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Serum hepatocyte growth factor and clinical outcome in biliary atresia
Serum Hepatocyte Growth Factor and Clinical Outcome in Biliary Atresia By Paisarn Vejchapipat, Apiradee Theamboonlers, Rapeepan Chaokhonchai, Voranush Chongsrisawat, Soottiporn Chittmittrapap, and Yong Poovorawan Bangkok, Thailand
Purpose: Biliary atresia (BA) remains one of the most intractable liver diseases leading to liver fibrosis. Serum hepatocyte growth factor (HGF) has been shown to increase in cirrhotic patients. The aim of this study was to investigate the possible role of HGF in BA. Methods: Serum levels of HGF were determined using an enzyme-linked immunosorbent assay from 28 BA patients and 25 healthy children. The patients were categorized into 3 groups according to their clinical outcomes (good, fair, and poor): group A (good), jaundice-free patients (total bilirubin [TB] ⬍ 2.0 mg%); group B (fair), patients with mild to moderate jaundice (TB, 2 to 10 mg%); and group C (poor), patients with marked jaundice (TB ⬎ 10 mg%). Unpaired t test and analysis of variance (ANOVA) with post-hoc tests were used. Data were expressed as mean and SEM. Results: Serum HGF levels in BA patients were higher than the controls (P ⫽ .02). Subgroup analysis found that there were 12 patients in group A, 8 patients in group B, and 8 patients in group C. The mean age of patients in groups A, B,
B
and C were 5.34 ⫾ 0.52, 7.45 ⫾ 1.98, and 5.49 ⫾ 1.57 years (P ⬎ .05). Serum HGF in controls and groups A, B, and C were 0.24 ⫾ 0.03, 0.28 ⫾ 0.04, 0.36 ⫾ 0.09, and 0.56 ⫾ 0.07 ng/mL, respectively. Serum HGF levels in BA patients with poor outcome were higher than patients with good outcome (P ⫽ .02). There was no difference in serum HGF of BA patients with fair outcome compared with other groups. Conclusions: Serum HGF is elevated in BA. Furthermore, BA patients with poor outcome have significantly elevated HGF compared with patients with good outcome. Serum HGF levels may be predictive of prognosis with respect to the progression of liver dysfunction. However, the results of HGF in patients with fair outcome are inconclusive, probably because of the small sample size. Further studies are needed to elucidate the detailed mechanisms. J Pediatr Surg 39:1045-1049. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Biliary atresia, hepatocyte growth factor.
ILIARY ATRESIA (BA), the absence of patent extrahepatic bile ducts, still remains one of the most intractable liver diseases in children. When patients with BA are left without any surgical management, the majority will die of hepatic decompensation, esophageal variceal bleeding, or infection.1,2 Currently, it is accepted that hepatic portoenterostomy or Kasai operation at the early age is indispensable to the successful management of infants with BA,3 especially in countries in which a liver transplantation program is not widely established. A wide spectrum of the results of Kasai operation has been published. According to a report from Japan, of more than 900 patients who underwent the surgical correction, 59% of patients were jaundice free, 21% had their jaundice decreased, whereas in 20% jaundice persisted.4 Hence, despite Kasai operation, a number of patients with BA finally have end-stage cirrhosis caused by progressive hepatic fibrosis.1,3,4 However, the exact cause of the fibrosis or cirrhosis in BA patients is still unclear. During the last 15 years, hepatocyte growth factor (HGF) has been identified as the most potent mitogen for primary hepatocytes.5 The effects of HGF are mediated by a specific receptor encoded by the c-met proto-oncogene.6 HGF has subsequently been shown to be a multifunctional cytokine. The functional properties of HGF
include regeneration, antifibrosis, and cytoprotection.7 Recently, HGF has been clarified to have an antifibrogenic property in dimethylnitrosamine-induced hepatic fibrosis in rats.8 It has been shown that serum HGF levels increased in patients with damaged liver9 including hepatic resection10 and liver tumors after chemotherapy.11 Currently, there is little information available regarding the role of HGF in the pathophysiology of BA. Because progressive hepatic fibrosis is an important development in patients with BA together with the possible role of HGF in fibrogenesis, there may be some links between the pathophysiology of BA and the levels of serum HGF. The aims of the study were to evaluate the serum HGF levels in BA patients and to investigate
Journal of Pediatric Surgery, Vol 39, No 7 (July), 2004: pp 1045-1049
1045
From the Department of Surgery and Viral Hepatitis Research Unit, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Supported by the Thailand Research Fund and Center of Excellence, Viral Hepatitis Research Unit, Chulalongkorn University. Address reprint requests to Professor Yong Poovorawan, MD, Viral Hepatitis Research Unit, Department of Pediatrics, Chulalongkorn Hospital, Rama IV Rd, Patumwan, Bangkok, Thailand 10330. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3907-0009$30.00/0 doi:10.1016/j.jpedsurg.2004.03.052
VEJCHAPIPAT ET AL
3.27 ⫾ 0.13 3.66 ⫾ 0.35 4.21 ⫾ 0.42 — 4.03 ⫾ 0.11 3.09 ⫾ 0.24 2.83 ⫾ 0.21 — 129.42 ⫾ 17.21 133.75 ⫾ 47.88 110.00 ⫾ 20.64 — 132.83 ⫾ 23.04 148.88 ⫾ 35.07 214.63 ⫾ 41.33 — 527.92 ⫾ 65.93 657.63 ⫾ 66.14 529.63 ⫾ 57.47 — 0.41 ⫾ 0.13 4.20 ⫾ 0.77 18.03 ⫾ 1.05 — 1.03 ⫾ 0.17 5.30 ⫾ 0.84 21.83 ⫾ 1.02 —
HGF (ng/mL)
12 8 8 28 25
NOTE. Data are expressed as mean and SEM.
8/4 3/5 4/4 15/13 5.34 ⫾ 0.52 7.45 ⫾ 1.98 5.49 ⫾ 1.57 5.99 ⫾ 0.74 7.56 ⫾ 0.82
Globulin (g%) Albumin (g%) ALT (IU/l) AST (IU/l) AP (IU/l) DB (mg%) TB (mg%) Age (yr) Sex (M/F)
A B C Total Control
The demographic data are shown in Table 1. There was no difference of age and gender between normal healthy children and the BA patients. The patients had significantly higher levels of serum HGF than healthy
No. of Patients
The Comparisons Between BA Patients and Healthy Children
Group
RESULTS
Age at Kasai Operation ⬍2/⬎2 mo (No.)
This study was approved by the Ethical Committee of the Faculty of Medicine, Chulalongkorn University. All parents of children with BA and healthy children had been informed of the purpose of the study. The written informed consents were obtained. Twenty-eight BA patients were recruited into the study during annual routine follow-up between May 2001 and August 2001. All patients attending the department and agreeing to participate in the study were included. The control group comprised 25 healthy children among those who participated in an evaluation of hepatitis B vaccination during the same period. Blood samples were collected from the same samples that would be used for the evaluation of hepatitis B vaccination. Hence, no children had their blood taken for only HGF determination. Briefly, peripheral venous whole blood was drawn with a sterile syringe, transferred to a centrifuge tube, allowed to clot, and then centrifuged at 4°C. The serum was stored at ⫺70°C until they could be assayed. Serum HGF was determined by a commercially available kit (IMMUNIS HGF EIA, Institute of Immunology Co, Japan). Principle of HGF determination is Enzyme Immuno Assay (EIA) using mouse monoclonal antibodies. The procedure consists of 2-step antigen–antibody reaction and enzyme coloring reaction. The serum HGF levels were expressed as nanograms per milliliter. In addition, in BA patients, liver function tests (LFT) including total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and globulin were performed using an automated chemical analyser (Hitachi 911) at the central laboratory of the hospital. Of 28 BA patients, none in this study underwent liver transplantation or showed symptoms and signs of fever or ascending cholangitis at the time of blood sampling. Either Kasai operation or supportive treatment (2 cases owing to significant liver cirrhosis and portal hypertension at the time of the laparotomy) was offered. The patients were subsequently divided into 3 groups according to the clinical outcome and levels of total bilirubin (TB): group A (good outcome), jaundice-free patients (TB ⬍ 2.0 mg%); group B (fair outcome), patients with mild to moderate jaundice (TB, 2 to 10 mg%); and group C (poor outcome), patients with marked jaundice (TB ⬎ 10 mg%). Patients in group C exhibited symptoms and signs of portal hypertension including ascites and esophageal varices. All patients of group C had their bilirubin decreased but were never jaundice free during the early postoperative period. The increase in jaundice and signs of liver deterioration became apparent later only after 2 or 3 years of age. This is different from patients having failed Kasai operation who would die within 2 to 3 years of age unless liver transplantation was performed. The comparison of serum HGF between 28 BA patients and 25 healthy children was performed by unpaired t test. Fisher’s Exact tests were used to analyze the difference of gender between control and BA patients. The relation between serum HGF and the clinical outcome of BA was analyzed using 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Data of HGF levels are expressed as mean and SEM in terms of nanograms per milliliter. Significant differences were established at P ⬍ .05. For all statistical analyses, GraphPad Prism version 3.02 (GraphPad Software Inc, California) was used.
Table 1. Demographic Data, Laboratory Findings, and Serum HGF Levels Among Different Groups of Children With Biliary Atresia and Controls
MATERIALS AND METHODS
8/4 3/5 2/6 13/15 11/14
whether there is an association between serum HGF and clinical outcome in BA patients after surgical treatment.
0.28 ⫾ 0.04 0.36 ⫾ 0.09 0.56 ⫾ 0.07 0.38 ⫾ 0.04 0.24 ⫾ 0.03
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HGF AND BILIARY ATRESIA
Fig 1. Serum HGF levels between biliary atresia patients and normal controls. Data are expressed as mean and SEM. @P ⴝ .02 compared with control.
children (0.24 ⫾ 0.03 v. 0.38 ⫾ 0.04; P ⫽ .02) as shown in Fig 1. Subgroup Analysis of BA Patients With Different Clinical Outcome Further subgroup analysis of 28 BA patients found that there were 12 patients with good outcome (group A), 8 patients with fair outcome (group B), and 8 patients with poor outcome (group C). All patients had BA type III (atresia of right and left hepatic duct) diagnosed. The demographic data, LFT, and HGF levels are shown in Table 1. There were no significant differences in age, alkaline phosphatase, AST, and ALT among the 3 groups. However, patients with good outcome had higher albumin levels compared with other groups (P ⬍ .005). Serum HGF levels in BA patients with poor outcome were significantly higher than those with good outcome (P ⫽ .02) as shown in Fig 2. However, there was no difference in serum HGF of BA patients with fair outcome compared with other groups. Furthermore, there was no difference in HGF levels between control and BA patients with good outcome.
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has been improved dramatically after the introduction of Kasai operation.1,3 However, the procedure is not entirely promising because even after a successful Kasai operation, a number of patients still progress to liver fibrosis.13 Persistent intrahepatic bile duct destruction and periportal fibrosis that lead to chronic liver damage are the main pathologic changes in BA. Its mechanism is still unclear.14 Hepatocyte growth factor (HGF) is well known as a pleiotropic substance with mitogenic, motogenic, morphogenic, and tumor suppressor activities.6,7,11,15 The role of HGF, especially in liver diseases, has been investigated widely, including its relation to regeneration, antifibrosis, cytoprotection, and differentiation. Immunohistochemistry and messenger RNA analyses show that HGF is synthesized in a number of different tissues, including liver, lungs, kidneys, and brain. In liver, HGF is produced by nonparenchymal cells, such as Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells.7,16 HGF receptor has been characterized as the c-met proto-oncogene product, a member of tyrosine kinase class of cell surface receptors.17 The binding of HGF to c-met induces autophosphorylation, which triggers the signal transduction pathways inside the target cells through a multifunctional docking site.18 C-met is widely distributed, mainly in epithelial cells in various organs, including liver, kidneys, and brain.7 In liver, the expression of c-met has been confirmed on the surfaces of hepatocytes19 and biliary epithelial cells.20 The effect of HGF on hepatic fibrosis was first reported by Yasuda et al,8 who administered a deletion variant of HGF that has the same biological activity as HGF to rats with dimethylnitrosamine-induced liver fibrosis. Liver histology was improved, and collagen content was decreased. The mechanism is likely to be caused by the suppression of stellate cell activation as shown by decreases in the mRNA levels of procollagen and transforming growth factor beta 1.21 Liver fibrosis is also known to be a bad prognostic factor for patients with BA.1,3 Although there have been a number of reports
DISCUSSION
Biliary atresia (BA) is a disease that results in progressive sclerosing fibrous obliteration of the extrahepatic bile ducts. It is the most common cause of surgical jaundice in infants. Without surgical correction, cholestasis leads to hepatic fibrosis and death within a few years.1,3,4,12 The livers of infants with BA are enlarged and firm with dark green discoloration. Later, nodular degeneration of cirrhosis develops. Microscopically, the hepatic histology is typical of bile pigment in hepatocytes and canaliculi, widening of the portal tracts, and proliferation of bile ductules.2,3 If the obstruction is unrelieved, portal and periportal fibrosis will progress, and cirrhosis will then be inevitable. The outcome of BA
Fig 2. Serum HGF levels among different groups of biliary atresia patients. Data are expressed as mean and SEM. *P ⴝ .02 compared with group C. **P < .05 compared with control and group A.
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regarding the relationship between serum HGF levels and various liver diseases,7,22 the study of HGF and biliary atresia receives little attention. Only few reports of the association between serum HGF and nephromegaly in BA patients have been described.23,24 According to our knowledge, there is no published article investigating the relation between HGF levels and the clinical outcome of biliary atresia. From the study, we showed that serum HGF in BA patients was higher than those of age- and gendercomparable normal children at the average age of 6 years. The serum HGF levels in healthy children of this study were not different from those of other reports.9,25 Because thrombin is an HGF activator, and HGF production is augmented by injurin, interleukin-1, and tumor necrosis factor, it is possible that elevated serum HGF might be caused by infection and inflammation. However, none of the BA patients in this study showed symptoms and signs of fever or ascending cholangitis at the time of serum collection; therefore, the elevated serum HGF in BA patients is unlikely to be caused by the acute phase response from the inflammatory process as described by Maeda et al.22 Elevated serum HGF has been reported in a number of liver diseases including acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary biliary cirrhosis, and fulminant hepatic failure.21,25,26 It might be possible that elevated HGF levels reflect the significant liver damage in BA patients or, alternatively, that the high HGF levels are caused by the imbalance between HGF clearance and HGF production. In addition, because there are other organs apart from liver that can produce HGF, the major sources of elevated serum HGF in this study may be extrahepatic organs. More studies are needed to elucidate this observation. Further subgroup analysis among BA patients according to clinical outcome showed that patients with good outcome had higher albumin levels implying a better nutritional status and liver function than the other 2 groups. Interestingly, there is no difference in serum HGF between healthy children and BA patients with good outcome. Therefore, the serum HGF should not be considered as a diagnostic marker for biliary atresia, although we found that BA patients, regardless of clinical outcome, had higher HGF levels than controls. Our results also showed that BA patients with poor outcome had significantly higher levels of serum HGF compared with patients with good outcome. Because there was no difference in age of patients among all groups, the elevated serum HGF detected is not caused by the variation of age. Exogenous HGF has been reported to stimulate liver regeneration in damaged liver, such as jaundiced27 and cirrhotic liver with impaired
VEJCHAPIPAT ET AL
regenerative ability.28 The mechanism that underlies serum HGF elevation in BA requires further investigation. In this study, HGF levels in serum seem to increase in parallel to the clinical outcome that reflects the degree of liver damage. It is possible that serum HGF levels are increased in response to liver damage. Although the specific mechanism for our observation has not been elucidated, we hypothesize that serum HGF levels are predictive of prognosis with respect to the progression of liver dysfunction. The study of serum HGF in patients pre- and postoperatively might answer our hypothesis. However, the results of HGF in patients with fair outcome are inconclusive. This is probably because of the wide variation within the group and small sample size. Because the liver is the major organ through which HGF is eliminated from the systemic circulation, the elevated serum HGF levels in BA patients are possibly caused by either the attempt to regenerate new hepatocytes in fibrotic liver, or the decrease in HGF clearance, or both processes. In addition, elevated serum HGF might enhance biliary ductular proliferation in BA because of the presence of c-met receptor on biliary epithelium.20 Thus, regulatory mechanisms of serum HGF in BA patients seem to be complex. It is not easy to explain the mechanisms of our observation from the study of serum HGF levels alone. Further research such as the expression of c-met receptors of hepatocytes and biliary epithelial cells in BA is required to elucidate whether the association between serum HGF and the clinical outcome has significant role in the pathophysiology of progressive liver fibrosis in BA patients. At least, our observation clearly shows that the association between serum HGF and clinical outcome in BA is significant enough to give serious consideration. BA patients had higher levels of serum HGF compared with control. In addition, BA patients with poor outcome have significantly elevated HGF compared with patients with good outcome. Serum HGF levels may be predictive of prognosis with respect to the progression of liver dysfunction. However, the results of HGF in patients with fair outcome are inconclusive, probably because of the small sample size. We speculate that HGF plays an important role in the progression of liver dysfunction in biliary atresia. The detailed mechanisms from our observation need to be investigated further. ACKNOWLEDGMENTS The authors thank the entire staff of the Viral Hepatitis Research Unit, Chulalongkorn University and Hospital for efforts in the current study. The authors thank Associate Professor Bidhya Chandrakamol for operating on most of the BA patients in the study. The authors also thank venerable Dr Mettanonado Bhikkhu of Wat Rajaorasaram, Bangkok for reviewing the manuscript.
HGF AND BILIARY ATRESIA
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REFERENCES 1. Ohi R: Surgical treatment of biliary atresia in the liver transplantation era. Surg Today 28:1229-1232, 1998 2. Davenport M, Gonde C, Redkar R, et al: Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg 36:1017-1025, 2001 3. Nio M, Ohi R: Biliary atresia. Sem Pediatr Surg 9:177-186, 2000 4. Ohi R: Surgery for biliary atresia. Liver 21:175-182, 2001 5. Nakamura T, Nawa K, Ichihara A, et al: Purification and subunit structure of hepatocyte growth factor. FEBS Lett 224:311-316, 1987 6. Tsubouchi H: Hepatocyte growth factor for liver disease. Hepatology 30:333-334, 1999 7. Kaido T, Imamura M: Hepatocyte growth factor: Clinical implications in hepatobiliary pancreatic surgery. J Hepatobiliary Pancreat Surg 8:65-75, 2001 8. Yasuda H, Imai E, Shiota A, et al: Antifibrotic effect of a deletion variant of hepatocyte growth factor on liver fibrosis in rats. Hepatology 24:636-642, 1996 9. Tsubouchi H, Niitani Y, Hirono S, et al: Levels of the human hepatocyte growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay. Hepatology 13:1-5, 1991 10. Tomiya T, Tani M, Yamada S, et al: Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Gastroenterology 103:1621-1624, 1992 11. von Schweinitz D, Faundez A, Teichmann B, et al: Hepatocyte growth-factor-scatter factor can stimulate post-operative tumor-cell proliferation in childhood hepatoblastoma. Int J Cancer 85:151-159, 2000 12. Narkewicz MR: Biliary atresia: An update on our understanding of the disorder. Curr Opin Pediatr 13:435-440, 2001 13. Tagge DU, Tagge EP, Drongowski RA, et al: A long-term experience with biliary atresia-reassessment of prognostic factors. Ann Surg 214:590-598, 1991 14. Liu C, Chiu J, Chin T, et al: Expression of Fas ligand on bile ductule epithelium in biliary atresia—A poor prognostic factor. J Pediatr Surg 35:1591-1596, 2000 15. Kosai KI, Matsumoto K, Funakoshi H, et al: Hepatocyte growth factor prevents endotoxin-induced lethal hepatic failure in mice. Hepatology 30:151-159, 1999 16. Schmidt C, Bladt F, Goedecke S, et al: Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373:699-702, 1995
17. Bottaro DP, Rubin JS, Faletto DL, et al: Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251:802-804, 1991 18. Ponzetto C, Bardelli A, Zhen Z, et al: A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 77:261-271, 1994 19. Miyazawa K, Tsubouchi H, Naka D, et al: Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem Biophys Res Commun 163:967-973, 1989 20. Liu Z, Sakamoto T, Ezure T, et al: Interleukin-6, hepatocyte growth factor, and their receptors in biliary epithelial cells durig a type I ductular reaction in mice: interactions between the periductalinflammatory and stromal cells and the biliary epithelium. Hepatology 28: 1260-1268, 1998 21. Gressner AM, Skrtic S: Role of cytokines in liver fibrosis, in Andus T, Rogler G, Adler G (eds): Falk Symposium 113: Cytokines and Cell Homeostasis in the Gastrointestinal Tract. Dordrecht, Kulwer Academic Publishers, 1999, pp 171-191 22. Hioki O, Watanabe A, Minermura M, et al: Clinical significance of serum hepatocyte growth factor levels in liver diseases. J Med 24:35-46, 1993 23. Tsau YK, Jou ST, Ni YH: Nephromegaly relates to hepatocyte growth factor dysregulation in biliary atresia. Pediatr Nephrol 17:554559, 2002 24. Tsau YK, Chen CH, Chang MH, et al: Nephromegaly and elevated hepatocyte growth factor in children with biliary atresia. Am J Kidney Dis 29:188-192, 1997 25. Shiota A, Okano J, Kawasaki H, et al: Serum hepatocyte growth factor levels in liver diseases: clinical implications. Hepatology 21:106112, 1995 26. Yamagamim H, Moriyama M, Matsumura H, et al: Serum concentrations of human hepatocyte growth factor is a useful indicator for predicting the occurrence of hepatocellular carcinomas in C-viral chroic liver diseases. Cancer 95:824-834, 2002 27. Yoshikawa A, Kaido T, Seto S, et al: Hepatocyte growth factor promotes liver regeneration with prompt improvement of hyperbilirubinemia in hepatectomized cholestatic rats. J Surg Res 78:54-59, 1998 28. Kaido T, Seto S, Yamaoka S, et al: Perioperative continuous hepatocyte growth factor supply prevents postoperative liver failure in rats with liver cirrhosis. J Surg Res 74:173-178, 1998
Purpose: Biliary atresia (BA) remains one of the most intractable liver diseases leading to liver fibrosis. Serum hepatocyte growth factor (HGF) has been shown to increase in cirrhotic patients. The aim of this study was to investigate the possible role of HGF in BA. Methods: Serum levels of HGF were determined using an enzyme-linked immunosorbent assay from 28 BA patients and 25 healthy children. The patients were categorized into 3 groups according to their clinical outcomes (good, fair, and poor): group A (good), jaundice-free patients (total bilirubin [TB] ⬍ 2.0 mg%); group B (fair), patients with mild to moderate jaundice (TB, 2 to 10 mg%); and group C (poor), patients with marked jaundice (TB ⬎ 10 mg%). Unpaired t test and analysis of variance (ANOVA) with post-hoc tests were used. Data were expressed as mean and SEM. Results: Serum HGF levels in BA patients were higher than the controls (P ⫽ .02). Subgroup analysis found that there were 12 patients in group A, 8 patients in group B, and 8 patients in group C. The mean age of patients in groups A, B,
B
and C were 5.34 ⫾ 0.52, 7.45 ⫾ 1.98, and 5.49 ⫾ 1.57 years (P ⬎ .05). Serum HGF in controls and groups A, B, and C were 0.24 ⫾ 0.03, 0.28 ⫾ 0.04, 0.36 ⫾ 0.09, and 0.56 ⫾ 0.07 ng/mL, respectively. Serum HGF levels in BA patients with poor outcome were higher than patients with good outcome (P ⫽ .02). There was no difference in serum HGF of BA patients with fair outcome compared with other groups. Conclusions: Serum HGF is elevated in BA. Furthermore, BA patients with poor outcome have significantly elevated HGF compared with patients with good outcome. Serum HGF levels may be predictive of prognosis with respect to the progression of liver dysfunction. However, the results of HGF in patients with fair outcome are inconclusive, probably because of the small sample size. Further studies are needed to elucidate the detailed mechanisms. J Pediatr Surg 39:1045-1049. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Biliary atresia, hepatocyte growth factor.
ILIARY ATRESIA (BA), the absence of patent extrahepatic bile ducts, still remains one of the most intractable liver diseases in children. When patients with BA are left without any surgical management, the majority will die of hepatic decompensation, esophageal variceal bleeding, or infection.1,2 Currently, it is accepted that hepatic portoenterostomy or Kasai operation at the early age is indispensable to the successful management of infants with BA,3 especially in countries in which a liver transplantation program is not widely established. A wide spectrum of the results of Kasai operation has been published. According to a report from Japan, of more than 900 patients who underwent the surgical correction, 59% of patients were jaundice free, 21% had their jaundice decreased, whereas in 20% jaundice persisted.4 Hence, despite Kasai operation, a number of patients with BA finally have end-stage cirrhosis caused by progressive hepatic fibrosis.1,3,4 However, the exact cause of the fibrosis or cirrhosis in BA patients is still unclear. During the last 15 years, hepatocyte growth factor (HGF) has been identified as the most potent mitogen for primary hepatocytes.5 The effects of HGF are mediated by a specific receptor encoded by the c-met proto-oncogene.6 HGF has subsequently been shown to be a multifunctional cytokine. The functional properties of HGF
include regeneration, antifibrosis, and cytoprotection.7 Recently, HGF has been clarified to have an antifibrogenic property in dimethylnitrosamine-induced hepatic fibrosis in rats.8 It has been shown that serum HGF levels increased in patients with damaged liver9 including hepatic resection10 and liver tumors after chemotherapy.11 Currently, there is little information available regarding the role of HGF in the pathophysiology of BA. Because progressive hepatic fibrosis is an important development in patients with BA together with the possible role of HGF in fibrogenesis, there may be some links between the pathophysiology of BA and the levels of serum HGF. The aims of the study were to evaluate the serum HGF levels in BA patients and to investigate
Journal of Pediatric Surgery, Vol 39, No 7 (July), 2004: pp 1045-1049
1045
From the Department of Surgery and Viral Hepatitis Research Unit, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Supported by the Thailand Research Fund and Center of Excellence, Viral Hepatitis Research Unit, Chulalongkorn University. Address reprint requests to Professor Yong Poovorawan, MD, Viral Hepatitis Research Unit, Department of Pediatrics, Chulalongkorn Hospital, Rama IV Rd, Patumwan, Bangkok, Thailand 10330. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3907-0009$30.00/0 doi:10.1016/j.jpedsurg.2004.03.052
VEJCHAPIPAT ET AL
3.27 ⫾ 0.13 3.66 ⫾ 0.35 4.21 ⫾ 0.42 — 4.03 ⫾ 0.11 3.09 ⫾ 0.24 2.83 ⫾ 0.21 — 129.42 ⫾ 17.21 133.75 ⫾ 47.88 110.00 ⫾ 20.64 — 132.83 ⫾ 23.04 148.88 ⫾ 35.07 214.63 ⫾ 41.33 — 527.92 ⫾ 65.93 657.63 ⫾ 66.14 529.63 ⫾ 57.47 — 0.41 ⫾ 0.13 4.20 ⫾ 0.77 18.03 ⫾ 1.05 — 1.03 ⫾ 0.17 5.30 ⫾ 0.84 21.83 ⫾ 1.02 —
HGF (ng/mL)
12 8 8 28 25
NOTE. Data are expressed as mean and SEM.
8/4 3/5 4/4 15/13 5.34 ⫾ 0.52 7.45 ⫾ 1.98 5.49 ⫾ 1.57 5.99 ⫾ 0.74 7.56 ⫾ 0.82
Globulin (g%) Albumin (g%) ALT (IU/l) AST (IU/l) AP (IU/l) DB (mg%) TB (mg%) Age (yr) Sex (M/F)
A B C Total Control
The demographic data are shown in Table 1. There was no difference of age and gender between normal healthy children and the BA patients. The patients had significantly higher levels of serum HGF than healthy
No. of Patients
The Comparisons Between BA Patients and Healthy Children
Group
RESULTS
Age at Kasai Operation ⬍2/⬎2 mo (No.)
This study was approved by the Ethical Committee of the Faculty of Medicine, Chulalongkorn University. All parents of children with BA and healthy children had been informed of the purpose of the study. The written informed consents were obtained. Twenty-eight BA patients were recruited into the study during annual routine follow-up between May 2001 and August 2001. All patients attending the department and agreeing to participate in the study were included. The control group comprised 25 healthy children among those who participated in an evaluation of hepatitis B vaccination during the same period. Blood samples were collected from the same samples that would be used for the evaluation of hepatitis B vaccination. Hence, no children had their blood taken for only HGF determination. Briefly, peripheral venous whole blood was drawn with a sterile syringe, transferred to a centrifuge tube, allowed to clot, and then centrifuged at 4°C. The serum was stored at ⫺70°C until they could be assayed. Serum HGF was determined by a commercially available kit (IMMUNIS HGF EIA, Institute of Immunology Co, Japan). Principle of HGF determination is Enzyme Immuno Assay (EIA) using mouse monoclonal antibodies. The procedure consists of 2-step antigen–antibody reaction and enzyme coloring reaction. The serum HGF levels were expressed as nanograms per milliliter. In addition, in BA patients, liver function tests (LFT) including total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and globulin were performed using an automated chemical analyser (Hitachi 911) at the central laboratory of the hospital. Of 28 BA patients, none in this study underwent liver transplantation or showed symptoms and signs of fever or ascending cholangitis at the time of blood sampling. Either Kasai operation or supportive treatment (2 cases owing to significant liver cirrhosis and portal hypertension at the time of the laparotomy) was offered. The patients were subsequently divided into 3 groups according to the clinical outcome and levels of total bilirubin (TB): group A (good outcome), jaundice-free patients (TB ⬍ 2.0 mg%); group B (fair outcome), patients with mild to moderate jaundice (TB, 2 to 10 mg%); and group C (poor outcome), patients with marked jaundice (TB ⬎ 10 mg%). Patients in group C exhibited symptoms and signs of portal hypertension including ascites and esophageal varices. All patients of group C had their bilirubin decreased but were never jaundice free during the early postoperative period. The increase in jaundice and signs of liver deterioration became apparent later only after 2 or 3 years of age. This is different from patients having failed Kasai operation who would die within 2 to 3 years of age unless liver transplantation was performed. The comparison of serum HGF between 28 BA patients and 25 healthy children was performed by unpaired t test. Fisher’s Exact tests were used to analyze the difference of gender between control and BA patients. The relation between serum HGF and the clinical outcome of BA was analyzed using 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Data of HGF levels are expressed as mean and SEM in terms of nanograms per milliliter. Significant differences were established at P ⬍ .05. For all statistical analyses, GraphPad Prism version 3.02 (GraphPad Software Inc, California) was used.
Table 1. Demographic Data, Laboratory Findings, and Serum HGF Levels Among Different Groups of Children With Biliary Atresia and Controls
MATERIALS AND METHODS
8/4 3/5 2/6 13/15 11/14
whether there is an association between serum HGF and clinical outcome in BA patients after surgical treatment.
0.28 ⫾ 0.04 0.36 ⫾ 0.09 0.56 ⫾ 0.07 0.38 ⫾ 0.04 0.24 ⫾ 0.03
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HGF AND BILIARY ATRESIA
Fig 1. Serum HGF levels between biliary atresia patients and normal controls. Data are expressed as mean and SEM. @P ⴝ .02 compared with control.
children (0.24 ⫾ 0.03 v. 0.38 ⫾ 0.04; P ⫽ .02) as shown in Fig 1. Subgroup Analysis of BA Patients With Different Clinical Outcome Further subgroup analysis of 28 BA patients found that there were 12 patients with good outcome (group A), 8 patients with fair outcome (group B), and 8 patients with poor outcome (group C). All patients had BA type III (atresia of right and left hepatic duct) diagnosed. The demographic data, LFT, and HGF levels are shown in Table 1. There were no significant differences in age, alkaline phosphatase, AST, and ALT among the 3 groups. However, patients with good outcome had higher albumin levels compared with other groups (P ⬍ .005). Serum HGF levels in BA patients with poor outcome were significantly higher than those with good outcome (P ⫽ .02) as shown in Fig 2. However, there was no difference in serum HGF of BA patients with fair outcome compared with other groups. Furthermore, there was no difference in HGF levels between control and BA patients with good outcome.
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has been improved dramatically after the introduction of Kasai operation.1,3 However, the procedure is not entirely promising because even after a successful Kasai operation, a number of patients still progress to liver fibrosis.13 Persistent intrahepatic bile duct destruction and periportal fibrosis that lead to chronic liver damage are the main pathologic changes in BA. Its mechanism is still unclear.14 Hepatocyte growth factor (HGF) is well known as a pleiotropic substance with mitogenic, motogenic, morphogenic, and tumor suppressor activities.6,7,11,15 The role of HGF, especially in liver diseases, has been investigated widely, including its relation to regeneration, antifibrosis, cytoprotection, and differentiation. Immunohistochemistry and messenger RNA analyses show that HGF is synthesized in a number of different tissues, including liver, lungs, kidneys, and brain. In liver, HGF is produced by nonparenchymal cells, such as Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells.7,16 HGF receptor has been characterized as the c-met proto-oncogene product, a member of tyrosine kinase class of cell surface receptors.17 The binding of HGF to c-met induces autophosphorylation, which triggers the signal transduction pathways inside the target cells through a multifunctional docking site.18 C-met is widely distributed, mainly in epithelial cells in various organs, including liver, kidneys, and brain.7 In liver, the expression of c-met has been confirmed on the surfaces of hepatocytes19 and biliary epithelial cells.20 The effect of HGF on hepatic fibrosis was first reported by Yasuda et al,8 who administered a deletion variant of HGF that has the same biological activity as HGF to rats with dimethylnitrosamine-induced liver fibrosis. Liver histology was improved, and collagen content was decreased. The mechanism is likely to be caused by the suppression of stellate cell activation as shown by decreases in the mRNA levels of procollagen and transforming growth factor beta 1.21 Liver fibrosis is also known to be a bad prognostic factor for patients with BA.1,3 Although there have been a number of reports
DISCUSSION
Biliary atresia (BA) is a disease that results in progressive sclerosing fibrous obliteration of the extrahepatic bile ducts. It is the most common cause of surgical jaundice in infants. Without surgical correction, cholestasis leads to hepatic fibrosis and death within a few years.1,3,4,12 The livers of infants with BA are enlarged and firm with dark green discoloration. Later, nodular degeneration of cirrhosis develops. Microscopically, the hepatic histology is typical of bile pigment in hepatocytes and canaliculi, widening of the portal tracts, and proliferation of bile ductules.2,3 If the obstruction is unrelieved, portal and periportal fibrosis will progress, and cirrhosis will then be inevitable. The outcome of BA
Fig 2. Serum HGF levels among different groups of biliary atresia patients. Data are expressed as mean and SEM. *P ⴝ .02 compared with group C. **P < .05 compared with control and group A.
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regarding the relationship between serum HGF levels and various liver diseases,7,22 the study of HGF and biliary atresia receives little attention. Only few reports of the association between serum HGF and nephromegaly in BA patients have been described.23,24 According to our knowledge, there is no published article investigating the relation between HGF levels and the clinical outcome of biliary atresia. From the study, we showed that serum HGF in BA patients was higher than those of age- and gendercomparable normal children at the average age of 6 years. The serum HGF levels in healthy children of this study were not different from those of other reports.9,25 Because thrombin is an HGF activator, and HGF production is augmented by injurin, interleukin-1, and tumor necrosis factor, it is possible that elevated serum HGF might be caused by infection and inflammation. However, none of the BA patients in this study showed symptoms and signs of fever or ascending cholangitis at the time of serum collection; therefore, the elevated serum HGF in BA patients is unlikely to be caused by the acute phase response from the inflammatory process as described by Maeda et al.22 Elevated serum HGF has been reported in a number of liver diseases including acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary biliary cirrhosis, and fulminant hepatic failure.21,25,26 It might be possible that elevated HGF levels reflect the significant liver damage in BA patients or, alternatively, that the high HGF levels are caused by the imbalance between HGF clearance and HGF production. In addition, because there are other organs apart from liver that can produce HGF, the major sources of elevated serum HGF in this study may be extrahepatic organs. More studies are needed to elucidate this observation. Further subgroup analysis among BA patients according to clinical outcome showed that patients with good outcome had higher albumin levels implying a better nutritional status and liver function than the other 2 groups. Interestingly, there is no difference in serum HGF between healthy children and BA patients with good outcome. Therefore, the serum HGF should not be considered as a diagnostic marker for biliary atresia, although we found that BA patients, regardless of clinical outcome, had higher HGF levels than controls. Our results also showed that BA patients with poor outcome had significantly higher levels of serum HGF compared with patients with good outcome. Because there was no difference in age of patients among all groups, the elevated serum HGF detected is not caused by the variation of age. Exogenous HGF has been reported to stimulate liver regeneration in damaged liver, such as jaundiced27 and cirrhotic liver with impaired
VEJCHAPIPAT ET AL
regenerative ability.28 The mechanism that underlies serum HGF elevation in BA requires further investigation. In this study, HGF levels in serum seem to increase in parallel to the clinical outcome that reflects the degree of liver damage. It is possible that serum HGF levels are increased in response to liver damage. Although the specific mechanism for our observation has not been elucidated, we hypothesize that serum HGF levels are predictive of prognosis with respect to the progression of liver dysfunction. The study of serum HGF in patients pre- and postoperatively might answer our hypothesis. However, the results of HGF in patients with fair outcome are inconclusive. This is probably because of the wide variation within the group and small sample size. Because the liver is the major organ through which HGF is eliminated from the systemic circulation, the elevated serum HGF levels in BA patients are possibly caused by either the attempt to regenerate new hepatocytes in fibrotic liver, or the decrease in HGF clearance, or both processes. In addition, elevated serum HGF might enhance biliary ductular proliferation in BA because of the presence of c-met receptor on biliary epithelium.20 Thus, regulatory mechanisms of serum HGF in BA patients seem to be complex. It is not easy to explain the mechanisms of our observation from the study of serum HGF levels alone. Further research such as the expression of c-met receptors of hepatocytes and biliary epithelial cells in BA is required to elucidate whether the association between serum HGF and the clinical outcome has significant role in the pathophysiology of progressive liver fibrosis in BA patients. At least, our observation clearly shows that the association between serum HGF and clinical outcome in BA is significant enough to give serious consideration. BA patients had higher levels of serum HGF compared with control. In addition, BA patients with poor outcome have significantly elevated HGF compared with patients with good outcome. Serum HGF levels may be predictive of prognosis with respect to the progression of liver dysfunction. However, the results of HGF in patients with fair outcome are inconclusive, probably because of the small sample size. We speculate that HGF plays an important role in the progression of liver dysfunction in biliary atresia. The detailed mechanisms from our observation need to be investigated further. ACKNOWLEDGMENTS The authors thank the entire staff of the Viral Hepatitis Research Unit, Chulalongkorn University and Hospital for efforts in the current study. The authors thank Associate Professor Bidhya Chandrakamol for operating on most of the BA patients in the study. The authors also thank venerable Dr Mettanonado Bhikkhu of Wat Rajaorasaram, Bangkok for reviewing the manuscript.
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