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Hepatic tissue endothelin-1 levels in chronic liver disease correlate with disease severity and ascites
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2000 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 95, No. 1, 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(99)00743-1
Hepatic Tissue Endothelin-1 Levels in Chronic Liver Disease Correlate With Disease Severity and Ascites I. Alam, M.D., N. M. Bass, M.D., Ph.D., P. Bacchetti, Ph.D., L. Gee, and D. C. Rockey, M.D. Departments of Medicine and Biostatistics and the Liver Center, University of California, San Francisco, California; and the Department of Medicine and Liver Center, Duke University Medical Center, Durham, North Carolina
OBJECTIVES: Plasma endothelin-1 (ET-1) levels are increased in patients with cirrhosis and ET-1 production is increased in the liver itself during experimental injury. These data suggest a possible role for this vasoactive peptide in intrahepatic microcirculatory changes that contribute to the pathogenesis of portal hypertension in cirrhosis. Therefore the aims of this study were to determine whether ET-1 levels were abnormal in the livers of patients with cirrhosis and to investigate possible clinical correlates of altered hepatic ET-1 in cirrhosis. METHODS: Liver specimens were obtained from explants at the time of liver transplantation in 62 cirrhotic patients; 49 without pretransplantation transjugular intrahepatic portosystemic shunt (TIPS) and 13 with pretransplantation TIPS. The presence of ascites was evaluated by physical examination and ultrasonography. Control specimens consisted of livers with normal morphology obtained from patients who died from nonliver-related causes. Hepatic ET-1 was measured by enzyme immunoassay. RESULTS: Hepatic ET-1 levels in cirrhotics without (0.17 pg/mg liver tissue) or with TIPS (0.12 pg/mg) were higher than in control patients [0.04 pg/mg (p ⫽ 0.02 for ET-1 levels in cirrhotics with or without TIPS vs control)]. In cirrhotics without ascites who had not had TIPS, ET-1 levels (0.07 pg/mg [0.04 –1.00]) were similar to those of the controls. In contrast, ET-1 content was increased in cirrhotics with small (0.11 pg/mg; p ⫽ 0.0002) and moderate-to-large (0.69 pg/mg; p ⫽ 0.0002) amounts of ascites compared to patients without ascites. There was a modest correlation between ET-1 levels and Child-Pugh score (correlation coefficient 0.32; p ⫽ 0.03) and ET-1 levels were significantly higher in patients with Child-Pugh score of 13 or greater (0.88 pg/mg; p ⫽ 0.02) than in those with Child-Pugh score of 12 or less (0.16 pg/mg). CONCLUSIONS: Hepatic tissue ET-1 levels are increased in the liver of patients with cirrhosis. This increase appears to be proportional to the severity of both liver disease and ascites. These data raise a possible role for ET-1 in modulation of intrahepatic resistance in cirrhotic portal
hypertension. (Am J Gastroenterol 2000;95:199 –203. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION The endothelins compromise a family of potent vasoconstrictors (1). Three unique endothelin peptides, each consisting of 21 amino acids, have been identified and termed endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). The endothelins have been implicated in a wide variety of normal and pathological processes. Their different effects are in part related to the receptor to which they bind endothelin-A (ETA) or endothelin-B (ETB) (2). Their major function appears to be in the local control of vascular tone (3). ET-1 and ET-3 appear to induce vasoconstriction through stimulation of smooth muscle cells and vasodilation due to effects on endothelial cells, respectively. Circulating levels of immunoreactive ET-1 have been reported to be elevated in a number of diseases including hypertension, atherosclerosis, acute myocardial infarction, and acute renal failure (1). Current information concerning plasma endothelin levels in cirrhosis is conflicting. During the past several years, numerous investigators have shown that both plasma ET-1 and ET-3 concentrations are increased in cirrhotics with ascites, varices, and hepatorenal syndrome (4 –14). However, some studies have reported both normal (15) and reduced (16) plasma levels in cirrhosis. Data demonstrating that ET-1 is overexpressed in different cellular compartments in cirrhotic tissue (17, 18), and that ET-1 is higher in the hepatic vein than in portal vein in patients with portal hypertension (19), have led investigators to postulate that the increased ET-1 levels in patients with cirrhosis are related to increased production by the diseased liver. This is supported by the recent demonstration of increased production of ET-1 (preproET-1 mRNA and peptide) in the liver during experimental injury (18), and increased hepatic levels of ET-1 mRNA and ET-1 in cirrhotic rats with ascites (20). However, hepatic endothelin levels in patients with cirrhosis have not been reported. Therefore, the present study was undertaken to determine whether
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ET-1 levels were abnormal in the livers of patients with cirrhosis, and to establish potential clinical correlates of altered hepatic ET-1 in cirrhosis.
MATERIALS AND METHODS Liver specimens were obtained consecutively from explants at the time of orthotopic liver transplantation in 62 patients with cirrhosis during the period July 1993 to August 1995. Thirteen of these patients had undergone a prior transjugular intrahepatic portosystemic shunt (TIPS) procedure. The diagnosis of cirrhosis was based on histological findings. Patients with alcoholic liver disease had a longstanding history of alcohol abuse, i.e., a consumption exceeding 50 g/day for ⬎5 yr. They had all abstained from alcohol use for at least 6 months before transplantation. Patients with chronic liver disease secondarily to hepatitis C virus (HCV) and hepatitis B virus were anti-HCV antibody and hepatitis B surface antigen positive, respectively. Patients who had nonalcoholic, nonviral cirrhosis were classified as cholestatic (secondary to primary sclerosing cholangitis or primary biliary cirrhosis; with a total bilirubin of ⬎3 mg/dl) and others, which included genetic hemochromatosis and autoimmune hepatitis. The presence of ascites was evaluated by physical examination and roughly quantitated (none, small, and moderate-to-large) by ultrasonography or CT before transplantation. The cirrhotic patients did not have hepatocellular carcinoma, as assessed by ultrasound or CT and histology. All cirrhotic patients were on a low salt diet and the majority were receiving oral diuretics. Patients were excluded if they had no pretransplantation ultrasound or CT, or if complete clinical data were not available. Indications for TIPS were variceal hemorrhage (38%), refractory ascites (54%), and hepatorenal syndrome (8%). Normal hepatic tissue was obtained from patients who had died from nonliver-related causes (motor vehicle accident or in whom liver tissue adjacent to cancers was obtained) (n ⫽ 8). There was no evidence of liver abnormality in the control patients, as determined from histological examination. This study was approved by the Committee on Human Research of the University of California, San Francisco and fulfilled criteria for research as put forth in the Declaration of Helsinki. Hepatic tissue ET-1 (picograms per milligram) was measured by enzyme immunoassay (Peninsula Laboratories, Belmont, CA). In brief, tissue samples from flushed livers were snap frozen in liquid nitrogen. From these samples, 250 –500 mg were individually sonicated in 0.1 mol/L hydrochloric acid (HCl), boiled for 10 min at 1000°C and then centrifuged at 7000 g for 10 min at 40°C. Tris-HCl (1 mol/L; 2:1 volume) was added to supernatants, which were then subjected to enzyme immunoassay as described by the manufacturer. Known concentrations of ET-1 were used to generate a standard curve, which was used to determine peptide concentrations in unknown samples. The intra- and
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Table 1. Clinical Characteristics of the Study Group Variable Median age (yr) [range] Gender (%) Male Female Etiology (%) Alcohol alone HCV ⫾ alcohol HBV Cholestatic Others Child-Pugh class (%) B C Ascites (%) None Small Moderate–large
Without TIPS With TIPS (n ⫽ 49) (n ⫽ 13) 49 [28–68]
Controls (n ⫽ 8)
43 [29–61] 50 [27–60]
69 31
77 23
12 43 16 20 9
31 31 8 20 10
57 43
23 77
24 37 39
38 31 31
100 0
Patients with alcoholic liver disease had a long-standing history of alcohol abuse, (⬎50 g/day for ⬎5 yr), but had abstained from alcohol use for at least 6 months before transplantation. Ascites was evaluated by physical examination and confirmed (and quantitated) by ultrasonography. HCV ⫽ hepatitis C virus; HBV ⫽ hepatitis B virus; TIPS ⫽ transjugular intrahepatic portosystemic shunt.
interassay variability for determination of ET-1 were each routinely ⬍10%. Because the distribution of ET-1 levels were skewed and included several high level outliers, we reported ET-1 levels by using the median and range. Furthermore, statistical differences between subgroups were tested using the nonparametric Wilcoxon two-sample and Kruskal-Wallis tests. Exact p values for these tests were calculated whenever one of the groups being compared contained fewer than five subjects. Spearman rank correlations were calculated to measure and test the association between ET-1 and ChildPugh score (21). A p value of ⬍0.05 was considered statistically significant.
RESULTS Epidemiological and clinical characteristics of the study group are shown in Table 1. The median (range) age of cirrhotic patients without pretransplant TIPS was 49 yr (28 – 68 yr); 31% were women; 43% were Child-Pugh class C and 43% had hepatitis C virus ⫾ alcoholic liver disease. The median (range) age patients with pretransplantation TIPS was 43 yr (29 – 61 yr); 23% were women; 77% were Child-Pugh class C and 31% had hepatitis C virus ⫾ alcoholic liver disease. The median age of the controls was 50 yr (27– 60 yr) and all were men. There was no evidence of diffuse parenchymal liver disease in any control subject as assessed by routine histological examination of livers. Hepatic ET-1 levels in the patient groups and controls are shown in Table 2. ET-1 levels were significantly higher in cirrhotics without (0.17 pg/mg [0.04 –2.9]) or with TIPS (0.12 pg/mg [0.04 – 0.7]) than in the control patients (0.04 pg/mg [0 – 0.1]; p ⫽ 0.02 for both comparisons). ET-1 levels
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Table 2. Hepatic Endothelin-1 (ET-1) Levels in Cirrhotics and Controls Hepatic ET-1 Levels (pg/mg) Patient Group
Median
Range
Control (n ⫽ 4) Cirrhotics without TIPS (n ⫽ 49) Cirrhotics with TIPS (n ⫽ 13)
0.05 0.17*† 0.12*
0.04–0.1 0.04–2.9 0.04–0.7
Liver tissue was snap frozen immediately after explant removal at the time of surgery and ET-1 was measured by EIA as described in methods. Values in parentheses indicate the number of patients in each group. * p ⫽ 0.02 vs controls; † p ⫽ 0.5 vs cirrhotic patients with pretransplantation TIPS. TIPS ⫽ transjugular intrahepatic portosystemic shunt.
were lower in cirrhotic patients with TIPS compared with those without TIPS, although this difference did not reach statistical significance (p ⫽ 0.5). In cirrhotics without ascites who had not had TIPS, ET-1 levels (0.07 pg/mg [0.04 –1.00]) were similar to those of the controls (Table 3). In contrast, ET-1 content was increased in cirrhotics with small (0.11 pg/mg [0.04 – 0.96]; p ⫽ 0.0002) and moderate-to-large (0.69 pg/mg [0.04 –2.9]; p ⫽ 0.0002) amounts of ascites compared to patients with no ascites (Table 3 and Fig. 1). In cirrhotics without TIPS, there was a significant direct correlation between hepatic tissue ET-1 levels and ChildPugh score (Spearman r ⫽ 0.32; p ⫽ 0.03) (Fig. 2). Furthermore, ET-1 levels were significantly higher in patients with Child-Pugh score ⱖ13 than in those with Child-Pugh score ⱕ12 (0.88 [0.48 –2.9] vs 0.16 [0.04 –1.26]; p ⫽ 0.02) (Table 4).
DISCUSSION A number of studies have demonstrated that systemic ET-1 levels are increased in patients with cirrhosis (4 –14). However, ET-1 appears to be rapidly cleared from the circulation and plasma and serum levels are much lower than those that mediate biological effects, suggesting little physiologic role for circulating ET-1 (22). Moreover, experimental data have shown that ET-1 messenger RNA or peptide levels are increased in the liver after injury, implicating a paracrine or
Figure 1. Hepatic endothelin-1 (ET-1) levels and ascites in cirrhotics without a transjugular intrahepatic portosystemic shunt (TIPS). Liver tissue was snap frozen after removal of the explant at the time of surgery and ET-1 was measured by enzyme immunoassay as described in methods. ET-1 levels were higher in cirrhotics with small (p ⫽ 0.0002) and a moderate-to-large (p ⫽ 0.0002) amount of ascites than in patients with no ascites.
autocrine action for ET-1 (17, 18, 20, 23). For this reason, we have investigated hepatic, rather than systemic, ET-1. Our findings, in which we demonstrate increased concentrations of hepatic ET-1 in patients with cirrhosis, provide further support for the postulate that ET-1 is overproduced in the liver after injury. In this study we demonstrated that hepatic ET-1 levels were much higher in patients with a Child-Pugh score of 13 or greater than in those with a Child-Pugh score of 12 or less. A modest but statistically significant correlation was found between hepatic ET-1 levels and Child-Pugh score.
Table 3. Hepatic Endothelin-1 (ET-1) Levels in Controls and Cirrhotics (without transjugular intrahepatic portosystemic shunt) With Varying Amounts of Ascites Hepatic ET-1 Levels (pg/mg) Amount of Ascites
Median
Range
Controls (n ⫽ 4) None (n ⫽ 12) Small (n ⫽ 18) Moderate–large (n ⫽ 19)
0.05 0.07 0.11* 0.69*
0.04–0.1 0.04–1.0 0.04–0.96 0.04–2.9
Liver tissue was immediately snap frozen after explant removal at the time of surgery and ET-1 was measured by EIA as described in methods. The presence of ascites was evaluated by physical examination and confirmed by ultrasonography before transplantation. Values in parentheses indicate the number of patients in each group. * p ⫽ 0.0002 vs cirrhotic patients with no ascites.
Figure 2. Relationship between hepatic endothelin-1 (ET-1) levels and Child-Pugh score in cirrhotic patients without a transjugular intrahepatic portosystemic shunt (TIPS). ET-1 was measured by enzyme immunoassay in snap frozen explanted tissue as described in methods. The Spearman correlation coefficient for a relationship between ET-1 level and Child-Pugh score was 0.32 (p ⫽ 0.03).
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Table 4. Child-Pugh Score and Hepatic Endothelin-1 (ET-1) Levels in Cirrhotics Without Transjugular Intrahepatic Portosystemic Shunt Hepatic ET-1 Levels (pg/mg) Child-Pugh Score Controls (n ⫽ 4) ⱕ12 (n ⫽ 45) ⱖ13 (n ⫽ 4)
Median
Range
0.05 0.16 0.88*
0.04–0.1 0.04–1.26 0.48–2.9
Liver tissue was snap frozen after explant removal at the time of surgery and ET-1 was measured by EIA as described in methods. The severity of cirrhosis was classified according to Pugh’s modification of Child’s classification (21). Values in parentheses indicate the number of patients in each group. * p ⫽ 0.02 vs cirrhotic patients with Child-Pugh score ⬍12.
Therefore, these results suggest that the increase in intrahepatic ET-1 level is, at least in part, related to the underlying severity of cirrhosis. A similar observation has been previously reported by Tsai et al. (6) for plasma ET-1 levels. In their study, plasma ET-1 concentrations in Child class C patients were much higher than in Child class A or B patients, and there was a significant positive correlation between plasma ET-1 levels and Child-Pugh score. Potential mechanisms responsible for increased ET-1 production in chronic liver disease include endotoxemia (12, 24) and shear stress in response to altered blood flow (25, 26). Endotoxemia is common in patients with cirrhosis (27, 28) and Lin et al. (28) recently demonstrated that plasma endotoxin levels progressively increased in relation to the severity of cirrhosis as assessed by Child-Pugh score. Endotoxin has also been shown to enhance ET-1 production from cultured thoracic endothelial cells and hepatic endothelial cells (24, 29). It has also been suggested that hemodynamic forces have important effects on the structure and function of vascular endothelial cells. Shear stress stimulates ET production (25, 26) and in the setting of a hyperdynamic circulation, which is most prominent in those with advanced disease (30, 31) and could contribute to increased production of ET-1 in patients with cirrhosis. We found that hepatic ET-1 levels in cirrhotic patients with ascites were higher than in patients with cirrhosis without ascites. The increase in hepatic ET-1 content in cirrhotics with ascites compared to those without ascites is consistent with the finding that ET-1 levels were increased in proportion to the severity of cirrhosis. The 14-fold relative increase in hepatic ET levels observed in patients with moderate-to-large ascites as compared with healthy subjects is greater than that reported in the plasma of cirrhotic patients with ascites studied by Asbert et al. (10) and Uchihara et al. (12). The higher value in our study may be due to the fact that we measured intrahepatic levels, whereas the latter studies measured circulating levels (and did not quantitate the amount of ascites). Recently, Leivas et al. (20) also reported a selective increase in ET production in the liver of cirrhotic rats with ascites (but not in animals without ascites), indicating a possible link between ET and development of ascites. The mechanism that accounts for increased hepatic production of ET-1 in cirrhotics with ascites is
unknown, but could be related to factors that stimulate ET-1 production. For example, cirrhotic patients with ascites have high concentrations of endotoxin, angiotensin II, and antidiuretic hormone (32) and elevated ET levels could be related to an increased intrahepatic synthesis promoted by these substances (24, 33). Finally, we cannot exclude the possibility that ET-1 contributes to ascites formation, such that patients with high intrahepatic ET-1 levels are more likely to develop ascites. Although statistical differences could not be detected between hepatic ET-1 levels in patients with and without a previous TIPS procedure, there was a trend toward a decrease in ET-1 levels in patients with a TIPS. Parallel findings were reported by Martinet et al. (34), who reported that shunting for refractory ascites induced a marked decrease in ET-1 and big ET-1 in portal and renal veins. The mechanism by which TIPS could lead to decreased production of endothelin is not fully understood, but may be related to a reduction in pressure within the liver. Future work including study of increased numbers of patients should provide a clearer understanding of the complex nature and effect of TIPS on ET-1 production after portal decompression with TIPS. The data presented in this study have important implications for the pathogenesis of portal hypertension. Because ET acts principally as a local paracrine or autocrine agent (35), it is possible that locally produced ET contributes to increased intrahepatic vascular resistance and concomitant portal hypertension typical of patients with cirrhosis. Current evidence indicates that hepatic stellate cells may contribute to increased intrahepatic resistance by perisinusoidal contraction and constriction of the sinusoid (36, 37). Given data demonstrating that all hepatic cell types (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) express ET receptors, but are most abundant on stellate cells (38) and that ET-1 induces potent contraction of stellate cells (36), it appears that stellate cells are a major target of ET (37, 39). Therefore, overproduced ET after hepatic injury and cirrhosis may play an important role in the genesis of increased intrahepatic resistance.
ACKNOWLEDGMENTS This study was supported by grants to I.A. from the National Institutes of Health, Bethesda, MD, (T 32 DK 07007), to the UCSF Liver Center (P30 DK26743) and to DCR (DK 50574 and DK 02124). We thank Tamara Ryan for assistance in collecting the liver tissue specimens used in this study.
Reprint requests and correspondence: Don Rockey, M.D., Liver Center Laboratory, Duke University Medical Center, Sands Building, Room 336, Research Drive, Box 3083, Durham, NC 27710. Received Nov. 9, 1998; accepted July 7, 1999.
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Hepatic Tissue Endothelin-1 Levels in Chronic Liver Disease Correlate With Disease Severity and Ascites I. Alam, M.D., N. M. Bass, M.D., Ph.D., P. Bacchetti, Ph.D., L. Gee, and D. C. Rockey, M.D. Departments of Medicine and Biostatistics and the Liver Center, University of California, San Francisco, California; and the Department of Medicine and Liver Center, Duke University Medical Center, Durham, North Carolina
OBJECTIVES: Plasma endothelin-1 (ET-1) levels are increased in patients with cirrhosis and ET-1 production is increased in the liver itself during experimental injury. These data suggest a possible role for this vasoactive peptide in intrahepatic microcirculatory changes that contribute to the pathogenesis of portal hypertension in cirrhosis. Therefore the aims of this study were to determine whether ET-1 levels were abnormal in the livers of patients with cirrhosis and to investigate possible clinical correlates of altered hepatic ET-1 in cirrhosis. METHODS: Liver specimens were obtained from explants at the time of liver transplantation in 62 cirrhotic patients; 49 without pretransplantation transjugular intrahepatic portosystemic shunt (TIPS) and 13 with pretransplantation TIPS. The presence of ascites was evaluated by physical examination and ultrasonography. Control specimens consisted of livers with normal morphology obtained from patients who died from nonliver-related causes. Hepatic ET-1 was measured by enzyme immunoassay. RESULTS: Hepatic ET-1 levels in cirrhotics without (0.17 pg/mg liver tissue) or with TIPS (0.12 pg/mg) were higher than in control patients [0.04 pg/mg (p ⫽ 0.02 for ET-1 levels in cirrhotics with or without TIPS vs control)]. In cirrhotics without ascites who had not had TIPS, ET-1 levels (0.07 pg/mg [0.04 –1.00]) were similar to those of the controls. In contrast, ET-1 content was increased in cirrhotics with small (0.11 pg/mg; p ⫽ 0.0002) and moderate-to-large (0.69 pg/mg; p ⫽ 0.0002) amounts of ascites compared to patients without ascites. There was a modest correlation between ET-1 levels and Child-Pugh score (correlation coefficient 0.32; p ⫽ 0.03) and ET-1 levels were significantly higher in patients with Child-Pugh score of 13 or greater (0.88 pg/mg; p ⫽ 0.02) than in those with Child-Pugh score of 12 or less (0.16 pg/mg). CONCLUSIONS: Hepatic tissue ET-1 levels are increased in the liver of patients with cirrhosis. This increase appears to be proportional to the severity of both liver disease and ascites. These data raise a possible role for ET-1 in modulation of intrahepatic resistance in cirrhotic portal
hypertension. (Am J Gastroenterol 2000;95:199 –203. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION The endothelins compromise a family of potent vasoconstrictors (1). Three unique endothelin peptides, each consisting of 21 amino acids, have been identified and termed endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). The endothelins have been implicated in a wide variety of normal and pathological processes. Their different effects are in part related to the receptor to which they bind endothelin-A (ETA) or endothelin-B (ETB) (2). Their major function appears to be in the local control of vascular tone (3). ET-1 and ET-3 appear to induce vasoconstriction through stimulation of smooth muscle cells and vasodilation due to effects on endothelial cells, respectively. Circulating levels of immunoreactive ET-1 have been reported to be elevated in a number of diseases including hypertension, atherosclerosis, acute myocardial infarction, and acute renal failure (1). Current information concerning plasma endothelin levels in cirrhosis is conflicting. During the past several years, numerous investigators have shown that both plasma ET-1 and ET-3 concentrations are increased in cirrhotics with ascites, varices, and hepatorenal syndrome (4 –14). However, some studies have reported both normal (15) and reduced (16) plasma levels in cirrhosis. Data demonstrating that ET-1 is overexpressed in different cellular compartments in cirrhotic tissue (17, 18), and that ET-1 is higher in the hepatic vein than in portal vein in patients with portal hypertension (19), have led investigators to postulate that the increased ET-1 levels in patients with cirrhosis are related to increased production by the diseased liver. This is supported by the recent demonstration of increased production of ET-1 (preproET-1 mRNA and peptide) in the liver during experimental injury (18), and increased hepatic levels of ET-1 mRNA and ET-1 in cirrhotic rats with ascites (20). However, hepatic endothelin levels in patients with cirrhosis have not been reported. Therefore, the present study was undertaken to determine whether
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ET-1 levels were abnormal in the livers of patients with cirrhosis, and to establish potential clinical correlates of altered hepatic ET-1 in cirrhosis.
MATERIALS AND METHODS Liver specimens were obtained consecutively from explants at the time of orthotopic liver transplantation in 62 patients with cirrhosis during the period July 1993 to August 1995. Thirteen of these patients had undergone a prior transjugular intrahepatic portosystemic shunt (TIPS) procedure. The diagnosis of cirrhosis was based on histological findings. Patients with alcoholic liver disease had a longstanding history of alcohol abuse, i.e., a consumption exceeding 50 g/day for ⬎5 yr. They had all abstained from alcohol use for at least 6 months before transplantation. Patients with chronic liver disease secondarily to hepatitis C virus (HCV) and hepatitis B virus were anti-HCV antibody and hepatitis B surface antigen positive, respectively. Patients who had nonalcoholic, nonviral cirrhosis were classified as cholestatic (secondary to primary sclerosing cholangitis or primary biliary cirrhosis; with a total bilirubin of ⬎3 mg/dl) and others, which included genetic hemochromatosis and autoimmune hepatitis. The presence of ascites was evaluated by physical examination and roughly quantitated (none, small, and moderate-to-large) by ultrasonography or CT before transplantation. The cirrhotic patients did not have hepatocellular carcinoma, as assessed by ultrasound or CT and histology. All cirrhotic patients were on a low salt diet and the majority were receiving oral diuretics. Patients were excluded if they had no pretransplantation ultrasound or CT, or if complete clinical data were not available. Indications for TIPS were variceal hemorrhage (38%), refractory ascites (54%), and hepatorenal syndrome (8%). Normal hepatic tissue was obtained from patients who had died from nonliver-related causes (motor vehicle accident or in whom liver tissue adjacent to cancers was obtained) (n ⫽ 8). There was no evidence of liver abnormality in the control patients, as determined from histological examination. This study was approved by the Committee on Human Research of the University of California, San Francisco and fulfilled criteria for research as put forth in the Declaration of Helsinki. Hepatic tissue ET-1 (picograms per milligram) was measured by enzyme immunoassay (Peninsula Laboratories, Belmont, CA). In brief, tissue samples from flushed livers were snap frozen in liquid nitrogen. From these samples, 250 –500 mg were individually sonicated in 0.1 mol/L hydrochloric acid (HCl), boiled for 10 min at 1000°C and then centrifuged at 7000 g for 10 min at 40°C. Tris-HCl (1 mol/L; 2:1 volume) was added to supernatants, which were then subjected to enzyme immunoassay as described by the manufacturer. Known concentrations of ET-1 were used to generate a standard curve, which was used to determine peptide concentrations in unknown samples. The intra- and
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Table 1. Clinical Characteristics of the Study Group Variable Median age (yr) [range] Gender (%) Male Female Etiology (%) Alcohol alone HCV ⫾ alcohol HBV Cholestatic Others Child-Pugh class (%) B C Ascites (%) None Small Moderate–large
Without TIPS With TIPS (n ⫽ 49) (n ⫽ 13) 49 [28–68]
Controls (n ⫽ 8)
43 [29–61] 50 [27–60]
69 31
77 23
12 43 16 20 9
31 31 8 20 10
57 43
23 77
24 37 39
38 31 31
100 0
Patients with alcoholic liver disease had a long-standing history of alcohol abuse, (⬎50 g/day for ⬎5 yr), but had abstained from alcohol use for at least 6 months before transplantation. Ascites was evaluated by physical examination and confirmed (and quantitated) by ultrasonography. HCV ⫽ hepatitis C virus; HBV ⫽ hepatitis B virus; TIPS ⫽ transjugular intrahepatic portosystemic shunt.
interassay variability for determination of ET-1 were each routinely ⬍10%. Because the distribution of ET-1 levels were skewed and included several high level outliers, we reported ET-1 levels by using the median and range. Furthermore, statistical differences between subgroups were tested using the nonparametric Wilcoxon two-sample and Kruskal-Wallis tests. Exact p values for these tests were calculated whenever one of the groups being compared contained fewer than five subjects. Spearman rank correlations were calculated to measure and test the association between ET-1 and ChildPugh score (21). A p value of ⬍0.05 was considered statistically significant.
RESULTS Epidemiological and clinical characteristics of the study group are shown in Table 1. The median (range) age of cirrhotic patients without pretransplant TIPS was 49 yr (28 – 68 yr); 31% were women; 43% were Child-Pugh class C and 43% had hepatitis C virus ⫾ alcoholic liver disease. The median (range) age patients with pretransplantation TIPS was 43 yr (29 – 61 yr); 23% were women; 77% were Child-Pugh class C and 31% had hepatitis C virus ⫾ alcoholic liver disease. The median age of the controls was 50 yr (27– 60 yr) and all were men. There was no evidence of diffuse parenchymal liver disease in any control subject as assessed by routine histological examination of livers. Hepatic ET-1 levels in the patient groups and controls are shown in Table 2. ET-1 levels were significantly higher in cirrhotics without (0.17 pg/mg [0.04 –2.9]) or with TIPS (0.12 pg/mg [0.04 – 0.7]) than in the control patients (0.04 pg/mg [0 – 0.1]; p ⫽ 0.02 for both comparisons). ET-1 levels
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Table 2. Hepatic Endothelin-1 (ET-1) Levels in Cirrhotics and Controls Hepatic ET-1 Levels (pg/mg) Patient Group
Median
Range
Control (n ⫽ 4) Cirrhotics without TIPS (n ⫽ 49) Cirrhotics with TIPS (n ⫽ 13)
0.05 0.17*† 0.12*
0.04–0.1 0.04–2.9 0.04–0.7
Liver tissue was snap frozen immediately after explant removal at the time of surgery and ET-1 was measured by EIA as described in methods. Values in parentheses indicate the number of patients in each group. * p ⫽ 0.02 vs controls; † p ⫽ 0.5 vs cirrhotic patients with pretransplantation TIPS. TIPS ⫽ transjugular intrahepatic portosystemic shunt.
were lower in cirrhotic patients with TIPS compared with those without TIPS, although this difference did not reach statistical significance (p ⫽ 0.5). In cirrhotics without ascites who had not had TIPS, ET-1 levels (0.07 pg/mg [0.04 –1.00]) were similar to those of the controls (Table 3). In contrast, ET-1 content was increased in cirrhotics with small (0.11 pg/mg [0.04 – 0.96]; p ⫽ 0.0002) and moderate-to-large (0.69 pg/mg [0.04 –2.9]; p ⫽ 0.0002) amounts of ascites compared to patients with no ascites (Table 3 and Fig. 1). In cirrhotics without TIPS, there was a significant direct correlation between hepatic tissue ET-1 levels and ChildPugh score (Spearman r ⫽ 0.32; p ⫽ 0.03) (Fig. 2). Furthermore, ET-1 levels were significantly higher in patients with Child-Pugh score ⱖ13 than in those with Child-Pugh score ⱕ12 (0.88 [0.48 –2.9] vs 0.16 [0.04 –1.26]; p ⫽ 0.02) (Table 4).
DISCUSSION A number of studies have demonstrated that systemic ET-1 levels are increased in patients with cirrhosis (4 –14). However, ET-1 appears to be rapidly cleared from the circulation and plasma and serum levels are much lower than those that mediate biological effects, suggesting little physiologic role for circulating ET-1 (22). Moreover, experimental data have shown that ET-1 messenger RNA or peptide levels are increased in the liver after injury, implicating a paracrine or
Figure 1. Hepatic endothelin-1 (ET-1) levels and ascites in cirrhotics without a transjugular intrahepatic portosystemic shunt (TIPS). Liver tissue was snap frozen after removal of the explant at the time of surgery and ET-1 was measured by enzyme immunoassay as described in methods. ET-1 levels were higher in cirrhotics with small (p ⫽ 0.0002) and a moderate-to-large (p ⫽ 0.0002) amount of ascites than in patients with no ascites.
autocrine action for ET-1 (17, 18, 20, 23). For this reason, we have investigated hepatic, rather than systemic, ET-1. Our findings, in which we demonstrate increased concentrations of hepatic ET-1 in patients with cirrhosis, provide further support for the postulate that ET-1 is overproduced in the liver after injury. In this study we demonstrated that hepatic ET-1 levels were much higher in patients with a Child-Pugh score of 13 or greater than in those with a Child-Pugh score of 12 or less. A modest but statistically significant correlation was found between hepatic ET-1 levels and Child-Pugh score.
Table 3. Hepatic Endothelin-1 (ET-1) Levels in Controls and Cirrhotics (without transjugular intrahepatic portosystemic shunt) With Varying Amounts of Ascites Hepatic ET-1 Levels (pg/mg) Amount of Ascites
Median
Range
Controls (n ⫽ 4) None (n ⫽ 12) Small (n ⫽ 18) Moderate–large (n ⫽ 19)
0.05 0.07 0.11* 0.69*
0.04–0.1 0.04–1.0 0.04–0.96 0.04–2.9
Liver tissue was immediately snap frozen after explant removal at the time of surgery and ET-1 was measured by EIA as described in methods. The presence of ascites was evaluated by physical examination and confirmed by ultrasonography before transplantation. Values in parentheses indicate the number of patients in each group. * p ⫽ 0.0002 vs cirrhotic patients with no ascites.
Figure 2. Relationship between hepatic endothelin-1 (ET-1) levels and Child-Pugh score in cirrhotic patients without a transjugular intrahepatic portosystemic shunt (TIPS). ET-1 was measured by enzyme immunoassay in snap frozen explanted tissue as described in methods. The Spearman correlation coefficient for a relationship between ET-1 level and Child-Pugh score was 0.32 (p ⫽ 0.03).
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Table 4. Child-Pugh Score and Hepatic Endothelin-1 (ET-1) Levels in Cirrhotics Without Transjugular Intrahepatic Portosystemic Shunt Hepatic ET-1 Levels (pg/mg) Child-Pugh Score Controls (n ⫽ 4) ⱕ12 (n ⫽ 45) ⱖ13 (n ⫽ 4)
Median
Range
0.05 0.16 0.88*
0.04–0.1 0.04–1.26 0.48–2.9
Liver tissue was snap frozen after explant removal at the time of surgery and ET-1 was measured by EIA as described in methods. The severity of cirrhosis was classified according to Pugh’s modification of Child’s classification (21). Values in parentheses indicate the number of patients in each group. * p ⫽ 0.02 vs cirrhotic patients with Child-Pugh score ⬍12.
Therefore, these results suggest that the increase in intrahepatic ET-1 level is, at least in part, related to the underlying severity of cirrhosis. A similar observation has been previously reported by Tsai et al. (6) for plasma ET-1 levels. In their study, plasma ET-1 concentrations in Child class C patients were much higher than in Child class A or B patients, and there was a significant positive correlation between plasma ET-1 levels and Child-Pugh score. Potential mechanisms responsible for increased ET-1 production in chronic liver disease include endotoxemia (12, 24) and shear stress in response to altered blood flow (25, 26). Endotoxemia is common in patients with cirrhosis (27, 28) and Lin et al. (28) recently demonstrated that plasma endotoxin levels progressively increased in relation to the severity of cirrhosis as assessed by Child-Pugh score. Endotoxin has also been shown to enhance ET-1 production from cultured thoracic endothelial cells and hepatic endothelial cells (24, 29). It has also been suggested that hemodynamic forces have important effects on the structure and function of vascular endothelial cells. Shear stress stimulates ET production (25, 26) and in the setting of a hyperdynamic circulation, which is most prominent in those with advanced disease (30, 31) and could contribute to increased production of ET-1 in patients with cirrhosis. We found that hepatic ET-1 levels in cirrhotic patients with ascites were higher than in patients with cirrhosis without ascites. The increase in hepatic ET-1 content in cirrhotics with ascites compared to those without ascites is consistent with the finding that ET-1 levels were increased in proportion to the severity of cirrhosis. The 14-fold relative increase in hepatic ET levels observed in patients with moderate-to-large ascites as compared with healthy subjects is greater than that reported in the plasma of cirrhotic patients with ascites studied by Asbert et al. (10) and Uchihara et al. (12). The higher value in our study may be due to the fact that we measured intrahepatic levels, whereas the latter studies measured circulating levels (and did not quantitate the amount of ascites). Recently, Leivas et al. (20) also reported a selective increase in ET production in the liver of cirrhotic rats with ascites (but not in animals without ascites), indicating a possible link between ET and development of ascites. The mechanism that accounts for increased hepatic production of ET-1 in cirrhotics with ascites is
unknown, but could be related to factors that stimulate ET-1 production. For example, cirrhotic patients with ascites have high concentrations of endotoxin, angiotensin II, and antidiuretic hormone (32) and elevated ET levels could be related to an increased intrahepatic synthesis promoted by these substances (24, 33). Finally, we cannot exclude the possibility that ET-1 contributes to ascites formation, such that patients with high intrahepatic ET-1 levels are more likely to develop ascites. Although statistical differences could not be detected between hepatic ET-1 levels in patients with and without a previous TIPS procedure, there was a trend toward a decrease in ET-1 levels in patients with a TIPS. Parallel findings were reported by Martinet et al. (34), who reported that shunting for refractory ascites induced a marked decrease in ET-1 and big ET-1 in portal and renal veins. The mechanism by which TIPS could lead to decreased production of endothelin is not fully understood, but may be related to a reduction in pressure within the liver. Future work including study of increased numbers of patients should provide a clearer understanding of the complex nature and effect of TIPS on ET-1 production after portal decompression with TIPS. The data presented in this study have important implications for the pathogenesis of portal hypertension. Because ET acts principally as a local paracrine or autocrine agent (35), it is possible that locally produced ET contributes to increased intrahepatic vascular resistance and concomitant portal hypertension typical of patients with cirrhosis. Current evidence indicates that hepatic stellate cells may contribute to increased intrahepatic resistance by perisinusoidal contraction and constriction of the sinusoid (36, 37). Given data demonstrating that all hepatic cell types (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) express ET receptors, but are most abundant on stellate cells (38) and that ET-1 induces potent contraction of stellate cells (36), it appears that stellate cells are a major target of ET (37, 39). Therefore, overproduced ET after hepatic injury and cirrhosis may play an important role in the genesis of increased intrahepatic resistance.
ACKNOWLEDGMENTS This study was supported by grants to I.A. from the National Institutes of Health, Bethesda, MD, (T 32 DK 07007), to the UCSF Liver Center (P30 DK26743) and to DCR (DK 50574 and DK 02124). We thank Tamara Ryan for assistance in collecting the liver tissue specimens used in this study.
Reprint requests and correspondence: Don Rockey, M.D., Liver Center Laboratory, Duke University Medical Center, Sands Building, Room 336, Research Drive, Box 3083, Durham, NC 27710. Received Nov. 9, 1998; accepted July 7, 1999.
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