The hepatic apelin system: A new therapeutic target for liver disease

Authors

  • Alessandro Principe,

    1. Biochemistry and Molecular Genetics Service, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
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  • Pedro Melgar-Lesmes,

    1. Biochemistry and Molecular Genetics Service, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
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  • Guillermo Fernández-Varo,

    1. Biochemistry and Molecular Genetics Service, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
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  • Luis Ruiz del Arbol,

    1. Department of Gastroenterology, Hospital Ramón y Cajal, University of Alcala, Madrid, Spain
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  • Josefa Ros,

    1. Biochemistry and Molecular Genetics Service, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
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  • Manuel Morales-Ruiz,

    1. Biochemistry and Molecular Genetics Service, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
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  • Mauro Bernardi,

    1. Department of Internal Medicine, Policlinico Sant'Orsola-Malpighi, University of Bologna, Bologna, Italy
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  • Vicente Arroyo,

    1. Liver Unit, CIBEREHD, Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain
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  • Wladimiro Jiménez

    Corresponding author
    1. Biochemistry and Molecular Genetics Service, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
    2. Department of Physiology I, University of Barcelona, Barcelona, Spain
    • Servicio de Bioquímica y Genética Molecular, Hospital Clinic Universitari, Villarroel 170, Barcelona 08036, Spain
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    • fax: (3493)2275697.


  • Potential conflict of interest: Nothing to report.

Abstract

Apelin is a peptide that plays an important role in heart physiology and pathophysiology, inflammation, and angiogenesis. We evaluated whether the endogenous apelin system is involved in the pathogenesis of the hepatic remodeling and cardiovascular and renal complications occurring in advanced liver disease. The circulating levels of apelin, the messenger RNA (mRNA) and protein expression of apelin and apelin receptor, the immunohistological detection of apelin and apelin receptor, and the effects induced by the chronic administration of an apelin receptor antagonist on fibrosis and vessel density were evaluated in rats with cirrhosis and ascites and in control rats. The serum levels of apelin in patients with cirrhosis were also measured. Apelin levels were higher in rats with cirrhosis than in controls. Apelin mRNA showed a four-fold rise only in hepatic tissue, but not in the lung, heart, or kidney of rats with cirrhosis. These animals also showed hepatic apelin receptor mRNA levels 300 times higher than controls. Apelin was highly expressed by stellate cells, whereas apelin receptor was overexpressed in the hepatic parenchyma of animals with cirrhosis. Rats with cirrhosis treated with the apelin receptor antagonist showed diminished hepatic fibrosis and vessel density, improved cardiovascular performance, and renal function and lost ascites. Human patients also showed a marked increase in apelin levels. Conclusion: The selective hepatic activation of the apelin system, together with the drop in fibrosis and neoangiogenesis and the improvement in cardiovascular and excretory function resulting from apelin receptor blockade, points to the hepatic apelin system as a novel therapeutic target in liver disease. (HEPATOLOGY 2008.)

Apelin is the endogenous ligand of the angiotensin-like-receptor 1 (AGTRL1), a G-protein-coupled receptor that has been found to be involved in an array of physiologic events, such as water homeostasis,1 regulation of the cardiovascular tone,2 and cardiac contractility.3 Apelin and its receptor are widely expressed in the central nervous system and in peripheral tissues, especially in endothelial cells but also in leukocytes, enterocytes, adipocytes, and cardiomyocytes.4–8 AGTRL1 activation leads to inhibition of cyclic adenosine monophosphate production and activation of the Na+/H+ exchanger type 1.9 Through the former pathway, apelin enhances the vascular dilatation after the induction of endothelial nitric oxide (NO) synthase, in a molecular cascade leading to extracellular-signal regulated kinases and P70S6K activation.10, 11 On the other hand, the burst of Na+/H+ exchanger type 1 activity in cardiomyocytes leads to a dose-dependent increase in in vivo and in vitro myocardial contractility.3, 12 Recent studies also suggest a role for apelin in inflammation and angiogenesis since its expression is regulated by tumor necrosis factor–α,6 and it has been demonstrated that apelin could trigger vascular sprouting in absence of vascular endothelial growth factor (VEGF).13

Hepatic cirrhosis is among the most prevalent diseases in Western countries. The prognosis of these patients is grim, except for those who may benefit from liver transplantation. This is due to the multiple organic derangements, including renal failure, variceal bleeding, or bacterial peritonitis, developed by these individuals.14 Several clinical and experimental studies have demonstrated that the trigger for these disturbances is the existence of an important and progressively accentuated cardiocirculatory dysfunction, of which portal hypertension, arterial hypotension, high cardiac output (CO), and diminished systemic vascular resistance are the most relevant features.15 Thus, during the last decade the mechanisms leading to decreased arterial pressure and concomitant arterial vasodilation in cirrhosis have been a subject of major interest. Although it is generally believed that increased endothelial production of NO is of major importance in the pathogenesis of this phenomenon,15 the contribution of other recently identified endogenous systems has also been considered.16

In the current investigation, we assessed the circulating levels of apelin, the hemodynamic and renal effects induced by either the administration of apelin or the acute blockade of the apelin receptor, and the tissue expression of these peptides in rats with cirrhosis and ascites. Furthermore, we assessed whether chronic blockade of AGTRL1 may influence hepatic remodeling, fibrosis, and neoangiogenesis in rats with cirrhosis. Finally, the relationship between the degree of activation of the renin-angiotensin-aldosterone (ALDO) and sympathetic nervous systems and the circulating levels of apelin in patients with cirrhosis and ascites was also evaluated. The hypothesis of the investigation was that the endogenous apelin system is involved in the pathogenesis of the hepatic remodeling and cardiovascular and renal complications occurring in advanced liver disease.

Abbreviations

AGTRL1, angiotensin-like-receptor 1; ALDO, aldosterone; bw, body weight; CI, cardiac index; CO, cardiac output; MAP, mean arterial pressure; mRNA, messenger RNA; NO, nitric oxide; PP, portal pressure; SPP, splanchnic perfusion pressure; TPR, total peripheral resistance; VEGF, vascular endothelial growth factor

Patients and Methods

Induction of Cirrhosis in Rats.

Studies were performed in male adult Wistar rats (Charles River, Saint Aubin les Elseuf, France). Rats with cirrhosis and control rats were fed ad libitum with standard chow and distilled water containing phenobarbital (0.3 gm.L−1) as drinking fluid. Cirrhosis was induced by CCl4 as described elsewhere.17 Rats with cirrhosis and ascites were studied between 16 and 21 weeks when ascites had fully developed. Control rats were studied following a similar period of phenobarbital administration.

Hemodynamic and Renal Effects of Apelin Administration in Rats with Cirrhosis.

To assess the systemic and renal effects of apelin, 12 control rats and 12 rats with cirrhosis and ascites were randomly assigned to a hemodynamic (six rats with cirrhosis and six control rats) or a renal excretory function study (six rats with cirrhosis and six control rats) as described below. Thereafter, hemodynamic or renal parameters were measured in basal conditions and following the intravenous injection of Apelin-13 (15 μg/kg of body weight [kg.bw]; Sigma-Aldrich Chemie Gmbh, Steinherim, Germany).

Hemodynamic and Renal Effects of Apelin Receptor Blockade in Rats with Cirrhosis.

The systemic and renal effects of apelin receptor blockade were assessed in 12 control rats and 12 rats with cirrhosis and ascites. Animals were randomly assigned to a hemodynamic (six rats with cirrhosis and six control rats) or renal excretory function study (six rats with cirrhosis and six control rats) as described below. Thereafter, hemodynamic or renal parameters were measured in basal conditions and following the intravenous injection of the specific antagonist of apelin receptor F13A18 (15 μg/kg.bw, Phoenix Pharmaceuticals, Burlingame, CA).

Circulating Levels and Tissue Expression of Apelin and Apelin Receptor in Rats with Cirrhosis.

Serum concentration of apelin was measured in 24 control and 24 rats with cirrhosis and ascites. Blood samples were collected from the femoral vein; following centrifugation (1,800 g for 10 minutes, 4°C), plasma was frozen at −20°C in aliquots until further analysis. Liver, kidney, lung, and heart specimens were collected from 10 control and 10 rats with cirrhosis, washed in diethyl pyrocarbonate (0.1%)-treated phosphate buffered saline salt solution (in mM: NaCl 140, Na2HPO4 8.5, Na2HPO4.H2O 1.84, pH 7.4), immediately frozen in dry ice, and stored in liquid nitrogen to evaluate messenger expression of apelin and messenger RNA (mRNA) and protein abundance of AGTRL1.

Chronic Blockade of the Apelin Receptor, AGTRL1, in Rats with Cirrhosis.

Rats with cirrhosis and ascites were included in the protocol after developing stable ascites. Thereafter, CCl4 treatment was discontinued and animals were randomly assigned to one of the following groups: group A (n = 8), daily subcutaneous injection of the AGTRL1 antagonist, F13A18 (75 μg/kg.bw; Phoenix Pharmaceuticals) for 9 days beginning the second week after the detection of sustained ascites and group B (n = 8), daily subcutaneous injection of saline solution (1 mL/kg.bw). At the end of the treatment animals were anesthetized and mean arterial pressure (MAP), CO, and portal pressure (PP) were recorded as described below. Splanchnic perfusion pressure (SPP) was defined as MAP − PP. Thereafter, ascites volume was measured by laparotomy and liver samples from treated and untreated animals were fixed in 10% buffered formalin for further hematoxylin-eosin and immunostaining analysis.

Hemodynamic Studies.

Rats with cirrhosis and control rats were anesthetized with Inactin (100 mg/kg.bw; Sigma-Aldrich Chemie Gmbh) and prepared with a polyvinyl-50 catheter in the left femoral artery. A blood sample (2 mL) was taken to measure standard parameters of hepatic and renal function and plasma concentration of apelin. Packed cells were reconstituted to an equal volume with Ringer solution and reinfused over 3 minutes. Animals were prepared for measurements of hemodynamic parameters as described.19 Total peripheral resistance (TPR) was obtained using the formula TPR = MAP/CO. Hemodynamic parameters were allowed to equilibrate for 30 minutes and values of MAP, heart rate, CO, and TPR were recorded in basal conditions and 15 minutes after the intravenous administration of Apelin-13 or F13A. The cardiac index (CI) was calculated as CO/bw. Each value represents the average of two measurements.

Renal Excretory Function.

Rats with cirrhosis and ascites and control rats were placed in metabolic cages and the excretion of free water was determined once weekly (Tuesday) as described.20 When a moderate impairment in the renal ability to excrete free water was detected (% of water load excreted <85%) animals with cirrhosis were included in the protocol. After 24 hours, animals received an intravenous administration of Apelin-13 (15 μg/kg.bw) or F13A (15 μg/kg.bw) through the tail vein. Thereafter, animals were submitted to a second water overload, as previously described, and % of water excreted was determined in the 3-hour urine collection. After 24 hours, the animals were killed by isoflurane overdose and trunk blood was obtained by decapitation to measure standard parameters of hepatic and renal function and plasma concentration of apelin.

Messenger Expression of Apelin and AGTRL1 in Different Organs of Rats with Cirrhosis.

See online expanded experimental procedures in the Supplementary Materials.

AGTRL1 Protein Expression in Hepatic Tissue of Control and Rats with Cirrhosis.

See online expanded experimental procedures in the Supplementary Materials.

Immunodetection of Apelin, AGTRL1, and Von Willebrand Factor.

See online expanded experimental procedures in the Supplementary Materials.

Immunofluorescence.

See online expanded experimental procedures in the Supplementary Materials.

Fibrosis Quantification.

See online expanded experimental procedures in the Supplementary Materials.

Human Studies.

A total of 59 patients with cirrhosis and ascites and 10 healthy subjects were included in the study. After admission, patients were given a 50-70 mmol/day sodium diet without diuretics. At 8 AM of the fifth day, after overnight fasting and following 1 hour of bed rest, samples were obtained to measure liver and renal function, plasma renin activity, and plasma concentrations of ALDO, norepinephrine, and apelin. An antecubital vein was catheterized and blood samples were obtained under ice and centrifuged at 4°C and the serum was stored at −80°C until assayed.

Measurements and Statistical Analysis.

See online expanded experimental procedures in the Supplementary Materials.

Ethical Approval.

The study was performed according to the criteria of the Investigation and Ethics Committees of the Hospital Clínic Universitari and the Hospital Ramón y Cajal. Patients gave written informed consent to participate in the study, which was conducted according to the guidelines of Good Clinical Practice.

Results

Liver histology of all the animals treated with CCl4 included in the study had a finely granulated surface and histological examination showed the characteristic features of cirrhosis. Ascites volume ranged between 5 and 60 mL. Control rats showed no appreciable alterations in liver histology.

Increased Circulating Levels of Apelin in Cirrhotic Rats.

Cirrhotic rats were investigated after they had developed marked abnormalities in liver function tests. As anticipated, rats submitted to the cirrhosis induction protocol showed decreased serum albumin (25.3 ± 6.6 g/L, P < 0.001) increased activity of alanine aminotransferase (49.5 ± 6.2 U/L, P < 0.01), and enhanced serum concentration of bilirubin (1.48 ± 0.20 mg/dL, P < 0.001) in comparison to control rats (37.7 ± 1.2 g/L, 17.8 ± 7.1 U/L, and 0.18 ± 0.02 mg/dL, respectively). This marked hepatic dysfunction was associated with a significant increase in the circulating levels of apelin in comparison to healthy animals (1,235 ± 49 versus 930 ± 42 pg/mL, P < 0.001). In fact, only three out of the 24 rats with cirrhosis analyzed showed values of apelin within the range of control rats. No differences in serum creatinine were found between rats with cirrhosis and control rats (0.39 ± 0.01 versus 0.43 ± 0.03 mg/dL).

Apelin Is Involved in the Cardiocirculatory Dysfunction in Cirrhotic Rats.

To investigate the pathophysiological significance of the increased production of apelin in cirrhosis, we next assessed the hemodynamic and renal effects resulting from stimulating or inhibiting the apelin receptor in control and rats with cirrhosis. The results obtained in 12 control and 12 animals with cirrhosis following the intravenous administration of Apelin-13 are shown in Table 1. Rats were randomly assigned to either the hemodynamic or the renal response group. As expected, apelin administration to control rats resulted in a marked vasodilator effect as reflected by an approximately 30% reduction in TPR. This was associated with a concomitant increase in CI, thus resulting in no significant changes in MAP. By contrast, this characteristic hemodynamic response to the intravenous administration of apelin in healthy animals was not seen in cirrhotic rats. In fact, neither CI nor TPR experienced any noticeable change following the administration of the peptide. The renal response to a water load in basal conditions and after the administration of apelin is also shown in Table 1. Whereas apelin administration decreased water and sodium excretion in normal rats, the peptide did not further modify the already impaired ability to excrete a water load in cirrhotic animals.

Table 1. Hemodynamic and Renal Effects Induced by the Intravenous Administration of Apelin-13
 Control Rats (n = 12)Cirrhotic Rats (n = 12)
BasalPosttreatmentBasalPosttreatment
  • Apelin-13 dosage 15 μg/kg of body weight.

  • *

    P < 0.05,

  • **

    P < 0.01,

  • ***

    P < 0.005, in comparison to basal values (paired Student t test).

  • P < 0.001 in comparison to control rats (unpaired Student t test).

Hemodynamic study (n = 12)    
 Mean arterial pressure (mmHg)117 ± 3116 ± 496 ± 791 ± 7
 Cardiac index (mL.minute−1.kg−1)0.38 ± 0.010.54 ± 0.04***0.80 ± 0.060.92 ± 0.05
 Total peripheral resistance (mmHg.mL−1.minute)0.71 ± 0.050.49 ± 0.02**0.26 ± 0.020.21 ± 0.01
Renal study (n = 12)    
 Water excreted (%)99 ± 279 ± 11**72 ± 473 ± 4
 Urine sodium (μEq/minute)0.91 ± 0.190.36 ± 0.10*0.07 ± 0.020.04 ± 0.01

The hemodynamic and renal effects observed after AGTRL1 blockade were qualitatively opposite to those seen after the administration of Apelin-13 (Table 2). Actually, F13A administration did not modify any of the hemodynamic parameters assessed in control animals. By contrast, AGTRL1 blockade significantly improved cardiovascular function in cirrhotic rats, as indicated by a marked reduction in CI and a significant increase in TPR. The results observed on assessing renal function were in line with the cardiovascular findings. AGTRL1 blockade did not modify the renal ability to excrete a water load in control rats but significantly increased water and sodium excretion in cirrhotic rats with and ascites (Table 2).

Table 2. Hemodynamic and Renal Effects Induced by the Intravenous Administration of the Apelin Receptor Antagonist F13A
 Control Rats (n = 12)Cirrhotic Rats (n = 12)
BasalPosttreatmentBasalPosttreatment
  • F13A dosage 15 μg/kg body weight.

  • *

    P < 0.05;

  • **

    P < 0.01;

  • ***

    P < 0.005, in comparison to basal (paired Student t test) values.

  • P < 0.001 in comparison to control rats (unpaired Student t test).

Hemodynamic study (n = 12)    
 Mean arterial pressure (mmHg)115 ± 4120 ± 283 ± 491 ± 7
 Cardiac index (mL.minute−1.kg−1)0.41 ± 0.040.33 ± 0.030.80 ± 0.020.59 ± 0.02***
 Total peripheral resistance (mmHg.minute.mL−1)0.62 ± 0.040.80 ± 0.080.23 ± 0.020.32 ± 0.02**
Renal study (n = 12)    
 Water excreted (%)96 ± 296 ± 169 ± 289 ± 3**
 Urine sodium (μEq/minute)0.86 ± 0.191.04 ± 0.100.08 ± 0.040.29 ± 0.09*

The Apelin System Is Overactivated in the Liver of Cirrhotic Rats.

To further delineate whether the apelin system is selectively activated in cirrhosis, we measured mRNA expression of apelin and AGTRL1 in heart, kidney, lung, and liver of rats with cirrhosis and control rats (Fig. 1). No significant differences were found in the abundance of both messengers between hearts or kidneys of animals with cirrhosis and control animals. A decreased expression of apelin and apelin receptor was observed in the lung of rats with cirrhosis in comparison to controls, although values only reached statistical significance in the former case. However, an intense upregulation of the endogenous apelin system was observed in the liver of rats with cirrhosis. The amount of apelin and AGTRL1 transcripts was three-fold to four-fold and around 300-fold-higher in the hepatic tissue of rats with cirrhosis than in controls, respectively (Fig. 1). This overactivation of the hepatic apelin system in cirrhosis was also noted at the posttranslational level. AGTRL1 content in protein extracts obtained from hepatic homogenates of rats with cirrhosis was markedly higher than in control animals (Fig. 2).

Figure 1.

mRNA expression of apelin and AGTRL1 (apelin receptor) by real-time polymerase chain reaction (PCR). Liver, kidney, heart, and lung mRNAs taken from 10 control rats and 10 rats with cirrhosis were retrotranscribed and amplified. hypoxanthine phosphoribosyltransferase (HPRT) was used as the housekeeping gene.

Figure 2.

Protein expression of the apelin receptor, AGTRL1. Representative western blot of whole protein extracts taken from two cirrhotic and two control livers. Densitometric analysis in all animals (six rats with cirrhosis and six control rats) is shown at the top of the figure.

Apelin Is Produced by Stellate Cells.

In an attempt to identify the cellular source of the altered expression of apelin and AGTRL1, we performed histological immunolocalization of these peptides in the liver of cirrhotic and control rats. Apelin immunolabeling was almost undetectable in control rats, but in animals with cirrhosis was identified as linear staining in the portal tracts and fibrous septa and was more intense in the severely fibrotic tissue (Fig. 3). As occurred with the expression pattern of apelin, hepatic AGTRL1 detection was restricted to some perivenular areas in control animals, whereas in rats with cirrhosis, labeling-positive cells extended throughout all the hepatic parenchyma with the exception of the fibrotic areas (Fig. 3). Interestingly, apelin immunostaining colocalized with α–smooth muscle actin (Fig. 4). These findings indicate that activation of apelin production in the liver of rats with cirrhosis selectively occurs in myofibroblastic cell types located in the margin of the fibrous septa.

Figure 3.

Protein expression of apelin and apelin receptor (AGTRL1) was immunolocalized in liver sections of control (CT) and rats with cirrhosis and ascites (CH). Apelin expression was restricted to perivenular areas (arrows) in control rats, while a stronger signal was detected in rats with cirrhosis, especially across the fibrous septa. AGTRL1 was faintly detected in perivenular areas (arrow) in control rats, while a marked increase in its expression was detectable throughout the cirrhotic parenchyma, but not in the scar tissue. Original magnification: ×200.

Figure 4.

Immunofluorescent localization of apelin and α-smooth muscle actin (SMA) in livers of cirrhotic rats with ascites. Apelin (A, green), α-SMA (B, red), and cell nuclei (C, blue) fluorescent staining was performed using 4,6-diamidino-2-phenylindole (DAPI) and specific antibodies. Colocalization of α-SMA and apelin (arrow, yellow) is shown in the merge panel (D). b.v., blood vessel. Original magnification: ×100.

Effect of Chronic AGTRL1 Blockade on Systemic Hemodynamics, Hepatic Fibrosis, and Vessel Density in Cirrhotic Rats.

As anticipated, cirrhotic rats receiving vehicle had a marked hyperdynamic circulatory syndrome (MAP: 82 ± 3 mmHg; CI: 1.13 ± 0.1 mL.kg−1.minute−1; TPR: 0.20 ± 0.01 mmHg.mL−1.minute; PP: 14 ± 1 mmHg; and SPP: 70 ± 4 mmHg). Administration of the AGTRL1 antagonist for 9 days to rats with cirrhosis significantly improved systemic hemodynamics (MAP: 97 ± 3 mmHg, P < 0.05; CI: 0.85 ± 0.06 mL.kg−1.minute−1, P < 0.05; TPR: 0.32 ± 0.04 mmHg.mL−1.minute, P < 0.05) when there were no changes in PP (13 ± 1 mm Hg) but a significant increase in SPP (84 ± 4 mmHg, P < 0.05). At the end of the study, ascites volume significantly differs between nontreated and treated cirrhotic rats (42 ± 8 versus 16 ± 8 mL, P < 0.05). Furthermore, ascites was undetectable in four out of the eight rats with cirrhosis treated with the apelin receptor antagonist, whereas noticeable volumes of this liquid were detected in all cirrhotic rats treated with vehicle. No significant differences in creatinine clearance were observed between treated and nontreated rats with cirrhosis (0.54 ± 0.12 versus 0.59 ± 0.05 mL/minute). Anti-von Willebrand Factor antibody and Sirius red were used to identify vascular proliferation and collagen fibrils, respectively. Both groups of rats have pronounced vascular growth and abundant fibrosis (Fig. 5). However, liver sections obtained from cirrhotic rats receiving F13A showed an approximately 40% lower vessel density and 25% decrease of fibrosis area than sections of vehicle-treated cirrhotic rats (Fig. 5).

Figure 5.

Effect of apelin receptor AGTRL1 blockade on liver fibrosis and vessel density. Top panels: Sirius red staining of representative liver sections obtained from cirrhotic rats with ascites treated either with vehicle (n = 8) or F13A (n = 8). Bottom panels: Immunolocalization of von Willebrand Factor (vWF) was used to quantify vessel density in liver sections taken from rats with cirrhosis treated either with vehicle (n = 8) or F13A (n = 8). Bars on the right show the quantitative measurement of relative fibrosis and vessel density in all animals. Original magnification, ×100.

Patients with Cirrhosis Have Increased Circulating Levels of Apelin.

The demographic and laboratory data in healthy subjects and patients with cirrhosis are shown in Table 3. Marked hepatic failure was present in patients with cirrhosis, as indicated by high serum bilirubin and low serum albumin and prothrombin activity. Serum creatinine ranged between 0.64 and 3.26 mg/dL in patients with cirrhosis, although no statistical differences were found when compared with healthy subjects. As anticipated, patients displayed marked overactivity of the renin-angiotensin and sympathetic nervous systems (Table 3). Patients with cirrhosis also presented a striking increase in the circulating levels of apelin. In most patients apelin plasma levels were at least two-fold higher than in healthy subjects and in some individuals figures reached values 10-fold higher than in controls. An indirect significant relationship was found between apelin levels and serum creatinine (r = −0.351, P < 0.01), whereas no correlation was found between apelin concentration and plasma renin activity (r = 0.138), ALDO (r = 0.307), or norepinephrine (r = 0.173) in patients with cirrhosis.

Table 3. Demographic Data, Liver and Renal Function Test, and Endogenous Vasoactive Systems in Subjects with Cirrhosis and Control Subjects
 Healthy Subjects (n = 10)Subjects with Cirrhosis and Ascites (n = 59)
  • *

    P < 0.05;

  • P < 0.01;

  • P < 0.005; with respect to healthy subjects (unpaired Student t test).

Sex (M/F)5/549/10
Age (years)43 ± 1052 ± 12
Etiology (viral/alcohol)N/A32/27
Serum bilirrubin (mg/dL)0.38 ± 0.054.8 ± 0.7*
Serum albumin (g/L)40.2 ± 0.631.4 ± 0.1
Blood urea nitrogen (mg/dL)14.4 ± 0.945.42 ± 5.0*
Serum creatinine (mg/dL)0.75 ± 0.051.17 ± 0.1
Plasma renin activity (ng/mL.hour)0.14 ± 0.044.47 ± 0.5
Aldosterone (ng/dL)10.34 ± 1.486.48 ± 11.2
Norepinephrine (ng/mL)196 ± 17.4471 ± 34.7
Serum apelin (pg/mL)282 ± 11855 ± 75

Discussion

Hepatic architectural disruption is a major feature of patients with chronic liver disease. Actually, the liver of these patients undergoes an intense process of tissue remodeling characterized by chronic inflammation, neoangiogenesis, and fibrogenesis.21, 22 In end-stage liver disease, hepatic failure occurs, with liver transplantation being the only therapeutic alternative. Apelin is a recently described endogenous peptide that plays an important role in heart physiology, pathophysiology,7, 12 as well as inflammation4 and angiogenesis.13

The current study evaluated whether the endogenous apelin system is involved in the hepatic remodeling and the cardiovascular and renal complications occurring in advanced liver disease. First, we measured the circulating levels of apelin, which were markedly enhanced in rats with cirrhosis in comparison to control animals. This represents the first circumstantial evidence linking apelin activation to decompensated cirrhosis. The pathophysiological meaning of this finding was next evaluated by assessing the changes induced by either systemically challenging or blocking the apelin system in rats with cirrhosis.

Acute single intravenous administration of apelin to control animals resulted in a pronounced reduction in peripheral resistance, which is consistent with previous investigations demonstrating that this peptide possesses vasodilator properties, exerted through the NO signaling pathway.2, 23 This effect was not associated with significant modifications in the MAP of these animals due to the marked rise in CO induced by the peptide. In fact, apelin has been described as one of the most powerful endogenous positive inotropic substances known so far.3 Administration of this peptide also deteriorated renal excretory function in control animals. The impaired ability to excrete water and sodium observed after a water load was probably due to the decrease in peripheral resistance and/or a direct renal effect induced by apelin in these animals. This would be in line with an investigation describing the presence of AGTRL1 in the renal tissue of adult and neonate rats.9 However, contrary to control animals, cirrhotic rats with ascites exhibited marked resistance to the cardiovascular and renal effects of apelin, since this peptide did not modify the already impaired hemodynamic and excretory function of cirrhotic rats. Conversely, AGTRL1 blockade improved systemic hemodynamics and significantly ameliorated the renal excretory function in cirrhotic rats with ascites. The most interesting finding was that the administration of the antagonist had a marked negative inotropic effect that resulted in an almost 30% decrease in CI and a significant rise in TPR with no evident effects on MAP. F13A did not produce any significant change in control animals. As far as we know, these results are the first indication that the increased circulating levels of apelin are of pathogenic cardiovascular significance in cirrhosis.

Studies assessing tissue distribution of apelin and AGTRL1 in normal adult rats demonstrated that the highest expression of both messengers was in the lung and also found quite strong levels in the heart; however, the abundance of these transcripts was very faint in the liver.8, 9 Therefore, to establish whether cirrhosis is associated with an altered expression pattern of apelin and AGTRL1 mRNAs, we next extracted total mRNA from liver, lung, heart, and kidney from the two groups of animals. Only the hepatic tissue of cirrhotic rats showed significantly increased abundance of both messengers with respect to control rats. This difference was markedly striking in the case of AGTRL1 mRNA and was paralleled by higher abundance of the AGTRL1 protein in the hepatic tissue of cirrhotic rats than in controls. Our findings indicate that the hepatic apelin system is markedly and selectively activated in cirrhosis. These results are in line with the concept that the increased circulating levels of apelin in cirrhotic rats mainly result from an increased hepatic production rather than an impaired renal catabolism of this peptide. This is further supported by the absence of differences in serum creatinine between cirrhotic and control rats. Immunolocalization experiments revealed a strong positive signal for apelin and AGTRL1 in the liver of cirrhotic animals. In the former case the signal was located in stellate cells, whereas the apelin receptor was mainly identified in hepatocytes. These results indicate that, beyond its cardiotropic and vasodilator effects, apelin may also behave as a hepatic paracrine substance in the cirrhotic liver, secreted by the activated stellate cells interacting with the nearby parenchymal cells, and hence triggering proinflammatory and neoangiogenic signaling pathways. Fibrosis, therefore, can no longer be considered as a simple deposition of fibrillar extracellular matrix. Rather, hepatic remodeling should be considered as a process in which neoformed vessels are embedded in an active evolving scar tissue, where a complex interplay occurs between several cell types and soluble substances.

Previous investigations have shown that apelin and AGTRL1 are induced by tumor necrosis factor–α6 and VEGF,24 respectively. Activation of these peptides in the fibrotic liver results from the chronic inflammation and reduced oxygenation19 that takes place in the injured viscera. Therefore, we speculated that the hepatic apelin system could be an important mediator in the initiation and maintenance of the inflammatory and fibrogenic processes occurring in the fibrotic liver. To confirm this hypothesis, we disrupted the apelin signaling pathway by chronically blocking AGTRL1. The degree of hepatic fibrosis and angiogenesis of cirrhotic rats chronically treated with the receptor antagonist dropped 25% and 40%, respectively, as compared with cirrhotic rats receiving vehicle. Moreover, these animals also showed an improvement in both cardiovascular performance and renal function and consequently, lost ascites. However, no differences in PP were observed between rats treated and not treated with the AGTRL1 antagonist. This is likely due to the increased SPP resulting from the vasoconstrictor effect of the apelin antagonist. Thus, in cirrhotic rats receiving F13A, the reduction in fibrosis was not paralleled by an amelioration in portal hypertension because of the counteracting effect of the increase in SPP. These results, therefore, indicate that selective chronic inhibition of the apelin receptor improves recovery from cirrhosis and causes a faster reversal of neoangiogenesis in experimental cirrhosis.

While the mechanisms of action of the hepatic apelin system have not been fully clarified, impaired oxygen availability in the hepatic tissue could play a pivotal position in upregulating both components of this system. Direct regulation of apelin expression and secretion by hypoxia has been shown in rat cardiac myocytes,25 whereas hypoxia is also a well-known inducer of VEGF, which in turn stimulates AGTRL1 expression in endothelial cells of blood vessels.24 This suggests that under liver injury–associated hypoxia, the hepatic apelin system could play a central role in mediating inflammatory response and fibrogenesis.

Finally, we sought to determine whether apelin activation also occurs in humans with liver disease. Patients with cirrhosis and ascites also showed a highly significant increase in the circulating levels of apelin, with almost no overlap with values of healthy subjects, thereby supporting the concept that the hepatic apelin system is of pathogenic significance in cirrhosis. Moreover, the selective hepatic activation of the apelin system in cirrhotic rats, together with the important drop in fibrogenesis and angiogenesis resulting from AGTRL1 blockade, suggests that therapeutic agents interfering with this signaling pathway might not only help to stabilize decompensated patients waiting for transplant, but may also alter the natural course of the disease.

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