensive rats, an Hsp90 inhibitor, geldanamycin (GA), partially attenuated excessive vasodilation (Shah, et al. 1999). This observation suggests that Hsp90, at least in part, plays a role in elevated activation of eNOS, which causes overproduction of NO in the superior mesenteric artery in portal hypertensive rats.

#### **2.1.1.2 Tetrahydrobiopterin (BH4)**

eNOS requires BH4 for its activity (Cosentino & Katusic 1995; Mayer & Werner 1995). Cirrhosis increases circulating endotoxin, which elevates activity of guanosine triphosphate (GPT)-cyclohydrolase I, an enzyme that generates BH4. One study shows that increased levels of BH4, as a result of cirrhosis, enhance eNOS activity in the superior mesenteric artery (Wiest, et al. 2003). Thus, an increase in BH4 production in the superior mesenteric artery of cirrhotic rats is thought to be one of the mechanisms by which eNOS contributes to excessive arterial vasodilation.

#### **2.1.1.3 Akt/protein kinase B**

Akt, a serine/threonine kinase, can directly phosphorylate eNOS on Serine1177 (human) or Serine1179 (bovine) and activates eNOS, leading to NO production (Dimmeler, et al. 1999; Fulton, et al. 1999). We have shown that portal hypertension increases eNOS

The Molecules: Abnormal Vasculatures in the

Splanchnic and Systemic Circulation in Portal Hypertension 5

Fig. 3. Hemeoxygenase (OH) pathway in the arterial splanchnic and systemic circulation in cirrhosis and portal hypertension. HO-1 is an inducible isoform, while HO-2 is a constitutive isoform. Both nitric oxide (NO) and carbon monoxide (CO) activate soluble guanylate

In contrast to other studies, a study by Sacerdoti et al. (Sacerdoti, et al. 2004) reported that HO-2, not the inducible HO-1, was up-regulated in mesenteric arteries of cirrhotic rats. In their study, cirrhotic rats were generated by giving carbon tetrachloride (CCl4) in gavage for 8 to 10 weeks. Consistent with other studies, however, administration of an HO inhibitor, tin(Sn)-mesoporphyrin IX, ameliorated excessive arterial vasodilation in cirrhotic rats. Collectively, these observations may suggest that different experimental models of cirrhosis and portal hypertension cause different effects on HO pathway in the aorta and mesenteric

Studies with cirrhotic patients also showed an increase in plasma CO levels (De las Heras, et al. 2003; Tarquini, et al. 2009). Spontaneous bacterial peritonitis further accelerated blood CO levels in cirrhotic patients (De las Heras, et al. 2003). Furthermore, Tarquini et al. (Tarquini, et al. 2009) documented that plasma CO levels as well as HO expression and activity in polymorphonuclear cells were significantly increased in patients with viral hepatitis and the hyperdynamic circulatory syndrome. Importantly, plasma CO levels were directly correlated with the severity of the hyperdynamic circulatory syndrome. Collectively, these clinical studies with cirrhotic patients also suggest that enhanced circulating CO levels are associated

PGI2 is generated by the activity of cyclooxygenase (COX) in endothelial cells and facilitates smooth muscle relaxation by stimulating adenylate cyclase to produce cyclic adenosine monophosphate (Claesson, et al. 1977) (Figure 4). There are two isoforms of COX. COX-1 is a constitutively expressed form, and COX-2 is an inducible form (Smith, et al. 2000; Smith, et

PGI2 is an important mediator in the development of experimental and clinical portal hypertension (Hou, et al. 1998; Ohta, et al. 1995; Skill, et al. 2008). Increased COX-1

cyclase (sGC) in smooth muscle cells and facilitate vasodilation.

arteries, thus resulting in up-regulation of different types of HO isoforms.

with the development of the hyperdynamic circulatory syndrome.

**2.1.3 Prostacyclin (PGI2)** 

al. 1996).

phosphorylation by Akt in the superior mesenteric artery and that wortmannin, an inhibitor of the phosphatidylinositol-3-OH-kinase (PI3K)/Akt pathway, decreases NO production and excessive vasodilation in the superior mesenteric artery isolated from portal hypertensive rats (Iwakiri, et al. 2002). These observations suggest that Akt-dependent phosphorylation and activation of eNOS play a role in excessive NO production and the resulting vasodilation in the superior mesenteric artery of portal hypertensive rats.

Since eNOS is the major NOS that generates NO in arteries of the splanchnic and systemic circulation, understanding the mechanisms by which eNOS is activated in these arteries is essential and allows us to develop critical strategies to block excessive arterial vasodilation and the subsequent development of the hyperdynamic circulatory syndrome.

#### **2.1.2 Carbon monoxide (CO)**

CO is an end product of the heme oxygenase (HO) pathway and a potent vasodilatory molecule that functions in a similar mechanism to NO (Figure 3). It activates sGC in vascular smooth muscle cells and regulates the blood flow and resistance in several vascular beds (Naik & Walker 2003). HO has two isoforms, HO-1 and HO-2. HO-1, also known as heat shock protein 32, is an inducible isoform. HO-2, a ubiquitously expressed constitutive isoform, is also found in blood vessels (Ishizuka, et al. 1997; Zakhary, et al. 1996). In pathological conditions, HO activity increases markedly due to the up-regulation of HO-1 (Cruse & Lewis 1988). Several experimental and clinical studies have shown a possible relationship between HO pathway and several complications of cirrhosis and portal hypertension, such as cardiac dysfunction (Liu, et al. 2001), renal dysfunction (Miyazono, et al. 2002), hepatopulmonary syndrome (Carter, et al. 2002), spontaneous bacterial peritonitis (De las Heras, et al. 2003) and viral hepatitis (Tarquini, et al. 2009).

Increased portal pressure alone contributes to the activation of HO pathway in mesenteric arteries and other organs (Angermayr, et al. 2006; Fernandez & Bonkovsky 1999). In a study using rats with partial portal vein ligation, a surgical model that induces portal hypertension, HO-1 was up-regulated in the superior mesenteric arterial beds (Angermayr, et al. 2006). When rats with partial portal vein ligation were given an HO inhibitor, tin(Sn) mesoporphyrin IX, intraperitoneally immediately after surgery for the following 7 days, a significant reduction in portal pressure was observed in the HO inhibitor-treated group compared to the placebo group. However, the HO inhibition did not affect the formation of portosystemic collaterals in portal hypertensive rats (Angermayr, et al. 2006).

Like those surgically induced portal hypertensive rats, rats with cirrhosis exhibit enhanced HO pathway to mediate excessive vasodilation in arteries of the splanchnic and systemic circulation (Chen, et al. 2004; Tarquini, et al. 2009). Rats with bile duct ligation (a surgical model of biliary cirrhosis) showed an increase in HO-1 expression in both the superior mesenteric artery and the aorta, compared to sham-operated rats. In contrast, HO-2 expression did not differ between the two groups of rats. Importantly, aortic HO activities as well as blood CO levels were positively related to the degree of the hyperdynamic circulatory syndrome assessed by mean arterial pressure, cardiac input and peripheral vascular resistance. Acute administration of an HO inhibitor, zinc protoporphyrin (ZnPP), ameliorated the hyperdynamic circulatory syndrome in cirrhotic rats with 4 weeks after bile duct ligation (Chen, et al. 2004; Tarquini, et al. 2009).

phosphorylation by Akt in the superior mesenteric artery and that wortmannin, an inhibitor of the phosphatidylinositol-3-OH-kinase (PI3K)/Akt pathway, decreases NO production and excessive vasodilation in the superior mesenteric artery isolated from portal hypertensive rats (Iwakiri, et al. 2002). These observations suggest that Akt-dependent phosphorylation and activation of eNOS play a role in excessive NO production and the

Since eNOS is the major NOS that generates NO in arteries of the splanchnic and systemic circulation, understanding the mechanisms by which eNOS is activated in these arteries is essential and allows us to develop critical strategies to block excessive arterial vasodilation

CO is an end product of the heme oxygenase (HO) pathway and a potent vasodilatory molecule that functions in a similar mechanism to NO (Figure 3). It activates sGC in vascular smooth muscle cells and regulates the blood flow and resistance in several vascular beds (Naik & Walker 2003). HO has two isoforms, HO-1 and HO-2. HO-1, also known as heat shock protein 32, is an inducible isoform. HO-2, a ubiquitously expressed constitutive isoform, is also found in blood vessels (Ishizuka, et al. 1997; Zakhary, et al. 1996). In pathological conditions, HO activity increases markedly due to the up-regulation of HO-1 (Cruse & Lewis 1988). Several experimental and clinical studies have shown a possible relationship between HO pathway and several complications of cirrhosis and portal hypertension, such as cardiac dysfunction (Liu, et al. 2001), renal dysfunction (Miyazono, et al. 2002), hepatopulmonary syndrome (Carter, et al. 2002), spontaneous bacterial peritonitis

Increased portal pressure alone contributes to the activation of HO pathway in mesenteric arteries and other organs (Angermayr, et al. 2006; Fernandez & Bonkovsky 1999). In a study using rats with partial portal vein ligation, a surgical model that induces portal hypertension, HO-1 was up-regulated in the superior mesenteric arterial beds (Angermayr, et al. 2006). When rats with partial portal vein ligation were given an HO inhibitor, tin(Sn) mesoporphyrin IX, intraperitoneally immediately after surgery for the following 7 days, a significant reduction in portal pressure was observed in the HO inhibitor-treated group compared to the placebo group. However, the HO inhibition did not affect the formation of

Like those surgically induced portal hypertensive rats, rats with cirrhosis exhibit enhanced HO pathway to mediate excessive vasodilation in arteries of the splanchnic and systemic circulation (Chen, et al. 2004; Tarquini, et al. 2009). Rats with bile duct ligation (a surgical model of biliary cirrhosis) showed an increase in HO-1 expression in both the superior mesenteric artery and the aorta, compared to sham-operated rats. In contrast, HO-2 expression did not differ between the two groups of rats. Importantly, aortic HO activities as well as blood CO levels were positively related to the degree of the hyperdynamic circulatory syndrome assessed by mean arterial pressure, cardiac input and peripheral vascular resistance. Acute administration of an HO inhibitor, zinc protoporphyrin (ZnPP), ameliorated the hyperdynamic circulatory syndrome in cirrhotic rats with 4 weeks after bile

resulting vasodilation in the superior mesenteric artery of portal hypertensive rats.

and the subsequent development of the hyperdynamic circulatory syndrome.

(De las Heras, et al. 2003) and viral hepatitis (Tarquini, et al. 2009).

portosystemic collaterals in portal hypertensive rats (Angermayr, et al. 2006).

duct ligation (Chen, et al. 2004; Tarquini, et al. 2009).

**2.1.2 Carbon monoxide (CO)** 

Fig. 3. Hemeoxygenase (OH) pathway in the arterial splanchnic and systemic circulation in cirrhosis and portal hypertension. HO-1 is an inducible isoform, while HO-2 is a constitutive isoform. Both nitric oxide (NO) and carbon monoxide (CO) activate soluble guanylate cyclase (sGC) in smooth muscle cells and facilitate vasodilation.

In contrast to other studies, a study by Sacerdoti et al. (Sacerdoti, et al. 2004) reported that HO-2, not the inducible HO-1, was up-regulated in mesenteric arteries of cirrhotic rats. In their study, cirrhotic rats were generated by giving carbon tetrachloride (CCl4) in gavage for 8 to 10 weeks. Consistent with other studies, however, administration of an HO inhibitor, tin(Sn)-mesoporphyrin IX, ameliorated excessive arterial vasodilation in cirrhotic rats. Collectively, these observations may suggest that different experimental models of cirrhosis and portal hypertension cause different effects on HO pathway in the aorta and mesenteric arteries, thus resulting in up-regulation of different types of HO isoforms.

Studies with cirrhotic patients also showed an increase in plasma CO levels (De las Heras, et al. 2003; Tarquini, et al. 2009). Spontaneous bacterial peritonitis further accelerated blood CO levels in cirrhotic patients (De las Heras, et al. 2003). Furthermore, Tarquini et al. (Tarquini, et al. 2009) documented that plasma CO levels as well as HO expression and activity in polymorphonuclear cells were significantly increased in patients with viral hepatitis and the hyperdynamic circulatory syndrome. Importantly, plasma CO levels were directly correlated with the severity of the hyperdynamic circulatory syndrome. Collectively, these clinical studies with cirrhotic patients also suggest that enhanced circulating CO levels are associated with the development of the hyperdynamic circulatory syndrome.

#### **2.1.3 Prostacyclin (PGI2)**

PGI2 is generated by the activity of cyclooxygenase (COX) in endothelial cells and facilitates smooth muscle relaxation by stimulating adenylate cyclase to produce cyclic adenosine monophosphate (Claesson, et al. 1977) (Figure 4). There are two isoforms of COX. COX-1 is a constitutively expressed form, and COX-2 is an inducible form (Smith, et al. 2000; Smith, et al. 1996).

PGI2 is an important mediator in the development of experimental and clinical portal hypertension (Hou, et al. 1998; Ohta, et al. 1995; Skill, et al. 2008). Increased COX-1

The Molecules: Abnormal Vasculatures in the

circulatory syndrome in those rats (Lin, et al. 2011).

Splanchnic and Systemic Circulation in Portal Hypertension 7

endotoxemia decreased hepatic endocannabinoid levels and ameliorated the hyperdynamic

Fig. 5. Anandamide produced by circulating monocytes causes hypotension in cirrhotic rats and patients. Anandamide (arachidonyl ethanolamide) is an endogenous lipid ligand that belongs to endocannabinoids (Wagner, et al. 1997) and generated from arachidonic acid (AA). The bacterial endotoxin lipopolysaccharide (LPS) elicits production of anandamide in monocytes and endothelial cells (Varga, et al. 1998). Anandamide binds to CB1 receptors

Monocytes and platelets are the two major sources of endocannabinoids in endotoxemia (Batkai, et al. 2001; Ros, et al. 2002; Varga, et al. 1998). When monocytes and platelets were pre-exposed to LPS and then injected to normal rat recipients, hypotension was developed (Varga, et al. 1998). Hypotension was however prevented by pretreatment of recipient rats with a CB1 receptor antagonist, SR141716A. Thus, endotoxemia elicits production of

Anandamide levels were also elevated 2- to 3-fold and 16-fold in monocytes isolated from cirrhotic rats and patients, respectively, compared to their corresponding controls (Batkai, et al. 2001). Transplantation of monocytes isolated from cirrhotic rats or patients via intravenous injection, but not those monocytes from control rats, to normal recipient rats gradually caused the development of hypotension. In contrast, when normal recipient rats were pretreated with a CB1 receptor antagonist, SR141716A, the monocytes from the same cirrhotic rats or patients did not cause hypotension in those rats. Besides elevated anandamide levels, CB1 receptor levels were 3 times higher in hepatic arterial endothelial cells isolated from cirrhotic human livers than in those isolated from normal human livers. Importantly again, blocking CB1 receptor by SR141716A ameliorated arterial hypotension and the hyperdynamic circulatory syndrome in cirrhotic rats. Collectively, these results suggest that CB1 receptor can be a therapeutic target to ameliorate the hyperdynamic

located on endothelial cells and smooth muscle cells and causes hypotension.

endocannabinoids in monocytes and platelets, leading to hypotension.

circulatory syndrome in cirrhosis and portal hypertension.

expression contributed to increased arterial vasodilation in the splanchnic circulation in portal hypertensive rats (Hou, et al. 1998). COX-2, however, was not detected in the superior mesenteric artery of those rats. These observations suggested that COX-1, not COX-2, would be responsible for the increased vasodilation in the superior mesenteric artery of portal hypertensive rats. However, inhibiting COX-1 only neither decreased PGI2 levels nor ameliorated the hyperdynaic circulatory syndrome in portal hypertensive mice (Skill, et al. 2008). A study using both COX-1-/- and COX-2-/- mice in combination of selective COX-2 (NS398) and COX-1 (SC560) inhibitors, respectively, showed that blockade of both COX-1 and COX-2 ameliorated the hyperdynamic circulatory syndrome in portal hypertensive mice. Therefore, it is suggested that both COX-1 and COX-2 need to be suppressed to reduce PGI2 production and to ameliorate the hyperdynamic circulatory syndrome (Skill, et al. 2008). Similar to experimental portal hypertension, circulating PGI2 levels are also elevated in cirrhotic patients (Ohta, et al. 1995).

Fig. 4. Cyclooxygenase (COX) pathway in the arterial splanchnic and systemic circulation in cirrhosis and portal hypertension. COX-1 is a constitutive form, while COX-2 is inducible form. Both COX-1 and COX-2 seem to play a role in production of prostacyclin (PGI2), which activates adenylate cyclase (AC) in smooth muscle cells to produce cyclic adenosine monophosphate (cAMP), thereby leading to vasodilation.

#### **2.1.4 Endocannabinoids**

Endocannabinoid is a collective term used for a group of endogenous lipid ligands, including anandamide (arachidonyl ethanolamide) (Wagner, et al. 1997). Endocannabinoids bind to their receptors, CB1 receptors, and cause hypotension (Figure 5). The bacterial endotoxin lipopolysaccharide (LPS) elicits production of endocannabinoids (Varga, et al. 1998) and thus develops hypotension.

Cirrhotic patients are generally endotoxemia, which is characterized by elevated endotoxin/LPS levels in the blood. Thus, it is not surprising that circulating anandamide levels are elevated in cirrhotic patients (Caraceni, et al. 2010; Fernandez-Rodriguez, et al. 2004). Cirrhotic rats also exhibit endotoxemia. Thus, antibiotic treatment to suppress

expression contributed to increased arterial vasodilation in the splanchnic circulation in portal hypertensive rats (Hou, et al. 1998). COX-2, however, was not detected in the superior mesenteric artery of those rats. These observations suggested that COX-1, not COX-2, would be responsible for the increased vasodilation in the superior mesenteric artery of portal hypertensive rats. However, inhibiting COX-1 only neither decreased PGI2 levels nor ameliorated the hyperdynaic circulatory syndrome in portal hypertensive mice (Skill, et al. 2008). A study using both COX-1-/- and COX-2-/- mice in combination of selective COX-2 (NS398) and COX-1 (SC560) inhibitors, respectively, showed that blockade of both COX-1 and COX-2 ameliorated the hyperdynamic circulatory syndrome in portal hypertensive mice. Therefore, it is suggested that both COX-1 and COX-2 need to be suppressed to reduce PGI2 production and to ameliorate the hyperdynamic circulatory syndrome (Skill, et al. 2008). Similar to experimental portal hypertension, circulating PGI2 levels are also elevated

Fig. 4. Cyclooxygenase (COX) pathway in the arterial splanchnic and systemic circulation in cirrhosis and portal hypertension. COX-1 is a constitutive form, while COX-2 is inducible form. Both COX-1 and COX-2 seem to play a role in production of prostacyclin (PGI2), which activates adenylate cyclase (AC) in smooth muscle cells to produce cyclic adenosine

Endocannabinoid is a collective term used for a group of endogenous lipid ligands, including anandamide (arachidonyl ethanolamide) (Wagner, et al. 1997). Endocannabinoids bind to their receptors, CB1 receptors, and cause hypotension (Figure 5). The bacterial endotoxin lipopolysaccharide (LPS) elicits production of endocannabinoids (Varga, et al.

Cirrhotic patients are generally endotoxemia, which is characterized by elevated endotoxin/LPS levels in the blood. Thus, it is not surprising that circulating anandamide levels are elevated in cirrhotic patients (Caraceni, et al. 2010; Fernandez-Rodriguez, et al. 2004). Cirrhotic rats also exhibit endotoxemia. Thus, antibiotic treatment to suppress

monophosphate (cAMP), thereby leading to vasodilation.

**2.1.4 Endocannabinoids** 

1998) and thus develops hypotension.

in cirrhotic patients (Ohta, et al. 1995).

endotoxemia decreased hepatic endocannabinoid levels and ameliorated the hyperdynamic circulatory syndrome in those rats (Lin, et al. 2011).

Fig. 5. Anandamide produced by circulating monocytes causes hypotension in cirrhotic rats and patients. Anandamide (arachidonyl ethanolamide) is an endogenous lipid ligand that belongs to endocannabinoids (Wagner, et al. 1997) and generated from arachidonic acid (AA). The bacterial endotoxin lipopolysaccharide (LPS) elicits production of anandamide in monocytes and endothelial cells (Varga, et al. 1998). Anandamide binds to CB1 receptors located on endothelial cells and smooth muscle cells and causes hypotension.

Monocytes and platelets are the two major sources of endocannabinoids in endotoxemia (Batkai, et al. 2001; Ros, et al. 2002; Varga, et al. 1998). When monocytes and platelets were pre-exposed to LPS and then injected to normal rat recipients, hypotension was developed (Varga, et al. 1998). Hypotension was however prevented by pretreatment of recipient rats with a CB1 receptor antagonist, SR141716A. Thus, endotoxemia elicits production of endocannabinoids in monocytes and platelets, leading to hypotension.

Anandamide levels were also elevated 2- to 3-fold and 16-fold in monocytes isolated from cirrhotic rats and patients, respectively, compared to their corresponding controls (Batkai, et al. 2001). Transplantation of monocytes isolated from cirrhotic rats or patients via intravenous injection, but not those monocytes from control rats, to normal recipient rats gradually caused the development of hypotension. In contrast, when normal recipient rats were pretreated with a CB1 receptor antagonist, SR141716A, the monocytes from the same cirrhotic rats or patients did not cause hypotension in those rats. Besides elevated anandamide levels, CB1 receptor levels were 3 times higher in hepatic arterial endothelial cells isolated from cirrhotic human livers than in those isolated from normal human livers. Importantly again, blocking CB1 receptor by SR141716A ameliorated arterial hypotension and the hyperdynamic circulatory syndrome in cirrhotic rats. Collectively, these results suggest that CB1 receptor can be a therapeutic target to ameliorate the hyperdynamic circulatory syndrome in cirrhosis and portal hypertension.

The Molecules: Abnormal Vasculatures in the

Splanchnic and Systemic Circulation in Portal Hypertension 9

Fig. 6. Overview of endothelium-derived hyperpolarizing factor (EDHF) in the superior mesenteric artery. Shear stress generated by an increase in portal pressure increases endothelial Ca2+ concentration and produces hyperpolarization by activating ion channels, such as small conductance calcium-activated potassium channel (SK3) and intermediate conductance calcium-activated potassium channel (IK1). Hydrogen sulfide (H2S) is formed in vascular endothelial cells from cysteine by L-cystathionine-gamma-lyase (CSE). H2S causes hyperpolarization through activation of SK3, IK1 and ATP-sensitive potassium channel (KATP). Connexins (Cx) 37 and 40 are predominant gap junction proteins in endothelial cells and contribute to EDHF-mediated response. Connexin 43 (Cx43) is also present at the gap junction, but it does not play a major role in this context. Potassium ion (K+) activates Na+/K+-ATPase pump, preventing the effects of any substantial rise of potassium during endothelium-dependent hyperpolarization. Bradykinin, through its Gprotein coupled receptor (B2R), activates the metabolism of arachidonic acid (AA) via cytochrome P450 monooxygenase (P450). Bradykinin also activates phospholipase C (PLC)

that stimulates inositol trisphosphate (IP3) to increase cytosolic Ca2+ concentration. Epoxyeicosatrienoic acids (EETs) cause hyperpolarization/relaxation, acting through the

artery of cirrhotic rats (Dal-Ros, et al. 2010). However, Bolognesi et al. (Bolognesi, et al. 2011) presented that mesenteric arteries isolated from cirrhotic rats exhibited elevated Cx40 and Cx43 expression, which increased sensitivity to epoxyeicosatrienoic acids (EETs) in those

A proinflammatory cytokine, tumor necrosis factor (TNF), is produced by mononuclear cells upon activation by bacterial endotoxins. In cirrhosis and portal hypertension, therefore, TNF levels are elevated (Lopez-Talavera, et al. 1995; Mookerjee, et al. 2003). Inhibition of TNF action by an anti-TNF antibody resulted in a significant reduction in hepatic venous pressure gradient (HVPG) of patients with alcoholic hepatitis (Mookerjee, et al. 2003). Similarly, inhibition of TNF synthesis by thalidomide also prevented the development of the hyperdynamic circulatory syndrome in portal hypertensive rats (Lopez-Talavera, et al. 1996). The mechanism of TNF action in cirrhosis and portal hypertension is not fully

voltage-gated potassium channel (BKCa) and gap junction.

arteries and contributed to enhanced vasodilation.

**2.1.6 Tumor necrosis factor** 

understood.

#### **2.1.5 Endothelium-Derived Hyperpolarizing Factor (EDHF)**

Endothelium-derived hyperpolarizing factor (EDHF) is also an important vasodilatory molecule that regulates vascular tone (Cohen 2005; Feletou & Vanhoutte 2006; Feletou & Vanhoutte 2007; Griffith 2004). It is associated with hyperpolarization of vascular smooth muscle cells and facilitates vasodilation. The term EDHF might be confusing, since it implies a single molecule (Feletou & Vanhoutte 2006). Currently, it is not still fully characterized what molecule EDHF is. However, accumulating evidence suggests that HDHF could be multiple molecules, including PGI2 (Feletou & Vanhoutte 2006), NO (Cohen, et al. 1997; Plane, et al. 1998), epoxyeicosatrienoic acids (EETs) (Fleming 2004; Gauthier, et al. 2004; Li & Campbell 1997; Oltman, et al. 1998; Quilley & McGiff 2000; Widmann, et al. 1998), lipoxygenase [12-(s)-hydroxyeicosatetraenoic acid (12-S-HETE)] (Barlow, et al. 2000; Faraci, et al. 2001; Gauthier, et al. 2004; Pfister, et al. 1998; Zhang, et al. 2005; Zink, et al. 2001), hydrogen peroxide (H2O2) (Beny & von der Weid 1991; Chaytor, et al. 2003; Ellis, et al. 2003; Gluais, et al. 2005; Matoba, et al. 2002; Matoba, et al. 2003; Matoba, et al. 2000; Morikawa, et al. 2003; Shimokawa & Matoba 2004), potassium ions (K+), C-type natriuretic paptides (Banks, et al. 1996; Wei, et al. 1994) and hydrogen sulfide (Mustafa, et al. 2011). It has also been suggested that EDHF function may be mediated through direct coupling between endothelial and smooth muscle cells at myoendothelial gap junctions composed of connexins (Cohen 2005; Feletou & Vanhoutte 2007; Griffith 2004) (Figure 6).

Most recently, a study by Mustafa et al. (Mustafa, et al. 2011) suggested that H2S could be an EDHF. H2S is synthesized endogenously from L-cystathionine--lyase (CSE) and cystathionine--synthase (Hosoki, et al. 1997; Stipanuk & Beck 1982). The H2S-mediated vasodilation occurs through the opening of ATP-sensitive potassium channel (KATP channel) and is independent of the activation of cGMP pathway (Zhao, et al. 2001). In the superior mesenteric artery of mice lacking CSE, hyperpolarization is virtually abolished. Most interestingly, H2S covalently modifies (i.e., S-sulfhydrating) KATP channel and leads to relaxation of vessels.

EDHF seems to be more important in smaller arteries and arterioles than in larger arteries. This tendency has been recognized in a number of vascular beds, including mesenteric and cerebral arteries and arteries in ear and stomach (Tomioka, et al. 1999; Urakami-Harasawa, et al. 1997; You, et al. 1999).

It has not been established whether EDHF is involved in vasodilation and hypocontractility of arteries of the splanchnic and systemic circulation in cirrhosis and portal hypertension. Barriere et al. (Barriere & Lebrec 2000) reported that EDHF contributed to hypocontractility in the superior mesenteric artery isolated from cirrhotic rats when NO and PGI2 production were inhibited. This hypocontractility was abolished when the vessels were further treated with inhibitors of small conductance Ca2+-activated K+ channel (SK channel), such as apamin and charybdotoxin, suggesting that EDHF blunts contractile response in cirrhotic rats (Barriere & Lebrec 2000). In contrast, a study by Dal-Ros et al. (Dal-Ros, et al. 2010) showed that the contribution of EDHF to vasodilation in mesenteric arteries was even smaller in cirrhotic rats than in normal rats. It was speculated that decreased expression of connexins (Cx), such as Cx37, Cx40, and Cx43, as well as Ca2+-activated K+ channel contributed to this smaller contribution of EDHF to vasodilation in the superior mesenteric

Endothelium-derived hyperpolarizing factor (EDHF) is also an important vasodilatory molecule that regulates vascular tone (Cohen 2005; Feletou & Vanhoutte 2006; Feletou & Vanhoutte 2007; Griffith 2004). It is associated with hyperpolarization of vascular smooth muscle cells and facilitates vasodilation. The term EDHF might be confusing, since it implies a single molecule (Feletou & Vanhoutte 2006). Currently, it is not still fully characterized what molecule EDHF is. However, accumulating evidence suggests that HDHF could be multiple molecules, including PGI2 (Feletou & Vanhoutte 2006), NO (Cohen, et al. 1997; Plane, et al. 1998), epoxyeicosatrienoic acids (EETs) (Fleming 2004; Gauthier, et al. 2004; Li & Campbell 1997; Oltman, et al. 1998; Quilley & McGiff 2000; Widmann, et al. 1998), lipoxygenase [12-(s)-hydroxyeicosatetraenoic acid (12-S-HETE)] (Barlow, et al. 2000; Faraci, et al. 2001; Gauthier, et al. 2004; Pfister, et al. 1998; Zhang, et al. 2005; Zink, et al. 2001), hydrogen peroxide (H2O2) (Beny & von der Weid 1991; Chaytor, et al. 2003; Ellis, et al. 2003; Gluais, et al. 2005; Matoba, et al. 2002; Matoba, et al. 2003; Matoba, et al. 2000; Morikawa, et al. 2003; Shimokawa & Matoba 2004), potassium ions (K+), C-type natriuretic paptides (Banks, et al. 1996; Wei, et al. 1994) and hydrogen sulfide (Mustafa, et al. 2011). It has also been suggested that EDHF function may be mediated through direct coupling between endothelial and smooth muscle cells at myoendothelial gap junctions composed of

**2.1.5 Endothelium-Derived Hyperpolarizing Factor (EDHF)** 

connexins (Cohen 2005; Feletou & Vanhoutte 2007; Griffith 2004) (Figure 6).

relaxation of vessels.

et al. 1997; You, et al. 1999).

Most recently, a study by Mustafa et al. (Mustafa, et al. 2011) suggested that H2S could be an EDHF. H2S is synthesized endogenously from L-cystathionine--lyase (CSE) and cystathionine--synthase (Hosoki, et al. 1997; Stipanuk & Beck 1982). The H2S-mediated vasodilation occurs through the opening of ATP-sensitive potassium channel (KATP channel) and is independent of the activation of cGMP pathway (Zhao, et al. 2001). In the superior mesenteric artery of mice lacking CSE, hyperpolarization is virtually abolished. Most interestingly, H2S covalently modifies (i.e., S-sulfhydrating) KATP channel and leads to

EDHF seems to be more important in smaller arteries and arterioles than in larger arteries. This tendency has been recognized in a number of vascular beds, including mesenteric and cerebral arteries and arteries in ear and stomach (Tomioka, et al. 1999; Urakami-Harasawa,

It has not been established whether EDHF is involved in vasodilation and hypocontractility of arteries of the splanchnic and systemic circulation in cirrhosis and portal hypertension. Barriere et al. (Barriere & Lebrec 2000) reported that EDHF contributed to hypocontractility in the superior mesenteric artery isolated from cirrhotic rats when NO and PGI2 production were inhibited. This hypocontractility was abolished when the vessels were further treated with inhibitors of small conductance Ca2+-activated K+ channel (SK channel), such as apamin and charybdotoxin, suggesting that EDHF blunts contractile response in cirrhotic rats (Barriere & Lebrec 2000). In contrast, a study by Dal-Ros et al. (Dal-Ros, et al. 2010) showed that the contribution of EDHF to vasodilation in mesenteric arteries was even smaller in cirrhotic rats than in normal rats. It was speculated that decreased expression of connexins (Cx), such as Cx37, Cx40, and Cx43, as well as Ca2+-activated K+ channel contributed to this smaller contribution of EDHF to vasodilation in the superior mesenteric

Fig. 6. Overview of endothelium-derived hyperpolarizing factor (EDHF) in the superior mesenteric artery. Shear stress generated by an increase in portal pressure increases endothelial Ca2+ concentration and produces hyperpolarization by activating ion channels, such as small conductance calcium-activated potassium channel (SK3) and intermediate conductance calcium-activated potassium channel (IK1). Hydrogen sulfide (H2S) is formed in vascular endothelial cells from cysteine by L-cystathionine-gamma-lyase (CSE). H2S causes hyperpolarization through activation of SK3, IK1 and ATP-sensitive potassium channel (KATP). Connexins (Cx) 37 and 40 are predominant gap junction proteins in endothelial cells and contribute to EDHF-mediated response. Connexin 43 (Cx43) is also present at the gap junction, but it does not play a major role in this context. Potassium ion (K+) activates Na+/K+-ATPase pump, preventing the effects of any substantial rise of potassium during endothelium-dependent hyperpolarization. Bradykinin, through its Gprotein coupled receptor (B2R), activates the metabolism of arachidonic acid (AA) via cytochrome P450 monooxygenase (P450). Bradykinin also activates phospholipase C (PLC) that stimulates inositol trisphosphate (IP3) to increase cytosolic Ca2+ concentration. Epoxyeicosatrienoic acids (EETs) cause hyperpolarization/relaxation, acting through the voltage-gated potassium channel (BKCa) and gap junction.

artery of cirrhotic rats (Dal-Ros, et al. 2010). However, Bolognesi et al. (Bolognesi, et al. 2011) presented that mesenteric arteries isolated from cirrhotic rats exhibited elevated Cx40 and Cx43 expression, which increased sensitivity to epoxyeicosatrienoic acids (EETs) in those arteries and contributed to enhanced vasodilation.

#### **2.1.6 Tumor necrosis factor**

A proinflammatory cytokine, tumor necrosis factor (TNF), is produced by mononuclear cells upon activation by bacterial endotoxins. In cirrhosis and portal hypertension, therefore, TNF levels are elevated (Lopez-Talavera, et al. 1995; Mookerjee, et al. 2003). Inhibition of TNF action by an anti-TNF antibody resulted in a significant reduction in hepatic venous pressure gradient (HVPG) of patients with alcoholic hepatitis (Mookerjee, et al. 2003). Similarly, inhibition of TNF synthesis by thalidomide also prevented the development of the hyperdynamic circulatory syndrome in portal hypertensive rats (Lopez-Talavera, et al. 1996). The mechanism of TNF action in cirrhosis and portal hypertension is not fully understood.

The Molecules: Abnormal Vasculatures in the

investigated.

vasodilation.

**2.1.8 Bradykinin** 

**2.1.9 Urotensin II** 

Splanchnic and Systemic Circulation in Portal Hypertension 11

Portal hypertension alone, regardless of the presence of cirrhosis, increases adrenomedullin production. One clinical study showed that adrenomedullin and NO levels are elevated not only in patients with cirrhotic portal hypertension, but also in those patients with noncirrhotic portal hypertension (Tahan, et al. 2003). How an increase in portal pressure influences production of adrenomedullin is an interesting and important question to be

Fig. 7. Adrenomedullin causes vasodilation and hypotension. Adrenomedullin (AM) binds to and induces its signaling through the G-protein-coupled calcitonin receptor-like receptor

Bradykinin is a nine amino acid peptide and known to facilitate vasodilation (Antonio & Rocha 1962). Bradykinin leads to endothelium-dependent hyperpolarization through activation of phospholipase C (PLC), which could raise Ca2+ concentration and also stimulate production of EETs (Feletou & Vanhoutte 2006) (Figure 6). Bradykinin reduces sensitivity to glypressin (a long lasting vasopressin analogue) in both portal hypertensive

Urotensin II is a cyclic peptide and has a structural similarity to somatostatin. It can function both as a vasoconstrictor and a vasodilator depending on vascular beds (Coulouarn, et al.

and cirrhotic rats (Chen, et al. 2009; Chu, et al. 2000), thereby advancing vasodilation.

(CRLR)/receptor activity-modifying protein (RAMP)2 and 3. The vascular action of adrenomedullin was at first considered to be solely due to elevated cAMP production by activation of adenylate cyclase (AC) in endothelial cells, thereby causing endotheliumindependent vasodilation. Adrenomedullin-induced endothelium-dependent vasodialation is exerted mostly through activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which activates eNOS to produce NO. NO then diffuses into smooth muscle cells to activate soluble guanylate cyclase (sGC) and produce cyclic GMP (cGMP), leading to

TNF stimulates NOS activity by increasing BH4 production through stimulation of expression and activity of guanosine triphosphate-cyclohydrolase I, a key enzyme for the regulation of BH4 biosynthesis in endothelial cells (Katusic, et al. 1998; Rosenkranz-Weiss, et al. 1994). Enhanced BH4 production directly increases eNOS-derived NO production (Katusic, et al. 1998; Rosenkranz-Weiss, et al. 1994; Wever, et al. 1997). In biliary cirrhotic rats, it was demonstrated that TNF, through the activation of iNOS in the aorta and lung, plays a role in the development of the hyperdynamic circulatory syndrome and the hepatopulmonary syndrome (Sztrymf, et al. 2004).

#### **2.1.7 Adrenomedullin**

Adrenomedullin is an endogenous vasodilatory peptide consisting of 52 amino acid residues in human and 50 amino acid residues in the rat (Kitamura, et al. 1993; Kitamura, et al. 1993; Nuki, et al. 1993). The major producers of circulating adrenomedullin are vascular smooth muscle cells (Sugo, et al. 1994) and endothelial cells (Sugo, et al. 1995). Adrenomedullin binds to and induces its signaling through the G-protein-coupled calcitonin receptor-like receptor/receptor activity-modifying protein (RAMP)2 and 3, which are expressed in multiple tissues, including blood vessels, kidney, lung, atrium, gastrointestinal tract, spleen, endocrine glands, brain and heart. Receptor RAMP2 is essential for angiogenesis and vascular integrity (Ichikawa-Shindo, et al. 2008). Adrenomedullin expression is up-regulated by hypoxia (Nagata, et al. 1999; Wang, et al. 1995) and inflammation (Sugo, et al. 1995; Ueda, et al. 1999), both of which are associated with neovascularization.

The vasodilatory action of adrenomedullin was considered in the beginning to be solely due to elevated cAMP production, i.e., endothelium-independent vasodilation. However, endothelial denudation substantially reduced its vasodilatory action in rodent aortic rings (Hirata, et al. 1995; Nishimatsu, et al. 2001). Furthermore, this adrenomedullin-induced endothelium-dependent vasodilation was exerted mostly through activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway (Nishimatsu, et al. 2001). It has been well established that this pathway is involved in various important actions in endothelial cells, such as activation of eNOS. While one study demonstrated in a mouse model of ischemia that adrenomedullin-induced collateral vessel formation in ischemic tissues was eNOS-dependent (Abe, et al. 2003), no study has so far shown that adrenomedullin activates eNOS through Akt activation.

Several studies have reported that in liver cirrhotic patients, circulating adrenomedullin levels are elevated and are associated with increased levels of plasma nitrite (a stable NO metabolite) and plasma volume expansion (Guevara, et al. 1998; Kojima, et al. 1998; Tahan, et al. 2003). Furthermore, the increased circulating adrenomedullin levels in those patients are inversely related to peripheral resistance (Guevara, et al. 1998). These observations indicate that adrenomedullin may promote excessive vasodilation and the hyperdynamic circulatory syndrome in cirrhotic patients. It is not surprising, therefore, that administration of an anti-adrenomedullin antibody prevented the occurrence of the hyperdynamic circulatory syndrome in the early sepsis (Wang, et al. 1998) and ameliorated blunted contractile response to phenylephrine in the aorta isolated from cirrhotic rats (Kojima, et al. 2004).

TNF stimulates NOS activity by increasing BH4 production through stimulation of expression and activity of guanosine triphosphate-cyclohydrolase I, a key enzyme for the regulation of BH4 biosynthesis in endothelial cells (Katusic, et al. 1998; Rosenkranz-Weiss, et al. 1994). Enhanced BH4 production directly increases eNOS-derived NO production (Katusic, et al. 1998; Rosenkranz-Weiss, et al. 1994; Wever, et al. 1997). In biliary cirrhotic rats, it was demonstrated that TNF, through the activation of iNOS in the aorta and lung, plays a role in the development of the hyperdynamic circulatory syndrome and the

Adrenomedullin is an endogenous vasodilatory peptide consisting of 52 amino acid residues in human and 50 amino acid residues in the rat (Kitamura, et al. 1993; Kitamura, et al. 1993; Nuki, et al. 1993). The major producers of circulating adrenomedullin are vascular smooth muscle cells (Sugo, et al. 1994) and endothelial cells (Sugo, et al. 1995). Adrenomedullin binds to and induces its signaling through the G-protein-coupled calcitonin receptor-like receptor/receptor activity-modifying protein (RAMP)2 and 3, which are expressed in multiple tissues, including blood vessels, kidney, lung, atrium, gastrointestinal tract, spleen, endocrine glands, brain and heart. Receptor RAMP2 is essential for angiogenesis and vascular integrity (Ichikawa-Shindo, et al. 2008). Adrenomedullin expression is up-regulated by hypoxia (Nagata, et al. 1999; Wang, et al. 1995) and inflammation (Sugo, et al. 1995; Ueda, et al. 1999), both of which are associated

The vasodilatory action of adrenomedullin was considered in the beginning to be solely due to elevated cAMP production, i.e., endothelium-independent vasodilation. However, endothelial denudation substantially reduced its vasodilatory action in rodent aortic rings (Hirata, et al. 1995; Nishimatsu, et al. 2001). Furthermore, this adrenomedullin-induced endothelium-dependent vasodilation was exerted mostly through activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway (Nishimatsu, et al. 2001). It has been well established that this pathway is involved in various important actions in endothelial cells, such as activation of eNOS. While one study demonstrated in a mouse model of ischemia that adrenomedullin-induced collateral vessel formation in ischemic tissues was eNOS-dependent (Abe, et al. 2003), no study has so far shown that adrenomedullin activates

Several studies have reported that in liver cirrhotic patients, circulating adrenomedullin levels are elevated and are associated with increased levels of plasma nitrite (a stable NO metabolite) and plasma volume expansion (Guevara, et al. 1998; Kojima, et al. 1998; Tahan, et al. 2003). Furthermore, the increased circulating adrenomedullin levels in those patients are inversely related to peripheral resistance (Guevara, et al. 1998). These observations indicate that adrenomedullin may promote excessive vasodilation and the hyperdynamic circulatory syndrome in cirrhotic patients. It is not surprising, therefore, that administration of an anti-adrenomedullin antibody prevented the occurrence of the hyperdynamic circulatory syndrome in the early sepsis (Wang, et al. 1998) and ameliorated blunted contractile response to phenylephrine in the aorta isolated from cirrhotic rats (Kojima, et al.

hepatopulmonary syndrome (Sztrymf, et al. 2004).

**2.1.7 Adrenomedullin** 

with neovascularization.

eNOS through Akt activation.

2004).

Portal hypertension alone, regardless of the presence of cirrhosis, increases adrenomedullin production. One clinical study showed that adrenomedullin and NO levels are elevated not only in patients with cirrhotic portal hypertension, but also in those patients with noncirrhotic portal hypertension (Tahan, et al. 2003). How an increase in portal pressure influences production of adrenomedullin is an interesting and important question to be investigated.

Fig. 7. Adrenomedullin causes vasodilation and hypotension. Adrenomedullin (AM) binds to and induces its signaling through the G-protein-coupled calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)2 and 3. The vascular action of adrenomedullin was at first considered to be solely due to elevated cAMP production by activation of adenylate cyclase (AC) in endothelial cells, thereby causing endotheliumindependent vasodilation. Adrenomedullin-induced endothelium-dependent vasodialation is exerted mostly through activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which activates eNOS to produce NO. NO then diffuses into smooth muscle cells to activate soluble guanylate cyclase (sGC) and produce cyclic GMP (cGMP), leading to vasodilation.

#### **2.1.8 Bradykinin**

Bradykinin is a nine amino acid peptide and known to facilitate vasodilation (Antonio & Rocha 1962). Bradykinin leads to endothelium-dependent hyperpolarization through activation of phospholipase C (PLC), which could raise Ca2+ concentration and also stimulate production of EETs (Feletou & Vanhoutte 2006) (Figure 6). Bradykinin reduces sensitivity to glypressin (a long lasting vasopressin analogue) in both portal hypertensive and cirrhotic rats (Chen, et al. 2009; Chu, et al. 2000), thereby advancing vasodilation.

#### **2.1.9 Urotensin II**

Urotensin II is a cyclic peptide and has a structural similarity to somatostatin. It can function both as a vasoconstrictor and a vasodilator depending on vascular beds (Coulouarn, et al.

The Molecules: Abnormal Vasculatures in the

levels.

**2.2.2 Arterial wall thinning** 

circulatory abnormalities seem to follow (iwakiri 2011).

Splanchnic and Systemic Circulation in Portal Hypertension 13

When mild portal hypertension is generated in rats using partial portal vein ligation, an increase in portal pressure is too small to cause splanchnic arterial vasodilation. However, the level of vascular endothelial growth factor (VEGF) is significantly elevated in the intestinal microcirculation, followed by increased eNOS levels (Abraldes, et al. 2006). This model of mild portal hypertension may likely correspond to the portal pressure changes observed in early-stage cirrhosis, in which the progression of portal hypertension is generally slow. When portal pressure is further increased to a certain level, vasodilation develops in the arterial splanchnic circulation. Once vasodilation is established in the intestinal microcirculation and the arterial splanchnic circulation, arterial systemic

Like the above study using rats with partial portal vein ligation, portal pressure modulates intestinal VEGF and eNOS levels during the development of cirrhosis in rats (Huang, et al. 2011). We have shown that there is a significant positive correlation between portal pressure and intestinal VEGF levels (r2 = 0.4, p<0.005). While plasma VEGF levels were significantly elevated in cirrhotic rats with portal hypertension (63.7 pg/ml, p<0.01) compared to controls (8.5 pg/ml), no correlation was observed between portal hypertension and plasma VEGF

Endothelial NO plays a critical part in regulating the structure of the vessel wall (Rudic, et al. 1998). Studies using cirrhotic rats with ascites documented the occurrence of arterial wall thinning. Those rats exhibited decreased thickness of the vascular walls of the thoracic aorta, abdominal aorta, mesenteric arteries and renal artery (Fernandez-Varo, et al. 2007; Fernandez-Varo, et al. 2003). Administration of a NOS inhibitor significantly ameliorated wall thickness and attenuated the hyperdynamic circulatory syndrome, by increasing arterial pressure and peripheral resistance (Fernandez-Varo, et al. 2003). Since NO is predominantly derived from endothelial cells in these arteries, these observations suggest that increased eNOS-derived NO, at least in part, is responsible for this profound arterial wall thinning. Therefore, understanding the mechanisms of arterial wall thinning is important for the development of useful therapies for patients with portal hypertension.

**3. Key molecules and factors – Portosystemic collateral vessel formation** 

In addition to excessive arterial vasodilation/hypocontractility in the splanchnic and systemic circulation, the formation of portosystemic collateral vessels is also thought to exacerbate portal hypertension (Bosch 2007; Iwakiri & Groszmann 2006). The portosystemic collateral vessel formation is probably an adaptive response to increased portal pressure, which, by releasing the pressure, may transiently help to delay the progression of portal hypertension. However, these collateral vessels eventually contribute to an increase in the blood flow through the portal vein and advance portal hypertension (Langer & Shah 2006). In addition, the formation of these vessels can also lead to detrimental complications. Since the vessels are fragile, they tend to rupture easily, causing esophageal and gastric variceal bleeding. Furthermore, since these vessels have the portal blood bypass the liver, toxic substances carried by it, such as drugs, bacterial toxins and toxic metabolites, returns to the

1998). In the systemic vessels including the aorta and coronary artery, urotensin II serves as the strongest vasoconstrictor known (Ames, et al. 1999; Douglas, et al. 2000). In rat mesenteric arteries, however, urotensin II causes vasodilation (Bottrill, et al. 2000). In biliary cirrhotic rats, plasma urotensin II levels were increased, and hypocontractility/vasodilatation was advanced in mesenteric arteries. An urotensin II receptor antagonist, palosuran, improved this hypocontractility/vasodilatation, by increasing RhoA/Rho-kinase expression and Rho-kinase activity (thereby more contraction) and decreasing nitrite/nitrate levels (Trebicka, et al. 2008). These observations may suggest that elevated levels of urotensin II also lead to hypocontractility/excessive vasodilation in the mesenteric arteries of patients with cirrhosis and portal hypertension. Thus, blocking the urotensin II-mediated signaling pathway may be an effective way to treat those patients.

#### **2.1.10 Neuropeptide Y**

Neuropeptide Y is a sympathetic neurotransmitter and known to cause -adrenergic vasoconstriction (Tatemoto 1982; Tatemoto, et al. 1982). RhoA/Rho-kinase modulates various cellular functions such as cell contractility through phosphorylation of myosin light chain (Uehata, et al. 1997; Wang, et al. 2009). It was suggested that impaired RhoA/Rhokinase signaling was responsible for excessive vasodilation and vascular hypocontractility in biliary cirrhotic rats (Hennenberg, et al. 2006). Acute administration of neuropeptide Y improved arterial contractility in the mesenteric arteries of cirrhotic rats by restoring impaired RhoA/Rho-kinase signaling (Moleda, et al. 2011). These observations may suggest that neuropeptide Y can be used for the treatment of hypocontractility/excessive vasodilation of the arterial splanchnic circulation in cirrhosis and portal hypertension.

#### **2.2 Key factors**

An increase in portal pressure alone can induce excessive arterial vasodilation and hypocontractility in the splanchnic and systemic circulation. In addition, chronic liver cirrhosis and portal hypertension are known to cause arterial wall thinning in these circulations. This arterial wall thinning is a critical factor that maintains excessive arterial vasodilation and hypocontractility and facilitates the development of the hyperdynamic circulatory syndrome in advanced portal hypertension.

#### **2.2.1 Portal pressure**

Using rats with partial portal vein ligation (Abraldes, et al. 2006; Fernandez, et al. 2005; Fernandez, et al. 2004; iwakiri 2011), which enables induction of different degrees of portal hypertension in animals (Iwakiri & Groszmann 2006), Abraldes et al. (Abraldes, et al. 2006) showed that portal pressure is detected at different vascular beds depending on the stage of portal hypertension. A small increase in portal pressure is first detected by the intestinal microcirculation. Then, further increased portal pressure is sensed by the arterial splanchnic circulation (e.g., the mesenteric arteries), finally followed by the arterial systemic circulation (e.g., the aorta). Thus, the intestinal microcirculation functions as a "sensing organ" to portal pressure. It is postulated that mechanical forces generated as a result of increased portal pressure, presumably cyclic strains and shear stress, activate eNOS and thus lead to NO production (Abraldes, et al. 2006; Iwakiri, et al. 2002; Tsai, et al. 2003).

When mild portal hypertension is generated in rats using partial portal vein ligation, an increase in portal pressure is too small to cause splanchnic arterial vasodilation. However, the level of vascular endothelial growth factor (VEGF) is significantly elevated in the intestinal microcirculation, followed by increased eNOS levels (Abraldes, et al. 2006). This model of mild portal hypertension may likely correspond to the portal pressure changes observed in early-stage cirrhosis, in which the progression of portal hypertension is generally slow. When portal pressure is further increased to a certain level, vasodilation develops in the arterial splanchnic circulation. Once vasodilation is established in the intestinal microcirculation and the arterial splanchnic circulation, arterial systemic circulatory abnormalities seem to follow (iwakiri 2011).

Like the above study using rats with partial portal vein ligation, portal pressure modulates intestinal VEGF and eNOS levels during the development of cirrhosis in rats (Huang, et al. 2011). We have shown that there is a significant positive correlation between portal pressure and intestinal VEGF levels (r2 = 0.4, p<0.005). While plasma VEGF levels were significantly elevated in cirrhotic rats with portal hypertension (63.7 pg/ml, p<0.01) compared to controls (8.5 pg/ml), no correlation was observed between portal hypertension and plasma VEGF levels.

#### **2.2.2 Arterial wall thinning**

12 Portal Hypertension – Causes and Complications

1998). In the systemic vessels including the aorta and coronary artery, urotensin II serves as the strongest vasoconstrictor known (Ames, et al. 1999; Douglas, et al. 2000). In rat mesenteric arteries, however, urotensin II causes vasodilation (Bottrill, et al. 2000). In biliary cirrhotic rats, plasma urotensin II levels were increased, and hypocontractility/vasodilatation was advanced in mesenteric arteries. An urotensin II receptor antagonist, palosuran, improved this hypocontractility/vasodilatation, by increasing RhoA/Rho-kinase expression and Rho-kinase activity (thereby more contraction) and decreasing nitrite/nitrate levels (Trebicka, et al. 2008). These observations may suggest that elevated levels of urotensin II also lead to hypocontractility/excessive vasodilation in the mesenteric arteries of patients with cirrhosis and portal hypertension. Thus, blocking the urotensin II-mediated signaling pathway may be an effective way to treat those patients.

Neuropeptide Y is a sympathetic neurotransmitter and known to cause -adrenergic vasoconstriction (Tatemoto 1982; Tatemoto, et al. 1982). RhoA/Rho-kinase modulates various cellular functions such as cell contractility through phosphorylation of myosin light chain (Uehata, et al. 1997; Wang, et al. 2009). It was suggested that impaired RhoA/Rhokinase signaling was responsible for excessive vasodilation and vascular hypocontractility in biliary cirrhotic rats (Hennenberg, et al. 2006). Acute administration of neuropeptide Y improved arterial contractility in the mesenteric arteries of cirrhotic rats by restoring impaired RhoA/Rho-kinase signaling (Moleda, et al. 2011). These observations may suggest that neuropeptide Y can be used for the treatment of hypocontractility/excessive vasodilation of the arterial splanchnic circulation in cirrhosis and portal hypertension.

An increase in portal pressure alone can induce excessive arterial vasodilation and hypocontractility in the splanchnic and systemic circulation. In addition, chronic liver cirrhosis and portal hypertension are known to cause arterial wall thinning in these circulations. This arterial wall thinning is a critical factor that maintains excessive arterial vasodilation and hypocontractility and facilitates the development of the hyperdynamic

Using rats with partial portal vein ligation (Abraldes, et al. 2006; Fernandez, et al. 2005; Fernandez, et al. 2004; iwakiri 2011), which enables induction of different degrees of portal hypertension in animals (Iwakiri & Groszmann 2006), Abraldes et al. (Abraldes, et al. 2006) showed that portal pressure is detected at different vascular beds depending on the stage of portal hypertension. A small increase in portal pressure is first detected by the intestinal microcirculation. Then, further increased portal pressure is sensed by the arterial splanchnic circulation (e.g., the mesenteric arteries), finally followed by the arterial systemic circulation (e.g., the aorta). Thus, the intestinal microcirculation functions as a "sensing organ" to portal pressure. It is postulated that mechanical forces generated as a result of increased portal pressure, presumably cyclic strains and shear stress, activate eNOS and thus lead to NO

circulatory syndrome in advanced portal hypertension.

production (Abraldes, et al. 2006; Iwakiri, et al. 2002; Tsai, et al. 2003).

**2.1.10 Neuropeptide Y** 

**2.2 Key factors** 

**2.2.1 Portal pressure** 

Endothelial NO plays a critical part in regulating the structure of the vessel wall (Rudic, et al. 1998). Studies using cirrhotic rats with ascites documented the occurrence of arterial wall thinning. Those rats exhibited decreased thickness of the vascular walls of the thoracic aorta, abdominal aorta, mesenteric arteries and renal artery (Fernandez-Varo, et al. 2007; Fernandez-Varo, et al. 2003). Administration of a NOS inhibitor significantly ameliorated wall thickness and attenuated the hyperdynamic circulatory syndrome, by increasing arterial pressure and peripheral resistance (Fernandez-Varo, et al. 2003). Since NO is predominantly derived from endothelial cells in these arteries, these observations suggest that increased eNOS-derived NO, at least in part, is responsible for this profound arterial wall thinning. Therefore, understanding the mechanisms of arterial wall thinning is important for the development of useful therapies for patients with portal hypertension.

#### **3. Key molecules and factors – Portosystemic collateral vessel formation**

In addition to excessive arterial vasodilation/hypocontractility in the splanchnic and systemic circulation, the formation of portosystemic collateral vessels is also thought to exacerbate portal hypertension (Bosch 2007; Iwakiri & Groszmann 2006). The portosystemic collateral vessel formation is probably an adaptive response to increased portal pressure, which, by releasing the pressure, may transiently help to delay the progression of portal hypertension. However, these collateral vessels eventually contribute to an increase in the blood flow through the portal vein and advance portal hypertension (Langer & Shah 2006). In addition, the formation of these vessels can also lead to detrimental complications. Since the vessels are fragile, they tend to rupture easily, causing esophageal and gastric variceal bleeding. Furthermore, since these vessels have the portal blood bypass the liver, toxic substances carried by it, such as drugs, bacterial toxins and toxic metabolites, returns to the

The Molecules: Abnormal Vasculatures in the

communications exerted by these molecules.

cirrhosis and portal hypertension.

treatment of portal hypertension.

Vol.306, No. 1, pp.10-5.

**5. Conclusion** 

**6. References** 

**4.1 Future direction** 

Splanchnic and Systemic Circulation in Portal Hypertension 15

Both experimental and clinical studies of cirrhosis and portal hypertension have documented that a wide variety of molecules are involved in excessive arterial vasodilation/hypocontractility in the splanchnic and systemic circulation. This accumulation of knowledge allows us to further investigate molecular and cellular mechanisms in which these molecules exert excessive arterial vasodilation/ hypocontractility in cirrhosis and portal hypertension. In particular, it is interesting and important to address how changes in portal pressure, along with these molecules, influence the function and structure of vasculatures in the splanchnic and systemic circulation. Furthermore, it is not fully elucidated how these molecules are excessively induced in portal hypertension. Another important investigation would be to elucidate paracrine and autocrine regulations of vascular cells (e.g., endothelial cells, smooth muscle cells and fibroblasts) by these molecules. While the roles of these molecules in the vasculature per se have been described, few studies have investigated cell specific regulations and cell-cell

Among those molecules introduced in this chapter, there are at least two molecules that are particularly anticipated for further investigation in the context of excessive arterial vasodilation and portosystemic collateral vessel formation in portal hypertension. One is hydrogen sulfide (H2S). An increasing body of evidence suggests that H2S is a crucial vasodilatory molecule in the superior mesenteric artery. However, it is not known whether H2S is also involved in excessive arterial vasodilation in the splanchnic circulation in portal hypertension. Another molecule of interest is adrenomedullin. Studies using mice lacking adrenomedullin or its receptors indicated that adrenomedullin plays a critical role in the regulation of blood vessel integrity, including vascular stability and permeability (Caron & Smithies 2001; Fritz-Six, et al. 2008; Ichikawa-Shindo, et al. 2008; Shindo, et al. 2001). Given that increased vascular permeability and decreased vessel integrity are typical of vessels in cirrhosis and portal hypertension, this aspect of adrenomedullin should be explored in

To date, there are only limited options for the treatment of portal hypertension, despite the fact that portal hypertension leads to the most lethal complications of liver diseases such as gastro-oesophageal varices and ascites. Facing this situation, there is a strong need for studies of the vascular abnormalities associated with cirrhosis and portal hypertension (Shah 2009). These studies will have potential to lead us to develop novel targets for the

Abe, M, M Sata, H Nishimatsu, D Nagata, E Suzuki, Y Terauchi, T Kadowaki, N Minamino,

Abraldes, JG, Y Iwakiri, M Loureiro-Silva, O Haq, WC Sessa & RJ Groszmann. (2006). Mild

K Kangawa, H Matsuo, Y Hirata & R Nagai. (2003). Adrenomedullin augments collateral development in response to acute ischemia. *Biochem Biophys Res Commun*,

increases in portal pressure upregulate vascular endothelial growth factor and

systemic circulation and can cause portal-systemic encephalopathy and sepsis (Bosch 2007; Iwakiri & Groszmann 2006). The enlargement of pre-existing vessels as well as angiogenesis facilitate the development of these collateral vessels (Langer & Shah 2006; Sumanovski, et al. 1999). Studies have shown that vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) play critical roles in the development of portosystemic collateral vessels in cirrhosis and portal hypertension.

#### **3.1 Vascular Endothelial Growth Factor (VEGF)**

The process of angiogenesis is regulated by growth factors exhibiting vasodilatory activity, such as VEGF. How are these angiogenic growth factors elevated in cirrhosis and portal hypertension? One mechanism may be initiated by an increase in portal pressure. As described previously, studies using portal hypertensive rats showed that a sudden increase in portal pressure is signaled to the intestinal microcirculation and induces intestinal VEGF expression (Abraldes, et al. 2006; Fernandez, et al. 2005). This sudden increase in portal pressure may create local mechanical forces, such as cyclic strains and shear stress, which may trigger VEGF induction.

It has been documented that administration of anti-angiogenic agents, such as blockers of VEGF receptor-2 (SU5416, anti-VEGFR2 monoclonal antibody) (Fernandez, et al. 2005; Fernandez, et al. 2004) and inhibitors of receptor tyrosine kinases (Sorafenib and Sunitinib) (Mejias, et al. 2009; Tugues, et al. 2007), reduces the formation of portosystemic collateral vessels and decreases portal pressure.

#### **3.2 Placental growth factor**

In addition to VEGF, placental growth factor (PlGF), another member of the VEGF family, has also been found to be increased in the intestinal microcirculation of portal hypertensive mice (Van Steenkiste, et al. 2009). In portal hypertensive mice lacking PlGF or given an anti-PlGF monoclonal antibody, both portal pressure and portosystemic collateral vessel formation were decreased. Collectively, these VEGF and PIGF studies suggest that blocking angiogenic activities, thereby decreasing the formation of portosystemic collateral vessels, has potential for the treatment of portal hypertension.

#### **4. Summary**

There are two major factors that contribute to excessive arterial vasodilation/hypocontractility in arteries of the splanchnic and systemic circulation in portal hypertension. One is an intrinsic factor and the other is a structural factor. The intrinsic factor includes vasodilatory molecules such as NO, CO, PGI2, endocannabinoids, EDHF, adrenomedullin, TNF, bradykinin and urotensin II. Decreased response to vasoconstrictors, such as neuropeptide Y, also facilitates hypocontractility of mesenteric arterial beds in cirrhosis and portal hypertension. The structural factor includes thinning of arterial wall (Fernandez-Varo, et al. 2007; Fernandez-Varo, et al. 2003). NO plays a critical role for arterial wall thinning in cirrhotic rats. However, its mechanism is not clear. In addition to excessive arterial vasodilatation/hypocontractility in the splanchnic and systemic circulation, the development of portosystemic collateral vessels is also regarded as the major factor that worsens portal hypertension (Bosch 2007; Iwakiri & Groszmann 2006).

#### **4.1 Future direction**

14 Portal Hypertension – Causes and Complications

systemic circulation and can cause portal-systemic encephalopathy and sepsis (Bosch 2007; Iwakiri & Groszmann 2006). The enlargement of pre-existing vessels as well as angiogenesis facilitate the development of these collateral vessels (Langer & Shah 2006; Sumanovski, et al. 1999). Studies have shown that vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) play critical roles in the development of portosystemic collateral

The process of angiogenesis is regulated by growth factors exhibiting vasodilatory activity, such as VEGF. How are these angiogenic growth factors elevated in cirrhosis and portal hypertension? One mechanism may be initiated by an increase in portal pressure. As described previously, studies using portal hypertensive rats showed that a sudden increase in portal pressure is signaled to the intestinal microcirculation and induces intestinal VEGF expression (Abraldes, et al. 2006; Fernandez, et al. 2005). This sudden increase in portal pressure may create local mechanical forces, such as cyclic strains and shear stress, which

It has been documented that administration of anti-angiogenic agents, such as blockers of VEGF receptor-2 (SU5416, anti-VEGFR2 monoclonal antibody) (Fernandez, et al. 2005; Fernandez, et al. 2004) and inhibitors of receptor tyrosine kinases (Sorafenib and Sunitinib) (Mejias, et al. 2009; Tugues, et al. 2007), reduces the formation of portosystemic collateral

In addition to VEGF, placental growth factor (PlGF), another member of the VEGF family, has also been found to be increased in the intestinal microcirculation of portal hypertensive mice (Van Steenkiste, et al. 2009). In portal hypertensive mice lacking PlGF or given an anti-PlGF monoclonal antibody, both portal pressure and portosystemic collateral vessel formation were decreased. Collectively, these VEGF and PIGF studies suggest that blocking angiogenic activities, thereby decreasing the formation of portosystemic collateral vessels,

There are two major factors that contribute to excessive arterial vasodilation/hypocontractility in arteries of the splanchnic and systemic circulation in portal hypertension. One is an intrinsic factor and the other is a structural factor. The intrinsic factor includes vasodilatory molecules such as NO, CO, PGI2, endocannabinoids, EDHF, adrenomedullin, TNF, bradykinin and urotensin II. Decreased response to vasoconstrictors, such as neuropeptide Y, also facilitates hypocontractility of mesenteric arterial beds in cirrhosis and portal hypertension. The structural factor includes thinning of arterial wall (Fernandez-Varo, et al. 2007; Fernandez-Varo, et al. 2003). NO plays a critical role for arterial wall thinning in cirrhotic rats. However, its mechanism is not clear. In addition to excessive arterial vasodilatation/hypocontractility in the splanchnic and systemic circulation, the development of portosystemic collateral vessels is also regarded as the major factor that worsens portal hypertension (Bosch 2007; Iwakiri &

vessels in cirrhosis and portal hypertension.

may trigger VEGF induction.

**3.2 Placental growth factor** 

**4. Summary** 

Groszmann 2006).

vessels and decreases portal pressure.

has potential for the treatment of portal hypertension.

**3.1 Vascular Endothelial Growth Factor (VEGF)** 

Both experimental and clinical studies of cirrhosis and portal hypertension have documented that a wide variety of molecules are involved in excessive arterial vasodilation/hypocontractility in the splanchnic and systemic circulation. This accumulation of knowledge allows us to further investigate molecular and cellular mechanisms in which these molecules exert excessive arterial vasodilation/ hypocontractility in cirrhosis and portal hypertension. In particular, it is interesting and important to address how changes in portal pressure, along with these molecules, influence the function and structure of vasculatures in the splanchnic and systemic circulation. Furthermore, it is not fully elucidated how these molecules are excessively induced in portal hypertension. Another important investigation would be to elucidate paracrine and autocrine regulations of vascular cells (e.g., endothelial cells, smooth muscle cells and fibroblasts) by these molecules. While the roles of these molecules in the vasculature per se have been described, few studies have investigated cell specific regulations and cell-cell communications exerted by these molecules.

Among those molecules introduced in this chapter, there are at least two molecules that are particularly anticipated for further investigation in the context of excessive arterial vasodilation and portosystemic collateral vessel formation in portal hypertension. One is hydrogen sulfide (H2S). An increasing body of evidence suggests that H2S is a crucial vasodilatory molecule in the superior mesenteric artery. However, it is not known whether H2S is also involved in excessive arterial vasodilation in the splanchnic circulation in portal hypertension. Another molecule of interest is adrenomedullin. Studies using mice lacking adrenomedullin or its receptors indicated that adrenomedullin plays a critical role in the regulation of blood vessel integrity, including vascular stability and permeability (Caron & Smithies 2001; Fritz-Six, et al. 2008; Ichikawa-Shindo, et al. 2008; Shindo, et al. 2001). Given that increased vascular permeability and decreased vessel integrity are typical of vessels in cirrhosis and portal hypertension, this aspect of adrenomedullin should be explored in cirrhosis and portal hypertension.

#### **5. Conclusion**

To date, there are only limited options for the treatment of portal hypertension, despite the fact that portal hypertension leads to the most lethal complications of liver diseases such as gastro-oesophageal varices and ascites. Facing this situation, there is a strong need for studies of the vascular abnormalities associated with cirrhosis and portal hypertension (Shah 2009). These studies will have potential to lead us to develop novel targets for the treatment of portal hypertension.

#### **6. References**


The Molecules: Abnormal Vasculatures in the

No. 2, pp.615-9.

pp.15212-7.

6, pp.475-82.

*Tumor Res*, Vol.32, No. pp.1-16.

Splanchnic and Systemic Circulation in Portal Hypertension 17

Caron, KM & O Smithies. (2001). Extreme hydrops fetalis and cardiovascular abnormalities

Carter, EP, CL Hartsfield, M Miyazono, M Jakkula, KG Morris, Jr. & IF McMurtry. (2002).

syndrome. *Am J Physiol Lung Cell Mol Physiol*, Vol.283, No. 2, pp.L346-53. Chaytor, AT, DH Edwards, LM Bakker & TM Griffith. (2003). Distinct hyperpolarizing and

Chen, CT, CJ Chu, FY Lee, FY Chang, SS Wang, HC Lin, MC Hou, SL Wu, CC Chan, HC

oxide and bradykinin. *Hepatogastroenterology*, Vol.56, No. 94-95, pp.1261-7. Chen, YC, P Gines, J Yang, SN Summer, S Falk, NS Russell & RW Schrier. (2004). Increased

Chu, CJ, SL Wu, FY Lee, SS Wang, FY Chang, HC Lin, CC Chan & SD Lee. (2000).

Claesson, HE, JA Lindgren & S Hammarstrom. (1977). Elevation of adenosine 3',5'-

Cohen, RA. (2005). The endothelium-derived hyperpolarizing factor puzzle: a mechanism

Cohen, RA, F Plane, S Najibi, I Huk, T Malinski & CJ Garland. (1997). Nitric oxide is the

rabbit carotid artery. *Proc Natl Acad Sci U S A*, Vol.94, No. 8, pp.4193-8. Cosentino, F & ZS Katusic. (1995). Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries. *Circulation*, Vol.91, No. 1, pp.139-44. Coulouarn, Y, I Lihrmann, S Jegou, Y Anouar, H Tostivint, JC Beauvillain, JM Conlon, HA

the spinal cord. *Proc Natl Acad Sci U S A*, Vol.95, No. 26, pp.15803-8.

artery in rats. *Gastroenterology*, Vol.138, No. 4, pp.1574-84.

Cruse, JM & RE Lewis, Jr. (1988). Cellular and cytokine immunotherapy of cancer. *Prog Exp* 

Dal-Ros, S, M Oswald-Mammosser, T Pestrikova, C Schott, N Boehm, C Bronner, T

De las Heras, D, J Fernandez, P Gines, A Cardenas, R Ortega, M Navasa, JA Barbera, B

spontaneous bacterial peritonitis. *Hepatology*, Vol.38, No. 2, pp.452-9.

experimental cirrhosis in rats. *Hepatology*, Vol.39, No. 4, pp.1075-87.

mediation by prostaglandin I2. *FEBS Lett*, Vol.81, No. 2, pp.415-8.

without a mediator? *Circulation*, Vol.111, No. 6, pp.724-7.

in mice lacking a functional Adrenomedullin gene. *Proc Natl Acad Sci U S A*, Vol.98,

Regulation of heme oxygenase-1 by nitric oxide during hepatopulmonary

relaxant roles for gap junctions and endothelium-derived H2O2 in NOindependent relaxations of rabbit arteries. *Proc Natl Acad Sci U S A*, Vol.100, No. 25,

Huang & SD Lee. (2009). Splanchnic hyposensitivity to glypressin in a hemorrhagetransfused common bile duct-ligated rat model of portal hypertension: role of nitric

vascular heme oxygenase-1 expression contributes to arterial vasodilation in

Splanchnic hyposensitivity to glypressin in a haemorrhage/transfused rat model of portal hypertension: role of nitric oxide and bradykinin. *Clin Sci (Lond)*, Vol.99, No.

monophosphate levels in 3T3 fibroblasts by arachidonic acid: evidence for

mediator of both endothelium-dependent relaxation and hyperpolarization of the

Bern & H Vaudry. (1998). Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of

Chataigneau, B Geny & VB Schini-Kerth. (2010). Losartan prevents portal hypertension-induced, redox-mediated endothelial dysfunction in the mesenteric

Calahorra, M Guevara, R Bataller, W Jimenez, V Arroyo & J Rodes. (2003). Increased carbon monoxide production in patients with cirrhosis with and without

endothelial nitric oxide synthase in the intestinal microcirculatory bed, leading to a hyperdynamic state. *Am J Physiol Gastrointest Liver Physiol*, Vol.290, No. 5, pp.G980-7.


Ames, RS, HM Sarau, JK Chambers, RN Willette, NV Aiyar, AM Romanic, CS Louden, JJ

Angermayr, B, M Mejias, J Gracia-Sancho, JC Garcia-Pagan, J Bosch & M Fernandez. (2006).

Arnold, WP, CK Mittal, S Katsuki & F Murad. (1977). Nitric oxide activates guanylate

Banks, M, CM Wei, CH Kim, JC Burnett, Jr. & VM Miller. (1996). Mechanism of relaxations to C-type natriuretic peptide in veins. *Am J Physiol*, Vol.271, No. 5 Pt 2, pp.H1907-11. Barlow, RS, AM El-Mowafy & RE White. (2000). H(2)O(2) opens BK(Ca) channels via the

Barriere, E, Tazi, KA, Rona, JP, Pessione, F, Heller, J & D Lebrec, Moreau, R. (2000). Evidence

Batkai, S, Z Jarai, JA Wagner, SK Goparaju, K Varga, J Liu, L Wang, F Mirshahi, AD

Beny, JL & PY von der Weid. (1991). Hydrogen peroxide: an endogenous smooth muscle cell hyperpolarizing factor. *Biochem Biophys Res Commun*, Vol.176, No. 1, pp.378-84. Bolognesi, M, F Zampieri, M Di Pascoli, A Verardo, C Turato, F Calabrese, F Lunardi, P

Bosch, J. (2000). Complications of cirrhosis. 1. Portal hypertension. *Hepatology*, Vol.32, No. 1

Bosch, J. (2007). Vascular deterioration in cirrhosis: the big picture. *J Clin Gastroenterol*,

Bottrill, FE, SA Douglas, CR Hiley & R White. (2000). Human urotensin-II is an

Caraceni, P, A Viola, F Piscitelli, F Giannone, A Berzigotti, M Cescon, M Domenicali, S

endothelium-dependent vasodilator in rat small arteries. *Br J Pharmacol*, Vol.130,

Petrosino, E Giampalma, A Riili, G Grazi, R Golfieri, M Zoli, M Bernardi & V Di Marzo. (2010). Circulating and hepatic endocannabinoids and endocannabinoidrelated molecules in patients with cirrhosis. *Liver Int*, Vol.30, No. 6, pp.816-25.

artery from rats with cirrhosis. *Hepatology*, Vol.32, No. 5, pp.935-41.

state in advanced liver cirrhosis. *Nat Med*, Vol.7, No. 7, pp.827-32.

isolated mammalian heart. *Circ Res*, Vol.11, No. pp.910-5.

*Physiol Heart Circ Physiol*, Vol.279, No. 2, pp.H475-83.

rats. *Liver Int*, Vol.31, No. 6, pp.881-90.

Vol.41 Suppl 3, No. pp.S247-53.

Suppl, pp.141-56.

No. 8, pp.1865-70.

preparations. *Proc Natl Acad Sci U S A*, Vol.74, No. 8, pp.3203-7.

Vol.401, No. 6750, pp.282-6.

endothelial nitric oxide synthase in the intestinal microcirculatory bed, leading to a hyperdynamic state. *Am J Physiol Gastrointest Liver Physiol*, Vol.290, No. 5, pp.G980-7.

Foley, CF Sauermelch, RW Coatney, Z Ao, J Disa, SD Holmes, JM Stadel, JD Martin, WS Liu, GI Glover, S Wilson, DE McNulty, CE Ellis, NA Elshourbagy, U Shabon, JJ Trill, DW Hay, EH Ohlstein, DJ Bergsma & SA Douglas. (1999). Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. *Nature*,

Heme oxygenase attenuates oxidative stress and inflammation, and increases VEGF expression in portal hypertensive rats. *J Hepatol*, Vol.44, No. 6, pp.1033-9. Antonio, A & ESM Rocha. (1962). Coronary vasodilation produced by bradykinin on

cyclase and increases guanosine 3':5'-cyclic monophosphate levels in various tissue

PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle. *Am J* 

for an endothelium-derived hyperpolarizing factor in the superior mesenteric

Khanolkar, A Makriyannis, R Urbaschek, N Garcia, Jr., AJ Sanyal & G Kunos. (2001). Endocannabinoids acting at vascular CB1 receptors mediate the vasodilated

Pontisso, P Angeli, C Merkel, A Gatta & D Sacerdoti. (2011). Increased myoendothelial gap junctions mediate the enhanced response to epoxyeicosatrienoic acid and acetylcholine in mesenteric arterial vessels of cirrhotic


The Molecules: Abnormal Vasculatures in the

Vol.392, No. 6678, pp.821-4.

Vol.43, No. 2, pp.413-9.

2, No. pp.S38-40.

Vol.278, No. 1, pp.87-90.

Vol.130, No. 3, pp.838-54.

Vol.27, No. 1, pp.20-7.

*Res Commun*, Vol.237, No. 3, pp.527-31.

*Eur J Pharmacol*, Vol.513, No. 3, pp.219-24.

with ascites. *Hepatology*, Vol.28, No. 1, pp.39-44.

pp.40-50.

pp.373-6.

Splanchnic and Systemic Circulation in Portal Hypertension 19

Fritz-Six, KL, WP Dunworth, M Li & KM Caron. (2008). Adrenomedullin signaling is

Fulton, D, JP Gratton, TJ McCabe, J Fontana, Y Fujio, K Walsh, TF Franke, A

Garcia-Cardena, G, R Fan, V Shah, R Sorrentino, G Cirino, A Papapetropoulos & WC Sessa.

Gauthier, KM, JR Falck, LM Reddy & WB Campbell. (2004). 14,15-EET analogs:

Gauthier, KM, N Spitzbarth, EM Edwards & WB Campbell. (2004). Apamin-sensitive K+

Gluais, P, G Edwards, AH Weston, PM Vanhoutte & M Feletou. (2005). Hydrogen peroxide

Griffith, TM. (2004). Endothelium-dependent smooth muscle hyperpolarization: do gap

Groszmann, RJ. (1993). Hyperdynamic state in chronic liver diseases. *J Hepatol*, Vol.17 Suppl

Guevara, M, C Bru, P Gines, G Fernandez-Esparrach, P Sort, R Bataller, W Jimenez, V

Heinemann, A & RE Stauber. (1995). The role of inducible nitric oxide synthase in vascular

Hennenberg, M, E Biecker, J Trebicka, K Jochem, Q Zhou, M Schmidt, KH Jakobs, T

Hirata, Y, H Hayakawa, Y Suzuki, E Suzuki, H Ikenouchi, O Kohmoto, K Kimura, K

Hou, MC, PA Cahill, S Zhang, YN Wang, RJ Hendrickson, EM Redmond & JV Sitzmann.

bovine coronary arteries. *Pharmacol Res*, Vol.49, No. 6, pp.515-24.

necessary for murine lymphatic vascular development. *J Clin Invest*, Vol.118, No. 1,

Papapetropoulos & WC Sessa. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. *Nature*, Vol.399, No. 6736, pp.597-601. Furchgott, RF & JV Zawadzki. (1980). The obligatory role of endothelial cells in the

relaxation of arterial smooth muscle by acetylcholine. *Nature*, Vol.288, No. 5789,

(1998). Dynamic activation of endothelial nitric oxide synthase by Hsp90. *Nature*,

characterization of structural requirements for agonist and antagonist activity in

currents mediate arachidonic acid-induced relaxations of rabbit aorta. *Hypertension*,

and endothelium-dependent hyperpolarization in the guinea-pig carotid artery.

junctions provide a unifying hypothesis? *Br J Pharmacol*, Vol.141, No. 6, pp.881-903.

Arroyo & Rodes. (1998). Increased cerebrovascular resistance in cirrhotic patients

hyporeactivity of endotoxin-treated and portal hypertensive rats. *Eur J Pharmacol*,

Sauerbruch & J Heller. (2006). Defective RhoA/Rho-kinase signaling contributes to vascular hypocontractility and vasodilation in cirrhotic rats. *Gastroenterology*,

Kitamura, T Eto, K Kangawa & et al. (1995). Mechanisms of adrenomedullininduced vasodilation in the rat kidney. *Hypertension*, Vol.25, No. 4 Pt 2, pp.790-5. Hosoki, R, N Matsuki & H Kimura. (1997). The possible role of hydrogen sulfide as an

endogenous smooth muscle relaxant in synergy with nitric oxide. *Biochem Biophys* 

(1998). Enhanced cyclooxygenase-1 expression within the superior mesenteric artery of portal hypertensive rats: role in the hyperdynamic circulation. *Hepatology*,


Dimmeler, S, I Fleming, B Fisslthaler, C Hermann, R Busse & AM Zeiher. (1999). Activation

Douglas, SA, AC Sulpizio, V Piercy, HM Sarau, RS Ames, NV Aiyar, EH Ohlstein & RN

Ellis, A, M Pannirselvam, TJ Anderson & CR Triggle. (2003). Catalase has negligible

Feletou, M & PM Vanhoutte. (2006). Endothelium-derived hyperpolarizing factor: where are

Feletou, M & PM Vanhoutte. (2007). Endothelium-dependent hyperpolarizations: past

Fernandez, M & HL Bonkovsky. (1999). Increased heme oxygenase-1 gene expression in

Fernandez, M, JC Garcia-Pagan, M Casadevall, C Bernadich, C Piera, BJ Whittle, JM Pique, J

Fernandez, M, M Mejias, B Angermayr, JC Garcia-Pagan, J Rodes & J Bosch. (2005).

Fernandez, M, F Vizzutti, JC Garcia-Pagan, J Rodes & J Bosch. (2004). Anti-VEGF receptor-2

Fernandez-Rodriguez, CM, J Romero, TJ Petros, H Bradshaw, JM Gasalla, ML Gutierrez, JL

Fernandez-Varo, G, M Morales-Ruiz, J Ros, S Tugues, J Munoz-Luque, G Casals, V Arroyo, J

Fernandez-Varo, G, J Ros, M Morales-Ruiz, P Cejudo-Martin, V Arroyo, M Sole, F Rivera, J

Fleming, I. (2004). Cytochrome P450 epoxygenases as EDHF synthase(s). *Pharmacol Res*,

hypertensive mice. *Gastroenterology*, Vol.126, No. 3, pp.886-94.

hemodynamics in cirrhosis. *Liver Int*, Vol.24, No. 5, pp.477-83.

vessels of cirrhotic rats. *J Hepatol*, Vol.46, No. 3, pp.440-6.

we now? *Arterioscler Thromb Vasc Biol*, Vol.26, No. 6, pp.1215-25.

beliefs and present facts. *Ann Med*, Vol.39, No. 7, pp.495-516.

and small mesenteric artery. *Br J Pharmacol*, Vol.140, No. 7, pp.1193-200. Faraci, FM, CG Sobey, S Chrissobolis, DD Lund, DD Heistad & NL Weintraub. (2001).

*Nature*, Vol.399, No. 6736, pp.601-5.

pp.R246-53.

No. 6, pp.1672-9.

pp.1985-93.

Vol.49, No. 6, pp.525-33.

Vol.108, No. 5, pp.1487-95.

rats. *J Hepatol*, Vol.43, No. 1, pp.98-103.

monkey. *Br J Pharmacol*, Vol.131, No. 7, pp.1262-74.

of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.

Willette. (2000). Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus

inhibitory effects on endothelium-dependent relaxations in mouse isolated aorta

Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels. *Am J Physiol Regul Integr Comp Physiol*, Vol.281, No. 1,

liver cells and splanchnic organs from portal hypertensive rats. *Hepatology*, Vol.29,

Bosch & J Rodes. (1995). Evidence against a role for inducible nitric oxide synthase in the hyperdynamic circulation of portal-hypertensive rats. *Gastroenterology*,

Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive

monoclonal antibody prevents portal-systemic collateral vessel formation in portal

Lledo, C Santander, TP Fernandez, E Tomas, G Cacho & JM Walker. (2004). Circulating endogenous cannabinoid anandamide and portal, systemic and renal

Rodes & W Jimenez. (2007). Impaired extracellular matrix degradation in aortic

Rodes & W Jimenez. (2003). Nitric oxide synthase 3-dependent vascular remodeling and circulatory dysfunction in cirrhosis. *Am J Pathol*, Vol.162, No. 6,


The Molecules: Abnormal Vasculatures in the

*Physiol*, Vol.280, No. 1, pp.G68-74.

Vol.290, No. 3, pp.909-13.

pp.1224-30.

pp.453-63.

rats. *Hepatology*, Vol.23, No. 6, pp.1616-21.

6, pp.877-84.

Splanchnic and Systemic Circulation in Portal Hypertension 21

Li, PL & WB Campbell. (1997). Epoxyeicosatrienoic acids activate K+ channels in coronary

Lin, HC, YY Yang, TH Tsai, CM Huang, YT Huang, FY Lee, TT Liu & SD Lee. (2011). The

Liu, H, D Song & SS Lee. (2001). Role of heme oxygenase-carbon monoxide pathway in

Lopez-Talavera, JC, G Cadelina, J Olchowski, W Merrill & RJ Groszmann. (1996).

Lopez-Talavera, JC, WW Merrill & RJ Groszmann. (1995). Tumor necrosis factor alpha: a

Matoba, T, H Shimokawa, H Kubota, K Morikawa, T Fujiki, I Kunihiro, Y Mukai, Y

Matoba, T, H Shimokawa, K Morikawa, H Kubota, I Kunihiro, L Urakami-Harasawa, Y

Matoba, T, H Shimokawa, M Nakashima, Y Hirakawa, Y Mukai, K Hirano, H Kanaide & A

Mayer, B & ER Werner. (1995). In search of a function for tetrahydrobiopterin in the

Mejias, M, E Garcia-Pras, C Tiani, R Miquel, J Bosch & M Fernandez. (2009). Beneficial

portal hypertensive and cirrhotic rats. *Hepatology*, Vol.49, No. 4, pp.1245-56. Miyazono, M, C Garat, KG Morris, Jr. & EP Carter. (2002). Decreased renal heme oxygenase-

Moleda, L, J Trebicka, P Dietrich, E Gabele, C Hellerbrand, RH Straub, T Sauerbruch, J

Mookerjee, RP, S Sen, NA Davies, SJ Hodges, R Williams & R Jalan. (2003). Tumour necrosis

derangements in alcoholic hepatitis. *Gut*, Vol.52, No. 8, pp.1182-7.

with portal hypertension. *J Hepatol*, Vol.54, No. 6, pp.1145-53.

hypertensive rats. *Gastroenterology*, Vol.108, No. 3, pp.761-7.

factor in mice. *J Clin Invest*, Vol.106, No. 12, pp.1521-30.

*Renal Physiol*, Vol.283, No. 5, pp.F1123-31.

pronounced splanchnic vasoaction. *Gut*, No.

smooth muscle through a guanine nucleotide binding protein. *Circ Res*, Vol.80, No.

relationship between endotoxemia and hepatic endocannabinoids in cirrhotic rats

pathogenesis of cirrhotic cardiomyopathy in the rat. *Am J Physiol Gastrointest Liver* 

Thalidomide inhibits tumor necrosis factor alpha, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive

major contributor to the hyperdynamic circulation in prehepatic portal-

Hirakawa & A Takeshita. (2002). Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries. *Biochem Biophys Res Commun*,

Mukai, Y Hirakawa, T Akaike & A Takeshita. (2003). Electron spin resonance detection of hydrogen peroxide as an endothelium-derived hyperpolarizing factor in porcine coronary microvessels. *Arterioscler Thromb Vasc Biol*, Vol.23, No. 7,

Takeshita. (2000). Hydrogen peroxide is an endothelium-derived hyperpolarizing

biosynthesis of nitric oxide. *Naunyn Schmiedebergs Arch Pharmacol*, Vol.351, No. 5,

effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in

1 expression contributes to decreased renal function during cirrhosis. *Am J Physiol* 

Schoelmerich & R Wiest. (2011). Amelioration of portal hypertension and the hyperdynamic circulatory syndrome in cirrhotic rats by neuropeptide Y via

factor alpha is an important mediator of portal and systemic haemodynamic


Huang, HC, O Haq, T Utsumi, S Sethasine, JG Abraldes, RJ Groszmann & Y Iwakiri. (2011).

Ichikawa-Shindo, Y, T Sakurai, A Kamiyoshi, H Kawate, N Iinuma, T Yoshizawa, T

angiogenesis and vascular integrity. *J Clin Invest*, Vol.118, No. 1, pp.29-39. Ignarro, LJ, GM Buga, KS Wood, RE Byrns & G Chaudhuri. (1987). Endothelium-derived

Ishizuka, D, Y Shirai & K Hatakeyama. (1997). Duodenal obstruction caused by gallstone

Iwakiri, Y. (2011). Endothelial dysfunction in the regulation of cirrhosis and portal

Iwakiri, Y & RJ Groszmann. (2006). The hyperdynamic circulation of chronic liver diseases: from the patient to the molecule. *Hepatology*, Vol.43, No. 2 Suppl 1, pp.S121-31. Iwakiri, Y, MH Tsai, TJ McCabe, JP Gratton, D Fulton, RJ Groszmann & WC Sessa. (2002).

Katusic, ZS, A Stelter & S Milstien. (1998). Cytokines stimulate GTP cyclohydrolase I gene

Kitamura, K, K Kangawa, M Kawamoto, Y Ichiki, S Nakamura, H Matsuo & T Eto. (1993).

Kojima, H, S Sakurai, M Uemura, H Satoh, T Nakashima, N Minamino, K Kangawa, H

Kojima, H, T Tsujimoto, M Uemura, A Takaya, S Okamoto, S Ueda, K Nishio, S Miyamoto,

Kwon, S, Iwakiri, Y., Cadelina, G., and Groszmann, RJ. (2004). Neuronal Nitric Oxide

Chronic Portal Hypertensive Rats. *Hepatology*, Vol.40, No. 4, pp.184A. Langer, DA & VH Shah. (2006). Nitric oxide and portal hypertension: interface of

vasoreactivity and angiogenesis. *J Hepatol*, Vol.44, No. 1, pp.209-16.

hepatic portal hypertension. *J Hepatol*, Vol.43, No. 2, pp.258-65.

*Biochem Biophys Res Commun*, Vol.194, No. 2, pp.720-5.

*Mol Med*, Vol.(in press), No.

No. 1, pp.182-3.

hypertension. *Liver Int*, No.

*Vasc Biol*, Vol.18, No. 1, pp.27-32.

Vol.39, No. 7, pp.686-93.

*Hepatol*, Vol.28, No. 5, pp.840-6.

*Acad Sci U S A*, Vol.84, No. 24, pp.9265-9.

Intestinal and plasma VEGF levels in cirrhosis: The role of portal pressure. *J Cell* 

Koyama, J Fukuchi, S Iimuro, N Moriyama, H Kawakami, T Murata, K Kangawa, R Nagai & T Shindo. (2008). The GPCR modulator protein RAMP2 is essential for

relaxing factor produced and released from artery and vein is nitric oxide. *Proc Natl* 

impaction into an intraluminal duodenal diverticulum. *Am J Gastroenterol*, Vol.92,

Phosphorylation of eNOS initiates excessive NO production in early phases of portal hypertension. *Am J Physiol Heart Circ Physiol*, Vol.282, No. 6, pp.H2084-90. Jurzik, L, M Froh, RH Straub, J Scholmerich & R Wiest. (2005). Up-regulation of nNOS and

associated increase in nitrergic vasodilation in superior mesenteric arteries in pre-

expression in cultured human umbilical vein endothelial cells. *Arterioscler Thromb* 

Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. *Biochem Biophys Res Commun*, Vol.192, No. 2, pp.553-60. Kitamura, K, J Sakata, K Kangawa, M Kojima, H Matsuo & T Eto. (1993). Cloning and

characterization of cDNA encoding a precursor for human adrenomedullin.

Matsuo & H Fukui. (2004). Adrenomedullin contributes to vascular hyporeactivity in cirrhotic rats with ascites via a release of nitric oxide. *Scand J Gastroenterol*,

A Kubo, N Minamino, K Kangawa, H Matsuo & H Fukui. (1998). Significance of increased plasma adrenomedullin concentration in patients with cirrhosis. *J* 

Synthase Plays a Role in the Vasodilation Observed in the Splanchnic Circulation in


The Molecules: Abnormal Vasculatures in the

*Clin Invest*, Vol.101, No. 4, pp.731-6.

*Pharmacol Exp Ther*, Vol.308, No. 2, pp.636-43.

Sessa, WC. (2004). eNOS at a glance. *J Cell Sci*, Vol.117, No. Pt 12, pp.2427-9.

hyperpolarizing factor. *Pharmacol Res*, Vol.49, No. 6, pp.543-9.

adrenomedullin gene. *Circulation*, Vol.104, No. 16, pp.1964-71.

molecular biology. *Annu Rev Biochem*, Vol.69, No. pp.145-82.

experimental cirrhosis. *J Hepatol*, Vol.26, No. 5, pp.1120-7.

*Commun*, Vol.203, No. 1, pp.719-26.

*Commun*, Vol.207, No. 1, pp.25-32.

pp.2236-43.

No. 1, pp.4-5.

Splanchnic and Systemic Circulation in Portal Hypertension 23

Rosenkranz-Weiss, P, WC Sessa, S Milstien, S Kaufman, CA Watson & JS Pober. (1994).

Rudic, RD, EG Shesely, N Maeda, O Smithies, SS Segal & WC Sessa. (1998). Direct evidence

Sacerdoti, D, NG Abraham, AO Oyekan, L Yang, A Gatta & JC McGiff. (2004). Role of the

Shah, V. (2009). Therapy for portal hypertension: What is our pipeline? *Hepatology*, Vol.49,

Shah, V, R Wiest, G Garcia-Cardena, G Cadelina, RJ Groszmann & WC Sessa. (1999). Hsp90

Shindo, T, Y Kurihara, H Nishimatsu, N Moriyama, M Kakoki, Y Wang, Y Imai, A Ebihara,

Skill, NJ, NG Theodorakis, YN Wang, JM Wu, EM Redmond & JV Sitzmann. (2008). Role of

Smith, WL, RM Garavito & DL DeWitt. (1996). Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. *J Biol Chem*, Vol.271, No. 52, pp.33157-60. Sogni, P, AP Smith, A Gadano, D Lebrec & TW Higenbottam. (1997). Induction of nitric

Stipanuk, MH & PW Beck. (1982). Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat. *Biochem J*, Vol.206, No. 2, pp.267-77. Sugo, S, N Minamino, H Shoji, K Kangawa, K Kitamura, T Eto & H Matsuo. (1994).

Sugo, S, N Minamino, H Shoji, K Kangawa, K Kitamura, T Eto & H Matsuo. (1995).

Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. *J Clin Invest*, Vol.93, No. 5,

for the importance of endothelium-derived nitric oxide in vascular remodeling. *J* 

heme oxygenases in abnormalities of the mesenteric circulation in cirrhotic rats. *J* 

regulation of endothelial nitric oxide synthase contributes to vascular control in portal hypertension. *American Journal of Physiology*, Vol.277, No. 2 Pt 1, pp.G463-8. Shimokawa, H & T Matoba. (2004). Hydrogen peroxide as an endothelium-derived

T Kuwaki, KH Ju, N Minamino, K Kangawa, T Ishikawa, M Fukuda, Y Akimoto, H Kawakami, T Imai, H Morita, Y Yazaki, R Nagai, Y Hirata & H Kurihara. (2001). Vascular abnormalities and elevated blood pressure in mice lacking

cyclooxygenase isoforms in prostacyclin biosynthesis and murine prehepatic portal hypertension. *Am J Physiol Gastrointest Liver Physiol*, Vol.295, No. 5, pp.G953-64. Smith, WL, DL DeWitt & RM Garavito. (2000). Cyclooxygenases: structural, cellular, and

oxide synthase II does not account for excess vascular nitric oxide production in

Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-alpha. *Biochem Biophys Res* 

Interleukin-1, tumor necrosis factor and lipopolysaccharide additively stimulate production of adrenomedullin in vascular smooth muscle cells. *Biochem Biophys Res* 


Morales-Ruiz, M, W Jimenez, D Perez-Sala, J Ros, A Leivas, S Lamas, F Rivera & V Arroyo.

Morikawa, K, H Shimokawa, T Matoba, H Kubota, T Akaike, MA Talukder, M Hatanaka, T

Mustafa, AK, G Sikka, SK Gazi, J Steppan, SM Jung, AK Bhunia, VM Barodka, FK Gazi, RK

Nagata, D, Y Hirata, E Suzuki, M Kakoki, H Hayakawa, A Goto, T Ishimitsu, N Minamino,

Nishimatsu, H, E Suzuki, D Nagata, N Moriyama, H Satonaka, K Walsh, M Sata, K

Ohta, M, F Kishihara, M Hashizume, H Kawanaka, M Tomikawa, H Higashi, K Tanoue & K

Oltman, CL, NL Weintraub, M VanRollins & KC Dellsperger. (1998). Epoxyeicosatrienoic

Pfister, SL, N Spitzbarth, K Nithipatikom, WS Edgemond, JR Falck & WB Campbell. (1998).

Plane, F, KE Wiley, JY Jeremy, RA Cohen & CJ Garland. (1998). Evidence that different

Ros, J, J Claria, J To-Figueras, A Planaguma, P Cejudo-Martin, G Fernandez-Varo, R Martin-

coronary microcirculation. *Circ Res*, Vol.83, No. 9, pp.932-9.

cirrhosis in the rat. *Gastroenterology*, Vol.122, No. 1, pp.85-93.

with ascites. *Hepatology*, Vol.24, No. 6, pp.1481-6.

Vol.112, No. 12, pp.1871-9.

*Circ Res*, No.

1, pp.H220-8.

pp.245-51.

pp.30879-87.

Vol.21, No. 4, pp.121-4.

*Acids*, Vol.53, No. 1, pp.41-5.

(1996). Increased nitric oxide synthase expression in arterial vessels of cirrhotic rats

Fujiki, H Maeda, S Takahashi & A Takeshita. (2003). Pivotal role of Cu,Znsuperoxide dismutase in endothelium-dependent hyperpolarization. *J Clin Invest*,

Barrow, R Wang, LM Amzel, DE Berkowitz & SH Snyder. (2011). Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels.

Y Ono, K Kangawa, H Matsuo & M Omata. (1999). Hypoxia-induced adrenomedullin production in the kidney. *Kidney Int*, Vol.55, No. 4, pp.1259-67. Naik, JS & BR Walker. (2003). Heme oxygenase-mediated vasodilation involves vascular

smooth muscle cell hyperpolarization. *Am J Physiol Heart Circ Physiol*, Vol.285, No.

Kangawa, H Matsuo, A Goto, T Kitamura & Y Hirata. (2001). Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3 kinase/Akt-dependent pathway in rat aorta. *Circ Res*, Vol.89, No. 1, pp.63-70. Nuki, C, H Kawasaki, K Kitamura, M Takenaga, K Kangawa, T Eto & A Wada. (1993).

Vasodilator effect of adrenomedullin and calcitonin gene-related peptide receptors in rat mesenteric vascular beds. *Biochem Biophys Res Commun*, Vol.196, No. 1,

Sugimachi. (1995). Increased prostacyclin content in gastric mucosa of cirrhotic patients with portal hypertensive gastropathy. *Prostaglandins Leukot Essent Fatty* 

acids and dihydroxyeicosatrienoic acids are potent vasodilators in the canine

Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta. *J Biol Chem*, Vol.273, No. 47,

mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery. *Br J Pharmacol*, Vol.123, No. 7, pp.1351-8. Quilley, J & JC McGiff. (2000). Is EDHF an epoxyeicosatrienoic acid? *Trends Pharmacol Sci*,

Ruiz, V Arroyo, F Rivera, J Rodes & W Jimenez. (2002). Endogenous cannabinoids: a new system involved in the homeostasis of arterial pressure in experimental


The Molecules: Abnormal Vasculatures in the

Vol.12, No. 11, pp.1035-44.

6659, pp.518-21.

pp.1856-62.

pp.1223-33.

*Disease*, Vol.19, No. 4, pp.411-26.

Vol.277, No. 3 Pt 2, pp.H893-900.

Splanchnic and Systemic Circulation in Portal Hypertension 25

Van Steenkiste, C, A Geerts, E Vanheule, H Van Vlierberghe, F De Vos, K Olievier, C

Varga, K, JA Wagner, DT Bridgen & G Kunos. (1998). Platelet- and macrophage-derived

Wagner, JA, K Varga, EF Ellis, BA Rzigalinski, BR Martin & G Kunos. (1997). Activation of

Wang, P, ZF Ba, WG Cioffi, KI Bland & IH Chaudry. (1998). The pivotal role of

Wang, X, TL Yue, FC Barone, RF White, RK Clark, RN Willette, AC Sulpizio, NV Aiyar, RR

Wang, Y, XR Zheng, N Riddick, M Bryden, W Baur, X Zhang & HK Surks. (2009). ROCK

Wei, CM, S Hu, VM Miller & JC Burnett, Jr. (1994). Vascular actions of C-type natriuretic

Weigert, AL, PY Martin, M Niederberger, EM Higa, IF McMurtry, P Gines & RW Schrier.

Wever, RM, T van Dam, HJ van Rijn, F de Groot & TJ Rabelink. (1997). Tetrahydrobiopterin

Wiest, R, G Cadelina, S Milstien, RS McCuskey, G Garcia-Tsao & RJ Groszmann. (2003).

Wiest, R, S Das, G Cadelina, G Garcia-Tsao, S Milstien & RJ Groszmann. (1999). Bacterial

Wiest, R & RJ Groszmann. (1999). Nitric oxide and portal hypertension: its role in the

You, J, TD Johnson, SP Marrelli & RM Bryan, Jr. (1999). Functional heterogeneity of

oxide synthase. *Biochem Biophys Res Commun*, Vol.237, No. 2, pp.340-4. Widmann, MD, NL Weintraub, JL Fudge, LA Brooks & KC Dellsperger. (1998). Cytochrome

vasodilation in vivo. *Am J Physiol*, Vol.274, No. 1 Pt 2, pp.H283-9.

vasculature of cirrhotic rats. *Hepatology*, Vol.38, No. 6, pp.1508-15.

hypertension. *Gastroenterology*, Vol.137, No. 6, pp.2112-24 e1-6.

sepsis. *Arch Surg*, Vol.133, No. 12, pp.1298-304.

*Natl Acad Sci U S A*, Vol.92, No. 25, pp.11480-4.

muscle cells. *Circ Res*, Vol.104, No. 4, pp.531-40.

cells. *Biochem Biophys Res Commun*, Vol.205, No. 1, pp.765-71.

Casteleyn, D Laukens, M De Vos, JM Stassen, P Carmeliet & I Colle. (2009). Role of placental growth factor in mesenteric neoangiogenesis in a mouse model of portal

endogenous cannabinoids are involved in endotoxin-induced hypotension. *Faseb J*,

peripheral CB1 cannabinoid receptors in haemorrhagic shock. *Nature*, Vol.390, No.

adrenomedullin in producing hyperdynamic circulation during the early stage of

Ruffolo, Jr. & GZ Feuerstein. (1995). Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage. *Proc* 

isoform regulation of myosin phosphatase and contractility in vascular smooth

peptide in isolated porcine coronary arteries and coronary vascular smooth muscle

(1995). Endothelium-dependent vascular hyporesponsiveness without detection of nitric oxide synthase induction in aortas of cirrhotic rats. *Hepatology*, Vol.22, No. 6,

regulates superoxide and nitric oxide generation by recombinant endothelial nitric

P-450 pathway in acetylcholine-induced canine coronary microvascular

Bacterial translocation up-regulates GTP-cyclohydrolase I in mesenteric

translocation in cirrhotic rats stimulates eNOS-derived NO production and impairs mesenteric vascular contractility. *Journal of Clinical Investigation*, Vol.104, No. 9,

regulation of intrahepatic and splanchnic vascular resistance. *Seminars in Liver* 

endothelial P2 purinoceptors in the cerebrovascular tree of the rat. *Am J Physiol*,


Sumanovski, LT, E Battegay, M Stumm, M van der Kooij & CC Sieber. (1999). Increased

Sztrymf, B, A Rabiller, H Nunes, L Savale, D Lebrec, A Le Pape, V de Montpreville, M

Tahan, V, E Avsar, C Karaca, E Uslu, F Eren, S Aydin, H Uzun, HO Hamzaoglu, F Besisik, C

Tarquini, R, E Masini, G La Villa, G Barletta, M Novelli, R Mastroianni, RG Romanelli, F

Tatemoto, K. (1982). Isolation and characterization of peptide YY (PYY), a candidate gut

Tatemoto, K, M Carlquist & V Mutt. (1982). Neuropeptide Y--a novel brain peptide with

Tomioka, H, Y Hattori, M Fukao, A Sato, M Liu, I Sakuma, A Kitabatake & M Kanno. (1999).

Trebicka, J, L Leifeld, M Hennenberg, E Biecker, A Eckhardt, N Fischer, AS Probsting, C

Tsai, MH, Y Iwakiri, G Cadelina, WC Sessa & RJ Groszmann. (2003). Mesenteric

Ueda, S, K Nishio, N Minamino, A Kubo, Y Akai, K Kangawa, H Matsuo, Y Fujimura, A

Uehata, M, T Ishizaki, H Satoh, T Ono, T Kawahara, T Morishita, H Tamakawa, K

Urakami-Harasawa, L, H Shimokawa, M Nakashima, K Egashira & A Takeshita. (1997).

portal hypertension. *World J Gastroenterol*, Vol.9, No. 10, pp.2325-7.

4, pp.1044-9.

Vol.23, No. 5, pp.752-8.

Vol.104, No. 4, pp.891-7.

Vol.79, No. 8, pp.2514-8.

*Res*, Vol.36, No. 4, pp.311-20.

*Hepatology*, Vol.47, No. 4, pp.1264-76.

cirrhotic rats. *Hepatology*, Vol.46, No. 6, pp.1919-26.

*J Respir Crit Care Med*, Vol.160, No. 1, pp.132-6.

*Nature*, Vol.389, No. 6654, pp.990-4.

*Clin Invest*, Vol.100, No. 11, pp.2793-9.

No. 5858, pp.659-60.

angiogenesis in portal hypertensive rats: role of nitric oxide. *Hepatology*, Vol.29, No.

Mazmanian, M Humbert & P Herve. (2004). Prevention of hepatopulmonary syndrome and hyperdynamic state by pentoxifylline in cirrhotic rats. *Eur Respir J*,

Kalayci, A Okten & N Tozun. (2003). Adrenomedullin in cirrhotic and non-cirrhotic

Vizzutti, U Santosuosso & G Laffi. (2009). Increased plasma carbon monoxide in patients with viral cirrhosis and hyperdynamic circulation. *Am J Gastroenterol*,

hormone that inhibits pancreatic exocrine secretion. *Proc Natl Acad Sci U S A*,

structural similarities to peptide YY and pancreatic polypeptide. *Nature*, Vol.296,

Relaxation in different-sized rat blood vessels mediated by endothelium-derived hyperpolarizing factor: importance of processes mediating precontractions. *J Vasc* 

Clemens, F Lammert, T Sauerbruch & J Heller. (2008). Hemodynamic effects of urotensin II and its specific receptor antagonist palosuran in cirrhotic rats.

vasoconstriction triggers nitric oxide overproduction in the superior mesenteric artery of portal hypertensive rats. *Gastroenterology*, Vol.125, No. 5, pp.1452-61. Tugues, S, G Fernandez-Varo, J Munoz-Luque, J Ros, V Arroyo, J Rodes, SL Friedman, P

Carmeliet, W Jimenez & M Morales-Ruiz. (2007). Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in

Yoshioka, K Masui, N Doi, Y Murao & S Miyamoto. (1999). Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome. *Am* 

Yamagami, J Inui, M Maekawa & S Narumiya. (1997). Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension.

Importance of endothelium-derived hyperpolarizing factor in human arteries. *J* 


**2** 

*UK* 

**Cystic Fibrosis Liver Disease** 

Cystic fibrosis (CF) is the most common fatal autosomal recessive disorder in the white population with a frequency of 1 in 2500 live births. Inherited defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in abnormal regulation of salt and water movement across membranes. The overall feature of CF is that secretions are dehydrated due to water deprivation of luminal surfaces. This is true for the sinopulmonary tract, biliary tree and for reproductive tubes (vas deferens and fallopian tubes). Lung involvement is the main cause of morbidity and mortality, but this abnormal gene results in a multisystem disease affecting the liver, pancreas, sinuses, bones and reproductive system. Since its recognition in 1938 when life expectancy was 6 months (Davis, 2006), advances in medicine and a multidisciplinary team approach to patient care have increased the life expectancy of this condition such that children born today could expect to reach their 50s (Dodge et al., 2007; UK CF Trust 2009; USCF foundation 2009). The changing course of the disease has resulted in increasing relevance of other organ systems

The incidence of CF liver disease (CFLD) varies significantly among previous studies

2. Structural – as increased bile content in the liver tissues or fatty infiltration visualised

3. Decompensated liver disease – which includes portal hypertension, hypoalbuminaemia,

The authors of this chapter propose to call 'clinically significant disease' as hepatic abnormalities that necessitate treatment by pharmacological and non-pharmacological

In one study by Nash et al (2008), 30% of CF patients were found to be affected by clinically significant CFLD. Clinically significant disease in this study was defined as biochemical

ranging from 5-30%. This is probably due to variations in definition.

**1. Introduction** 

such as the liver.

methods.

**2. Epidemiology of CF Liver Disease** 

on liver ultrasound studies

ascites and impaired coagulation.

On the whole, CFLD can be classified into 3 stages:

1. Biochemical – manifested by impaired liver enzymes

Andrew Low and Nabil A. Jarad *Department of Respiratory Medicine,* 

*Bristol Royal Infirmary,* 

