- Email: [email protected]
Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development
Journal of Hepatology 39 (2003) 686–692 www.elsevier.com/locate/jhep
Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development Fre´de´ric Clotman1,*, Louis Libbrecht2, Lionel Gresh3, Moshe Yaniv3, Tania Roskams2, Guy G. Rousseau1, Fre´de´ric P. Lemaigre1 1
Hormone and Metabolic Research Unit, Universite´ catholique de Louvain and Institute of Cellular Pathology, Avenue Hippocrate 75, box 7529, B-1200 Brussels, Belgium 2 Laboratory of Morphology and Molecular Pathology, Katholieke Universiteit Leuven, Leuven, Belgium 3 Gene Expression and Disease Unit, Department of Developmental Biology, Pasteur Institute, Paris, France
Background/Aims: The portal tracts contain bile ducts associated with branches of the portal vein and of the hepatic artery. Hepatic artery malformations are found in diseases in which fetal biliary structures persist after birth (ductal plate malformations). Here we investigated how hepatic artery malformations relate to abnormal bile duct development. Methods: Hepatic artery and biliary development was analyzed in fetuses with Jeune syndrome or Meckel syndrome, which show ductal plate malformations. We also analyzed hepatic artery development in transgenic mice which exhibit biliary anomalies following inactivation of the genes for hepatocyte nuclear factor (HNF)-6 or HNF-1b, two transcription factors expressed in biliary cells, but not in arteries. Results: We show that arterial anomalies occurred in fetuses with Jeune syndrome or Meckel syndrome. We provide the first description of hepatic artery branch development in the mouse and show that inactivation of the Hnf6 or Hnf1b gene results in anomalies of the hepatic artery branches. In the transgenic mice and in the human syndromes, the biliary anomalies preceded the arterial anomalies. Conclusions: A primary defect in biliary epithelial cells is associated with hepatic artery malformations in mice. Our data provide a model to interpret and study hepatic artery anomalies in humans. q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Biliary epithelial cell; Hepatic artery; Ductal plate malformation; Jeune syndrome; Meckel syndrome; HNF-6; HNF-1b
1. Introduction In portal tracts, a branch of the portal vein is on average associated with two branches of the hepatic artery and two bile ducts [1]. During development the biliary epithelial cells (BEC) first form a bilayered structure (the ductal plate) around branches of the portal vein. Focal dilations appear between the two layers of the ductal plate and give rise to Received 14 April 2003; received in revised form 22 July 2003; accepted 25 July 2003 * Corresponding author. Tel.: þ 32-2-7647531; fax: þ 32-2-7647507. E-mail address: [email protected] (F. Clotman). Abbreviations: BEC, biliary epithelial cells; CK, cytokeratin; DPM, ductal plate malformations; HNF, hepatocyte nuclear factor; P, postnatal day; SMA, smooth muscle actin.
the bile ducts. These become incorporated into the portal mesenchyme, and the rest of the ductal plate involutes [2,3]. In humans, the branches of the hepatic artery start to form around 10 weeks of gestation, in the vicinity of the BEC, which are still in ductal plate conformation [4]. A cross-talk between bile ducts and arteries is suggested by the association of bile duct anomalies with malformations of the arteries in two human diseases characterized by ductal plate malformations (DPM), i.e. abnormal bile ducts and persistence of portions of the ductal plate after birth [5]. The early severe form of extrahepatic biliary atresia associates DPM with a destructive and sclerosing form of cholangitis [5] and with hyperplasia of the hepatic arteries [6 – 8]. In congenital hepatic fibrosis, fibrous enlargements of the portal tracts containing DPM are
0168-8278/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-8278(03)00409-4
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
687
associated with an increased number of arteries [5]. In these diseases, it is not known whether an anomaly of the bile ducts perturbs arterial development or whether the arterial defect perturbs development of the ducts. Our aim was to investigate how hepatic artery branch malformation relates to abnormal development of the bile ducts. To approach this question we determined whether this relationship holds true in two other human diseases characterized by DPM and studied transgenic mice which develop biliary anomalies.
with primary antibodies for 30 min at room temperature and mouse liver sections with primary antibodies overnight at 4 8C. For immunohistochemistry, anti-CK antibodies were detected with anti-rabbit or antimouse peroxidase-conjugated EnVision reagent (DAKO) for 30 or 90 min at room temperature, peroxidase was detected using 3,3diaminobenzidine tetrahydrochloride or 3-amino-9-ethylcarbazole (ICN Biochemicals, Aurora, USA) plus H2O2, and sections were counterstained with hematoxylin. For immunofluorescence, anti-CK antibodies were detected with anti-rabbit IgG coupled to Texas Red (Vector Laboratories, Burlingame, USA) and anti-SMA antibodies were detected using biotinylated sheep anti-mouse antibody (Roche, Basel, Switzerland) followed by streptavidin-Alexa Fluor 488 (Molecular Probes, Eugene, USA).
2. Materials and methods
3. Results
2.1. Human subjects
3.1. Abnormal development of intrahepatic arteries in fetuses with Jeune syndrome or Meckel syndrome
Liver biopsies were collected from fetuses with Meckel syndrome (gestational age: 17, 19, 20, 21, 24 or 40 weeks; n ¼ 7) or Jeune syndrome (gestational age: 24 or 35 weeks; n ¼ 2). Liver biopsies (gestational age: 13–40 weeks; n ¼ 25) without histopathological abnormalities were used as control. DPM was defined as excess of cytokeratin (CK)-positive fetal bile duct structures in ductal plate conformation [5,9]. Samples with anomalies were compared to normal samples from fetuses of about the same age. The number of portal tracts studied was as follows. Controls: 58 at 17 weeks of gestation, 60 at 20 weeks, 52 at 21 weeks, 70 at 24 weeks, 56 at 40 weeks. Meckel syndrome: 65 at 17 weeks, 81 and 107 at 19 weeks, 42 at 20 weeks, 70 at 21 weeks, 66 at 24 weeks, 85 at 40 weeks. Jeune syndrome: 49 at 24 weeks, 82 at 35 weeks. The biopsies were collected from spontaneous abortions, from abortions performed on medical indications and from autopsies. Informed consent was obtained from the parents and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institutional review committee. Diagnosis of Meckel syndrome was based on the minimal criteria defined in the literature [10,11], namely lethality, central nervous system malformation, bilaterally large multicystic kidneys, DPM in the liver and polydactyly. Two out seven cases had no polydactyly (20- and 21-week-old fetuses). Jeune syndrome was diagnosed based on the minimal criteria defined by Jeune et al. [12], namely narrow thorax, short ribs, short limb dwarfism, abnormalities of the bones in pelvis and limbs.
2.2. Knockout mice Hnf6 2 /2 mice were obtained by homologous recombination [13]. The genetic background of the Hnf6 2 /2 mice was 129SVJ x Swiss. The mice with a liver-specific inactivation of the Hnf1b gene [14] were in C57BL/6 x 129SVJ x FVB/N background. Eleven control mice [two at postnatal day (P) P1, three at P4, three at P7, one at P8 and two at P10], nine Hnf6 2 /2 mice (three at P1, three at P4, two at P7, one at P10) and three Hnf1b 2 /2 mice at P8 were studied. The number of sections through portal tracts analyzed was as follows. Control mice: 90 at P1, 180 at P4, 480 at P7, 702 at P10. Hnf6 2 /2 mice: 56 at P1, 120 at P4, 300 at P7, 480 at P10. Hnf1b 2 /2 mice: 487 at P8. All animals received humane care and the study protocol was approved by the institutional review committee.
2.3. Histochemistry, immunohistochemistry and immunofluorescence Human samples were fixed in formalin or B5-fixative and embedded in paraffin, and 4-mm sections were made. Mouse livers were frozen in liquid nitrogen-cooled isopentane. Cryosections (5 mm) were post-fixed for 10 min in acetone at 4 8C. Collagen was stained with Sirius red solution for 45 min at room temperature, followed by hematoxylin staining. For immunohistochemistry and immunofluorescence, primary antibodies were rabbit polyclonal anti-human keratin, mouse monoclonal anti-CK 19 and mouse monoclonal anti-human smooth muscle actin (SMA, DAKO, Carpinteria, USA). Sections were heated in a microwave oven for 2 £ 5 min at 750 W in citrate buffer pH 6.0. Human sample sections were incubated
Hyperplasia of the hepatic artery branches is associated with DPM in some human diseases [5 – 8]. To further substantiate this relationship we investigated whether artery malformations also develop in the liver of fetuses with Jeune syndrome (OMIM#208500) or Meckel syndrome (OMIM#249000 and #603194). These are autosomal recessive diseases affecting multiple organs including the liver, where DPM are observed. As shown here, we found that DPM in these syndromes is associated with defects of the hepatic artery branches. At 24 weeks of gestation, portal tracts in normal liver contained a single bile duct and were surrounded by involuting portions of the ductal plate (Fig. 1A). One hepatic artery was found in the vicinity of the bile duct (Fig. 1D), as expected [15]. In contrast, the portal tracts in fetuses with Jeune or Meckel syndrome were surrounded by abnormally extensive portions of the ductal plate and were devoid of tubular bile duct (Fig. 1B,C). Each portal tract contained one hepatic artery (Fig. 1E,F), indicating that the initial formation of the hepatic artery is normal in these syndromes. However, at later gestational ages there was an excessive number of hepatic artery branches. As expected, the portal tract from a 35-week normal fetus contained an involutive ductal plate and one incorporated bile duct in close vicinity of a single hepatic artery (Fig. 1G,J). In contrast, the portal tract from a 35-week Jeune syndrome fetus showed DPM with an excess of biliary structures, and there was no tubular bile duct (Fig. 1H). This fetus also showed numerous hepatic arteries in the portal mesenchyme (arrowheads in Fig. 1K). In addition, the DPM was surrounded by irregularly organized SMA-positive cells (Fig. 1K). In a 40-week fetus with Meckel syndrome, the portal tract contained an excess of biliary cells in ductal plate conformation (Fig. 1I) and again, an excess of hepatic arteries was observed (Fig. 1L). In both the Jeune and the Meckel syndromes, the amount of portal mesenchyme and collagen pointed to moderate fibrosis (Fig. 1H, I, K, L, and data not shown). We conclude from these observations that the liver of fetuses with Jeune syndrome or Meckel syndrome exhibits
688
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
Fig. 1. Ductal plate malformations (DPM) and excess of arteries in the portal tract of human fetuses with Jeune syndrome or Meckel syndrome. Portal tracts in the liver from 24-week-old (A –F) normal (A,D) or Jeune (B,E) or Meckel (C,F) syndrome-affected fetuses were stained for cytokeratin 19 (CK, A –C) or a-smooth muscle actin (SMA, D– F). The normal fetus shows the expected involuting ductal plate, one incorporated bile duct (A) and one artery (D). The Jeune or Meckel syndrome-affected fetuses show no bile duct and the BEC persist in ductal plate conformation (arrows in B and C). One artery is present in the portal tract of Jeune (arrowhead in E) or Meckel (arrowhead in F) syndrome-affected fetuses. Numerous SMA-positive cells are found around the DPM. Portal tracts were analyzed at a later stage of gestation, namely 35 weeks for normal fetuses (G,J) and Jeune syndrome fetuses (H,K), and 40 weeks for Meckel syndrome fetuses (I,L), by staining for CK (G –I) or SMA (J–L). In the normal fetus a bile duct is being incorporated into the mesenchyme and remnants of the involuting ductal plate are detected (G). A single artery is located close to the duct (J). The Jeune or Meckel syndrome-affected fetuses show typical DPM (arrows in H and I) and numerous arteries (arrowheads in K and L). a, artery; bd, bile duct; pv, portal vein. Original magnifications 250 3 (panels A, C, D, F, G, H, J and K) and 200 3 (panels B, E, I and L).
arterial anomalies and that these are preceded by biliary anomalies. We also conclude that the initial formation of the hepatic artery is normal in these syndromes, but that persistence of BEC in ductal plate conformation correlates with hepatic artery malformations. 3.2. Hnf6 2 /2 mice show type I ductal plate malformations The etiology of the Jeune syndrome or of the Meckel syndrome, or that of other diseases associating DPM with hepatic artery malformations, is not known at the molecular level. Therefore, the above observations that biliary defects precede arterial malformation cannot distinguish between an arterial anomaly caused by an intrinsic arterial defect, and an arterial anomaly induced by a biliary defect. To approach this problem we looked for
animal models presenting with DPM associated with hepatic artery defects. The transcription factor hepatocyte nuclear factor (HNF)-6 is expressed in the BEC. We had shown earlier that inactivation of the Hnf6gene in mice results in perturbed differentiation of the BEC and defective formation of the bile ducts, leading to persistence of ductal plate remnants at birth [16]. Here, we first attempted to determine whether the mouse Hnf6 2 /2 biliary phenotype corresponds to human type I or type II DPM, according to the classification of Sergi et al. [17]. At P10, in control mice, immunohistochemical stainings with an anti-CK antibody known to specifically label the BEC [18] showed tubular bile ducts incorporated into the portal mesenchyme (Fig. 2A). In contrast, in Hnf6 2 /2 mice, the BEC formed a discontinuous bilayered structure surrounding the portal vein (Fig. 2B). Some Hnf6 2 /2 mice showed cystic dilations between the BEC layers (Fig. 3E,F,H). In humans,
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
689
a weak staining restricted to the proximity of the portal vein and of the bile ducts (Fig. 2C). Hnf6 2 /2 liver sections showed stronger staining of the portal mesenchyme, which extended somewhat into the parenchyma (Fig. 2D), pointing to moderate portal fibrosis. Taken together, our data indicate that the Hnf6 2 /2 phenotype is closely related to human type I DPM. These mice are therefore a good model for studying such diseases. 3.3. Abnormal development of the hepatic artery branches in Hnf6 2 /2 and in Hnf1b 2 /2 livers
Fig. 2. Ductal plate malformations (DPM) and portal fibrosis in the liver of Hnf6 2 /2 mice. Cytokeratin (CK) immunostainings (A,B) and Sirius Red (SR) histochemical stainings (C,D) were performed on adjacent sections of control (A,C) or Hnf6 2 /2 (B,D) liver sections at postnatal day (P) 10. In control livers, incorporated bile ducts (A) are surrounded by portal mesenchyme that stains faintly with Sirius Red (C). In Hnf6 2 /2 livers, DPM characterized by the persistence of biliary cells in a ductal plate conformation (arrows) are found instead of bile ducts (B). These abnormal biliary structures are surrounded by portal mesenchyme that stains with Sirius Red, indicating the abnormal presence of collagen (D). In addition, the portal mesenchyme forms extensions into the parenchyma (arrowheads). bd, bile duct; pm, portal mesenchyme; pv, portal vein. Scale bar is 50 mm.
DPM is often associated with portal fibrosis, either moderate (Type I DPM) or pronounced (Type II DPM) [17]. We checked whether fibrosis was associated with DPM in the mouse using Sirius red staining, which detects accumulation of collagen. In control animals, we observed
Fig. 3. Abnormal development of the biliary tract in Hnf6 2 /2 and in Hnf1b 2 /2 mice is associated with perturbed development of the hepatic arteries. Cytokeratin (CK, red) and a-smooth muscle actin (SMA, green) double-immunofluorescence stainings were performed on control (A –D, I) and Hnf6 2 /2 (E –H) or Hnf1b 2 /2 (J) liver sections. (A) In control mice at P1, bile ducts are being incorporated in the portal mesenchyme and SMA-positive cells outside of the portal vein are barely detectable (inset). (B) At P4, most of the incorporated bile ducts are associated with one artery. (C) At P7, the thickness of the arterial wall has increased. (D) At P10, an artery with a thick wall is observed close to each bile duct. (E–H) In Hnf6 2 /2 livers the arteries either fail to develop or are hyperplastic. (E) At the early stages after birth, some Hnf6 2 /2 livers (illustrated at P1) show abnormal biliary structures associated with SMA-positive cells (arrowheads). (F) In others, as illustrated at P4, no SMA-positive cells are found in the vicinity of the abnormal biliary structures. (G) Hnf6 2 /2 livers show an excess of arteries (arrows) in the vicinity of the DPM, as illustrated at P7. In addition, SMA-positive cells (arrowheads) are observed around the abnormal biliary structures. (H) In some Hnf6 2 /2 livers, as illustrated at P10, the hepatic artery is absent and only a few SMApositive cells are found in the vicinity of the ductal plate malformation. (J) In Hnf1b 2 /2 livers at P8, the biliary epithelial cells form clusters around the portal tract. An excess of arteries (arrows), as well as SMApositive cells (arrowheads), are found close to these clusters. In contrast, in the corresponding control mice (I), the bile ducts are incorporated in the portal mesenchyme and one artery is found close to each duct. a, artery; bd, bile duct; pv, portal vein. Scale bar is 50 mm.
HNF-6 is expressed in the BEC, but not in the hepatic artery [16]. Therefore, any anomaly of the hepatic artery branches in Hnf6 2 /2 mice should result from a primary
690
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
defect in the BEC and not from an intrinsic arterial defect. We compared the formation of the arteries in control and in Hnf6 2 /2 livers. To our knowledge, the time-course of hepatic artery formation in the mouse had not been described. Therefore, we first analyzed the development of the hepatic arteries in wild-type mice. No artery formation was observed before birth (not shown). At P1, a very weak SMA staining (inset in Fig. 3A) was observed in the vicinity of bile ducts (Fig. 3A). An artery was detected from P4 onwards, appearing as SMA-positive cells surrounding an endothelium around a lumen (Fig. 3B). The intensity of the SMA staining and the thickness of the arterial wall progressively increased until P10 (Fig. 3C,D). In each portal tract the number of bile ducts was equal to that of the arteries (one or two), and the arteries were always located close to the bile ducts (Fig. 3B – D). The formation of the hepatic artery branches, like that of bile ducts, occurred according to a gradient from the hilum to the periphery. Consequently, in a given liver, portal tracts located close to the hilum contained arteries at various stages of development, while those located at a distance from the hilum were still devoid of arteries. In Hnf6 2 /2 mice the development of the hepatic arteries was severely perturbed. In four mice out of six, aged P4 – P10, an excessive number of arteries was observed near the DPM (illustrated at P7 in Fig. 3G). These individual artery branches were very similar in size and structure to those observed in the controls. The formation of these arteries started at P4 (data not shown), like in the controls. In addition, we found irregularly organized cells expressing SMA close to BEC that were either in ductal plate conformation or delineating cystic dilations (illustrated at P1 and P7, arrowheads in Fig 3E,G, respectively). Excess of arteries was observed in portal tracts close to the hilum. Peripheral portal tracts lacked arteries, as a result of immaturity or of arterial agenesis. Indeed, a distinct phenotype was observed in two out of six Hnf6 2 /2 animals. In these mice no artery was detectable (illustrated at P4 in Fig. 3F and P10 in Fig. 3H) in any portal tract. The transcription factor HNF-1b is, like HNF-6, expressed in the BEC and not in the hepatic artery [19]. We had previously shown that HNF-6 controls expression of HNF-1b in the BEC and that inactivation of the Hnf1b gene in liver induces defects in the development of the biliary tract similar to those of Hnf6 2 /2 mice [14,16]. Therefore, we investigated whether inactivation of the Hnf1b gene in the liver also induced abnormal development of the hepatic arteries. Out of three animals at P8, two showed an excessive number of arteries and expression of SMA in irregularly organized cells close to the BEC (Fig. 3J). Like in some Hnf6 2 /2 mice, one Hnf1b 2 /2 liver showed no detectable hepatic artery branch. Thus, the defects in hepatic artery development were identical in Hnf6 2 /2 and in Hnf1b 2 /2 livers. We concluded, first, that in normal mice the arteries develop after the bile ducts and that development of
the arteries is closely linked in space with that of the bile ducts. Second, in Hnf6 2 /2 and in Hnf1b 2 /2 livers, abnormal formation of the bile ducts and persistence of an excess of BEC in ductal plate conformation correlates with abnormal formation of the hepatic arteries. Since HNF-6 and HNF-1b are expressed in BEC and not in arteries, a primary defect in the BEC can induce hepatic artery malformations.
4. Discussion In some human diseases hepatic artery anomalies are associated with DPM. It is not known whether these arterial anomalies are caused by an intrinsic arterial defect or result from the biliary anomaly. We show here that in Jeune syndrome and in Meckel syndrome, two diseases with DPM, hepatic artery branch anomalies also occur and they appear after the biliary anomalies. This would favor a model in which arterial anomalies are induced by the biliary defects. Consistent with this, we find that the Hnf6 2 /2 and Hnf1b 2 /2 livers, which have a primary defect in the BEC, exhibit similar hepatic artery branch anomalies, and that these are preceded by DPM. Our data suggest that a signal originating from the bile ducts induces hepatic artery branch development. Since the arteries are separated from the ducts by mesenchymal cells, this signal most likely consists of secreted proteins. Candidate proteins are the VEGFs, the FGFs, the angiopoietins, TGF-b, the PDGFs and HGF (reviewed in [20]). Such proteins are expressed in the liver [4,21 –28]. Arterial development in humans occurs close to the BEC, but, unlike in mice, this starts at a stage when the BEC are still in ductal plate conformation. The mechanism that triggers artery formation may therefore differ in mice and humans, and this would explain why the initial formation of the artery branches is normal in human syndromes, while it is perturbed in the Hnf6 2 /2 or Hnf1b 2 /2 livers. Still, despite the different timing of artery formation in mice and humans, arterial hyperplasia occurs consecutively to the DPM. It is unlikely that the arterial anomalies are secondary to the developing fibrosis, since an increase in the number of hepatic arterial branches is not a general feature of biliary fibrosis or cirrhosis [29,30]. Also, in contrast to Blankenberg et al. [31] who analyzed fetuses with Meckel syndrome, we found no anomalies of the portal veins, neither in the human syndromes, nor in our transgenic mice (data not shown). It is therefore unlikely that perturbed portal blood flow causes the hepatic artery anomalies. Thus, we speculate that a signal inducing hepatic artery branch development is amplified in DPM by the excess of persistent biliary cells, resulting in the arterial anomaly. Our model is supported by the observations that pharmacologically-induced proliferation of the bile ducts induces proliferation of the hepatic arteries in the rat [32]. However, our transgenic mice more closely reflect the human diseases with DPM, in which cholangiocytes do not
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
proliferate but are in excess as a result from reduced apoptosis [33]. An additional finding of our work was the presence of SMA-positive cells along DPM in the liver of mutant mice, as well as in human patients. SMA-positive cells are normally found around the incorporated bile ducts, but not along the ductal plate (data not shown and [15]). In hepatic fibrosis, an increase in SMA expression has been described in myofibroblast-like cells [34 – 36]. These cells most probably arise from mesenchymal or stellate cells [37]. As Hnf6 and Hnf1b are expressed in the BEC, and not in mesenchymal or in stellate cells [16,38], abnormal SMA expression is not cell-autonomous, but is secondary to the biliary defect. We therefore propose that the persistence of an excess of BEC induces the phenotypic transition of unknown progenitors to portal myofibroblast-like cells. In Hnf1b 2 /2 livers the excess of BEC and of arteries is transient, since at 2 months of age the liver is characterized by bile duct paucity and absence of hepatic artery branches [14]. This suggests that the BEC are required to maintain the arteries. However, we can not formally exclude that the lack of arteries and ducts at 2 months of age may be secondary to the cholestatic syndrome. As the Hnf6 2 /2 mice die at P10, they are not suitable for long-term studies. The presence of hepatic artery malformations should be investigated in other animal models of congenital biliary diseases with DPM. These include the Inv-mouse [39], the cpk mouse [40,41], and the PCK rat [42]. This could provide additional models to study bile duct and hepatic artery development under normal and pathological conditions.
Acknowledgements The authors thank P. Aertsen and M.-A. Gueuning for technical help, C. Coffinier for the floxed Hnf1b mice, and M. Pontoglio, the members of the HORM unit and Prof. V. Desmet for discussions. This work was supported by grants from the Belgian State Program on Interuniversity Poles of Attraction from the D.G. Higher Education and Scientific Research of the French Community of Belgium, from the Fund for Scientific Medical Research (Belgium), and from the Human Frontier Science Program (grant no. RG0303/2000-M to F.L.). L.G. was supported by a fellowship from the French Ministry of Education and Research.
References [1] Crawford AR, Lin X-Z, Crawford JM. The normal adult human liver biopsy: a quantitative reference standard. Hepatology 1998;28: 323–331. [2] Desmet VJ, Roskams T. Cholestatic syndromes of infancy and childhood. In: Zakim D, Boyer TD, editors. Hepatology, a textbook of liver disease, 4th ed. Philadelphia: Saunders; 2002. p. 1481–1539.
691
[3] Crawford JM. Development of the intrahepatic biliary tree. Semin Liver Dis 2002;22:213– 226. [4] Gouysse G, Couvelard A, Frachon S, Bouvier R, Nejjari M, Dauge MC, et al. Relationship between vascular development and vascular differentiation during liver organogenesis in humans. J Hepatol 2002; 37:730–740. [5] Desmet VJ. Congenital diseases of intrahepatic bile ducts: variations on the theme ‘ductal plate malformation’. Hepatology 1992;16: 1069– 1083. [6] Stowens D. Congenital biliary atresia. Am J Gastroenterol 1959;32: 577–590. [7] Brough AJ, Bernstein J. Liver biopsy in the diagnosis of infantile obstructive jaundice. Pediatrics 1969;43:519 –526. [8] Ho CW, Shioda K, Shirasaki K, Takahashi S, Tokimatsu S, Maeda K. The pathogenesis of biliary atresia: a morphological study of the hepatobiliary system and the hepatic artery. J Pediatr Gastroenterol Nutr 1993;16:53–60. [9] Jorgensen MJ. The ductal plate malformation. Acta Pathol Microbiol Scand 1977;1–87. [10] Optiz JM, Howe JJ. The Meckel syndrome (dysencephalia splanchnocystica, the Gruber syndrome). Birth Defects 1969;2:167–179. [11] Salonen R, Paavola P. Meckel syndrome. J Med Genet 1998;35: 497–501. [12] Jeune M, Beraud C, Carron R. Dystrophie thoracique asphyxiante de caracte`re familial. Arch Fr Pediatr 1955;12:886–891. [13] Jacquemin P, Durviaux S, Jensen J, Godfraind C, Gradwohl G, Guillemot F, et al. The transcription factor HNF-6 regulates pancreatic endocrine cell differentiation and controls the expression of the pro-endocrine gene ngn-3. Mol Cell Biol 2000;20:4445–4454. [14] Coffinier C, Gresh L, Fiette L, Tronche F, Schutz G, Babinet C, et al. Bile system morphogenesis defects and liver dysfunction upon targeted deletion of Hnf1beta. Development 2002;129: 1829– 1838. [15] Libbrecht L, Cassiman D, Desmet V, Roskams T. The correlation between portal myofibroblasts and development of intrahepatic bile ducts and arterial branches in human liver. Liver 2002;22:252–258. [16] Clotman F, Lannoy VJ, Reber M, Cereghini S, Cassiman D, Jacquemin P, et al. The onecut transcription factor Hnf6 is required for normal development of the biliary tract. Development 2002;129: 1819–1828. [17] Sergi C, Adam S, Kahl P, Otto HF. Study of the malformation of ductal plate of the liver in Meckel syndrome and review of other syndromes presenting with this anomaly. Pediatr Dev Pathol 2000;3: 568–583. [18] Van Eyken P, Sciot R, Callea F, Van der Steen K, Moerman P, Desmet VJ. The development of the intrahepatic bile ducts in man: a keratinimmunohistochemical study. Hepatology 1988;8:1586–1595. [19] Coffinier C, Thepot D, Babinet C, Yaniv M, Barra J. Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development 1999;126:4785–4794. [20] Conway EM, Collen D, Carmeliet P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2001;49:507–521. [21] Rosmorduc O, Wendum D, Corpechot C, Galy B, Sebbagh N, Raleigh J, et al. Hepatocellular hypoxia-induced vascular endothelial growth factor expression and angiogenesis in experimental biliary cirrhosis. Am J Pathol 1999;155:1065–1073. [22] Sato T, El-Assal ON, Ono T, Yamanoi A, Dhar DK, Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver. J Hepatol 2001;34: 690–698. [23] Lau AL, Kumar TR, Nishimori K, Bonadio J, Matzuk MM. Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration. Mol Cell Biol 2000;20: 6127– 6137. [24] Ishikawa KS, Masui T, Ishikawa K, Shiojiri N. Immunolocalization of hepatocyte growth factor and its receptor (c-Met) during mouse liver development. Histochem Cell Biol 2001;116:453–462.
692
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
[25] Malizia G, Brunt EM, Peters MG, Rizzo A, Broekelmann TJ, McDonald JA. Growth factor and procollagen type I gene expression in human liver disease. Gastroenterology 1995;108:145 –156. [26] Grappone C, Pinzani M, Parola M, Pellegrini G, Caligiuri A, DeFranco R, et al. Expression of platelet-derived growth factor in newly formed cholangiocytes during experimental biliary fibrosis in rats. J Hepatol 1999;31:100–109. [27] Faiz Kabir Uddin Ahmed A, Ohtani H, Nio M, Funaki N, Iwami D, Kumagai S. In situ expression of fibrogenic growth factors and their receptors in biliary atresia: comparison between early and late stages. J Pathol 2000;192:73–80. [28] Chow NH, Hsu PI, Lin XZ, Yang HB, Chan SH, Cheng KS, et al. Expression of vascular endothelial growth factor in normal liver and hepatocellular carcinoma: an immunohistochemical study. Hum Pathol 1997;28(6):698 –703. [29] Wanless IR. Vascular disorders. In: MacSween RNM, Burt AD, Portmann BC, Ishak KG, Scheuer PJ, Anthony PP, editors. Pathology of the liver. London: Churchill Livingstone; 2002. p. 539– 574. [30] Crawford JM. Liver cirrhosis. In: MacSween RNM, Burt AD, Portmann BC, Ishak KG, Scheuer PJ, Anthony PP, editors. Pathology of the liver. London: Churchill Livingstone; 2002. p. 575 –620. [31] Blankenberg TA, Ruebner BH, Ellis WG, Bernstein J, Dimmick JE. Pathology of renal and hepatic anomalies in Meckel syndrome. Am J Med Genet Suppl. 1987;3:395–410. [32] Masyuk TV, Ritman EL, LaRusso NF. Hepatic artery and portal vein remodelling in rat liver: vascular response to selective cholangiocyte proliferation. Am J Pathol 2003;162:1175–1182. [33] Sergi C, Kahl P, Otto HF. Contribution of apoptosis and apoptosisrelated proteins to the malformation of the primitive intrahepatic biliary system in Meckel syndrome. Am J Pathol 2000;156: 1589–1598.
[34] Nouchi T, Tanaka Y, Tsukada T, Sato C, Marumo F. Appearance of alpha-smooth-muscle-actin-positive cells in hepatic fibrosis. Liver 1991;11:100–105. [35] Seifert WF, Roholl PJ, Blauw B, van der Ham F, van Thiel-De Ruiter CF, Seifert-Bock I, et al. Fat-storing cells and myofibroblasts are involved in the initial phase of carbon tetrachloride-induced hepatic fibrosis in BN/BiRij rats. Int J Exp Pathol 1994;75: 131 –146. [36] Cassiman D, Libbrecht L, Desmet V, Denef C, Roskams T. Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers. J Hepatol 2002;36:200– 209. [37] Cassiman D, Roskams T. Beauty is in the eye of the beholder: emerging concepts and pitfalls in hepatic stellate cell research. J Hepatol 2002;37:527. [38] Coffinier C, Barra J, Babinet C, Yaniv M. Expression of the vHNF1/ HNF1beta homeoprotein gene during mouse organogenesis. Mech Dev 1999;89:211 –213. [39] Mazziotti MV, Willis LK, Heuckeroth RO, LaRegina MC, Swanson PE, Overbeek PA, et al. Anomalous development of the hepatobiliary system in the Inv mouse. Hepatology 1999;30:372–378. [40] Guay-Woodford LM, Green WJ, Lindsey JR, Beier DR. Germline and somatic loss of function of the mouse cpk gene causes biliary ductal pathology that is genetically modulated. Hum Mol Genet 2000;9: 769 –778. [41] Hou X, Mrug M, Yoder BK, Lefkowitz EJ, Kremmidiotis G, D’Eustachio P, et al. Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease. J Clin Invest 2002;109:533– 540. [42] Sanzen T, Harada K, Yasoshima M, Kawamura Y, Ishibashi M, Nakanuma Y. Polycystic kidney rat is a novel animal model of Caroli’s disease associated with congenital hepatic fibrosis. Am J Pathol 2001;158:1605– 1612.
Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development Fre´de´ric Clotman1,*, Louis Libbrecht2, Lionel Gresh3, Moshe Yaniv3, Tania Roskams2, Guy G. Rousseau1, Fre´de´ric P. Lemaigre1 1
Hormone and Metabolic Research Unit, Universite´ catholique de Louvain and Institute of Cellular Pathology, Avenue Hippocrate 75, box 7529, B-1200 Brussels, Belgium 2 Laboratory of Morphology and Molecular Pathology, Katholieke Universiteit Leuven, Leuven, Belgium 3 Gene Expression and Disease Unit, Department of Developmental Biology, Pasteur Institute, Paris, France
Background/Aims: The portal tracts contain bile ducts associated with branches of the portal vein and of the hepatic artery. Hepatic artery malformations are found in diseases in which fetal biliary structures persist after birth (ductal plate malformations). Here we investigated how hepatic artery malformations relate to abnormal bile duct development. Methods: Hepatic artery and biliary development was analyzed in fetuses with Jeune syndrome or Meckel syndrome, which show ductal plate malformations. We also analyzed hepatic artery development in transgenic mice which exhibit biliary anomalies following inactivation of the genes for hepatocyte nuclear factor (HNF)-6 or HNF-1b, two transcription factors expressed in biliary cells, but not in arteries. Results: We show that arterial anomalies occurred in fetuses with Jeune syndrome or Meckel syndrome. We provide the first description of hepatic artery branch development in the mouse and show that inactivation of the Hnf6 or Hnf1b gene results in anomalies of the hepatic artery branches. In the transgenic mice and in the human syndromes, the biliary anomalies preceded the arterial anomalies. Conclusions: A primary defect in biliary epithelial cells is associated with hepatic artery malformations in mice. Our data provide a model to interpret and study hepatic artery anomalies in humans. q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Biliary epithelial cell; Hepatic artery; Ductal plate malformation; Jeune syndrome; Meckel syndrome; HNF-6; HNF-1b
1. Introduction In portal tracts, a branch of the portal vein is on average associated with two branches of the hepatic artery and two bile ducts [1]. During development the biliary epithelial cells (BEC) first form a bilayered structure (the ductal plate) around branches of the portal vein. Focal dilations appear between the two layers of the ductal plate and give rise to Received 14 April 2003; received in revised form 22 July 2003; accepted 25 July 2003 * Corresponding author. Tel.: þ 32-2-7647531; fax: þ 32-2-7647507. E-mail address: [email protected] (F. Clotman). Abbreviations: BEC, biliary epithelial cells; CK, cytokeratin; DPM, ductal plate malformations; HNF, hepatocyte nuclear factor; P, postnatal day; SMA, smooth muscle actin.
the bile ducts. These become incorporated into the portal mesenchyme, and the rest of the ductal plate involutes [2,3]. In humans, the branches of the hepatic artery start to form around 10 weeks of gestation, in the vicinity of the BEC, which are still in ductal plate conformation [4]. A cross-talk between bile ducts and arteries is suggested by the association of bile duct anomalies with malformations of the arteries in two human diseases characterized by ductal plate malformations (DPM), i.e. abnormal bile ducts and persistence of portions of the ductal plate after birth [5]. The early severe form of extrahepatic biliary atresia associates DPM with a destructive and sclerosing form of cholangitis [5] and with hyperplasia of the hepatic arteries [6 – 8]. In congenital hepatic fibrosis, fibrous enlargements of the portal tracts containing DPM are
0168-8278/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-8278(03)00409-4
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
687
associated with an increased number of arteries [5]. In these diseases, it is not known whether an anomaly of the bile ducts perturbs arterial development or whether the arterial defect perturbs development of the ducts. Our aim was to investigate how hepatic artery branch malformation relates to abnormal development of the bile ducts. To approach this question we determined whether this relationship holds true in two other human diseases characterized by DPM and studied transgenic mice which develop biliary anomalies.
with primary antibodies for 30 min at room temperature and mouse liver sections with primary antibodies overnight at 4 8C. For immunohistochemistry, anti-CK antibodies were detected with anti-rabbit or antimouse peroxidase-conjugated EnVision reagent (DAKO) for 30 or 90 min at room temperature, peroxidase was detected using 3,3diaminobenzidine tetrahydrochloride or 3-amino-9-ethylcarbazole (ICN Biochemicals, Aurora, USA) plus H2O2, and sections were counterstained with hematoxylin. For immunofluorescence, anti-CK antibodies were detected with anti-rabbit IgG coupled to Texas Red (Vector Laboratories, Burlingame, USA) and anti-SMA antibodies were detected using biotinylated sheep anti-mouse antibody (Roche, Basel, Switzerland) followed by streptavidin-Alexa Fluor 488 (Molecular Probes, Eugene, USA).
2. Materials and methods
3. Results
2.1. Human subjects
3.1. Abnormal development of intrahepatic arteries in fetuses with Jeune syndrome or Meckel syndrome
Liver biopsies were collected from fetuses with Meckel syndrome (gestational age: 17, 19, 20, 21, 24 or 40 weeks; n ¼ 7) or Jeune syndrome (gestational age: 24 or 35 weeks; n ¼ 2). Liver biopsies (gestational age: 13–40 weeks; n ¼ 25) without histopathological abnormalities were used as control. DPM was defined as excess of cytokeratin (CK)-positive fetal bile duct structures in ductal plate conformation [5,9]. Samples with anomalies were compared to normal samples from fetuses of about the same age. The number of portal tracts studied was as follows. Controls: 58 at 17 weeks of gestation, 60 at 20 weeks, 52 at 21 weeks, 70 at 24 weeks, 56 at 40 weeks. Meckel syndrome: 65 at 17 weeks, 81 and 107 at 19 weeks, 42 at 20 weeks, 70 at 21 weeks, 66 at 24 weeks, 85 at 40 weeks. Jeune syndrome: 49 at 24 weeks, 82 at 35 weeks. The biopsies were collected from spontaneous abortions, from abortions performed on medical indications and from autopsies. Informed consent was obtained from the parents and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institutional review committee. Diagnosis of Meckel syndrome was based on the minimal criteria defined in the literature [10,11], namely lethality, central nervous system malformation, bilaterally large multicystic kidneys, DPM in the liver and polydactyly. Two out seven cases had no polydactyly (20- and 21-week-old fetuses). Jeune syndrome was diagnosed based on the minimal criteria defined by Jeune et al. [12], namely narrow thorax, short ribs, short limb dwarfism, abnormalities of the bones in pelvis and limbs.
2.2. Knockout mice Hnf6 2 /2 mice were obtained by homologous recombination [13]. The genetic background of the Hnf6 2 /2 mice was 129SVJ x Swiss. The mice with a liver-specific inactivation of the Hnf1b gene [14] were in C57BL/6 x 129SVJ x FVB/N background. Eleven control mice [two at postnatal day (P) P1, three at P4, three at P7, one at P8 and two at P10], nine Hnf6 2 /2 mice (three at P1, three at P4, two at P7, one at P10) and three Hnf1b 2 /2 mice at P8 were studied. The number of sections through portal tracts analyzed was as follows. Control mice: 90 at P1, 180 at P4, 480 at P7, 702 at P10. Hnf6 2 /2 mice: 56 at P1, 120 at P4, 300 at P7, 480 at P10. Hnf1b 2 /2 mice: 487 at P8. All animals received humane care and the study protocol was approved by the institutional review committee.
2.3. Histochemistry, immunohistochemistry and immunofluorescence Human samples were fixed in formalin or B5-fixative and embedded in paraffin, and 4-mm sections were made. Mouse livers were frozen in liquid nitrogen-cooled isopentane. Cryosections (5 mm) were post-fixed for 10 min in acetone at 4 8C. Collagen was stained with Sirius red solution for 45 min at room temperature, followed by hematoxylin staining. For immunohistochemistry and immunofluorescence, primary antibodies were rabbit polyclonal anti-human keratin, mouse monoclonal anti-CK 19 and mouse monoclonal anti-human smooth muscle actin (SMA, DAKO, Carpinteria, USA). Sections were heated in a microwave oven for 2 £ 5 min at 750 W in citrate buffer pH 6.0. Human sample sections were incubated
Hyperplasia of the hepatic artery branches is associated with DPM in some human diseases [5 – 8]. To further substantiate this relationship we investigated whether artery malformations also develop in the liver of fetuses with Jeune syndrome (OMIM#208500) or Meckel syndrome (OMIM#249000 and #603194). These are autosomal recessive diseases affecting multiple organs including the liver, where DPM are observed. As shown here, we found that DPM in these syndromes is associated with defects of the hepatic artery branches. At 24 weeks of gestation, portal tracts in normal liver contained a single bile duct and were surrounded by involuting portions of the ductal plate (Fig. 1A). One hepatic artery was found in the vicinity of the bile duct (Fig. 1D), as expected [15]. In contrast, the portal tracts in fetuses with Jeune or Meckel syndrome were surrounded by abnormally extensive portions of the ductal plate and were devoid of tubular bile duct (Fig. 1B,C). Each portal tract contained one hepatic artery (Fig. 1E,F), indicating that the initial formation of the hepatic artery is normal in these syndromes. However, at later gestational ages there was an excessive number of hepatic artery branches. As expected, the portal tract from a 35-week normal fetus contained an involutive ductal plate and one incorporated bile duct in close vicinity of a single hepatic artery (Fig. 1G,J). In contrast, the portal tract from a 35-week Jeune syndrome fetus showed DPM with an excess of biliary structures, and there was no tubular bile duct (Fig. 1H). This fetus also showed numerous hepatic arteries in the portal mesenchyme (arrowheads in Fig. 1K). In addition, the DPM was surrounded by irregularly organized SMA-positive cells (Fig. 1K). In a 40-week fetus with Meckel syndrome, the portal tract contained an excess of biliary cells in ductal plate conformation (Fig. 1I) and again, an excess of hepatic arteries was observed (Fig. 1L). In both the Jeune and the Meckel syndromes, the amount of portal mesenchyme and collagen pointed to moderate fibrosis (Fig. 1H, I, K, L, and data not shown). We conclude from these observations that the liver of fetuses with Jeune syndrome or Meckel syndrome exhibits
688
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
Fig. 1. Ductal plate malformations (DPM) and excess of arteries in the portal tract of human fetuses with Jeune syndrome or Meckel syndrome. Portal tracts in the liver from 24-week-old (A –F) normal (A,D) or Jeune (B,E) or Meckel (C,F) syndrome-affected fetuses were stained for cytokeratin 19 (CK, A –C) or a-smooth muscle actin (SMA, D– F). The normal fetus shows the expected involuting ductal plate, one incorporated bile duct (A) and one artery (D). The Jeune or Meckel syndrome-affected fetuses show no bile duct and the BEC persist in ductal plate conformation (arrows in B and C). One artery is present in the portal tract of Jeune (arrowhead in E) or Meckel (arrowhead in F) syndrome-affected fetuses. Numerous SMA-positive cells are found around the DPM. Portal tracts were analyzed at a later stage of gestation, namely 35 weeks for normal fetuses (G,J) and Jeune syndrome fetuses (H,K), and 40 weeks for Meckel syndrome fetuses (I,L), by staining for CK (G –I) or SMA (J–L). In the normal fetus a bile duct is being incorporated into the mesenchyme and remnants of the involuting ductal plate are detected (G). A single artery is located close to the duct (J). The Jeune or Meckel syndrome-affected fetuses show typical DPM (arrows in H and I) and numerous arteries (arrowheads in K and L). a, artery; bd, bile duct; pv, portal vein. Original magnifications 250 3 (panels A, C, D, F, G, H, J and K) and 200 3 (panels B, E, I and L).
arterial anomalies and that these are preceded by biliary anomalies. We also conclude that the initial formation of the hepatic artery is normal in these syndromes, but that persistence of BEC in ductal plate conformation correlates with hepatic artery malformations. 3.2. Hnf6 2 /2 mice show type I ductal plate malformations The etiology of the Jeune syndrome or of the Meckel syndrome, or that of other diseases associating DPM with hepatic artery malformations, is not known at the molecular level. Therefore, the above observations that biliary defects precede arterial malformation cannot distinguish between an arterial anomaly caused by an intrinsic arterial defect, and an arterial anomaly induced by a biliary defect. To approach this problem we looked for
animal models presenting with DPM associated with hepatic artery defects. The transcription factor hepatocyte nuclear factor (HNF)-6 is expressed in the BEC. We had shown earlier that inactivation of the Hnf6gene in mice results in perturbed differentiation of the BEC and defective formation of the bile ducts, leading to persistence of ductal plate remnants at birth [16]. Here, we first attempted to determine whether the mouse Hnf6 2 /2 biliary phenotype corresponds to human type I or type II DPM, according to the classification of Sergi et al. [17]. At P10, in control mice, immunohistochemical stainings with an anti-CK antibody known to specifically label the BEC [18] showed tubular bile ducts incorporated into the portal mesenchyme (Fig. 2A). In contrast, in Hnf6 2 /2 mice, the BEC formed a discontinuous bilayered structure surrounding the portal vein (Fig. 2B). Some Hnf6 2 /2 mice showed cystic dilations between the BEC layers (Fig. 3E,F,H). In humans,
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
689
a weak staining restricted to the proximity of the portal vein and of the bile ducts (Fig. 2C). Hnf6 2 /2 liver sections showed stronger staining of the portal mesenchyme, which extended somewhat into the parenchyma (Fig. 2D), pointing to moderate portal fibrosis. Taken together, our data indicate that the Hnf6 2 /2 phenotype is closely related to human type I DPM. These mice are therefore a good model for studying such diseases. 3.3. Abnormal development of the hepatic artery branches in Hnf6 2 /2 and in Hnf1b 2 /2 livers
Fig. 2. Ductal plate malformations (DPM) and portal fibrosis in the liver of Hnf6 2 /2 mice. Cytokeratin (CK) immunostainings (A,B) and Sirius Red (SR) histochemical stainings (C,D) were performed on adjacent sections of control (A,C) or Hnf6 2 /2 (B,D) liver sections at postnatal day (P) 10. In control livers, incorporated bile ducts (A) are surrounded by portal mesenchyme that stains faintly with Sirius Red (C). In Hnf6 2 /2 livers, DPM characterized by the persistence of biliary cells in a ductal plate conformation (arrows) are found instead of bile ducts (B). These abnormal biliary structures are surrounded by portal mesenchyme that stains with Sirius Red, indicating the abnormal presence of collagen (D). In addition, the portal mesenchyme forms extensions into the parenchyma (arrowheads). bd, bile duct; pm, portal mesenchyme; pv, portal vein. Scale bar is 50 mm.
DPM is often associated with portal fibrosis, either moderate (Type I DPM) or pronounced (Type II DPM) [17]. We checked whether fibrosis was associated with DPM in the mouse using Sirius red staining, which detects accumulation of collagen. In control animals, we observed
Fig. 3. Abnormal development of the biliary tract in Hnf6 2 /2 and in Hnf1b 2 /2 mice is associated with perturbed development of the hepatic arteries. Cytokeratin (CK, red) and a-smooth muscle actin (SMA, green) double-immunofluorescence stainings were performed on control (A –D, I) and Hnf6 2 /2 (E –H) or Hnf1b 2 /2 (J) liver sections. (A) In control mice at P1, bile ducts are being incorporated in the portal mesenchyme and SMA-positive cells outside of the portal vein are barely detectable (inset). (B) At P4, most of the incorporated bile ducts are associated with one artery. (C) At P7, the thickness of the arterial wall has increased. (D) At P10, an artery with a thick wall is observed close to each bile duct. (E–H) In Hnf6 2 /2 livers the arteries either fail to develop or are hyperplastic. (E) At the early stages after birth, some Hnf6 2 /2 livers (illustrated at P1) show abnormal biliary structures associated with SMA-positive cells (arrowheads). (F) In others, as illustrated at P4, no SMA-positive cells are found in the vicinity of the abnormal biliary structures. (G) Hnf6 2 /2 livers show an excess of arteries (arrows) in the vicinity of the DPM, as illustrated at P7. In addition, SMA-positive cells (arrowheads) are observed around the abnormal biliary structures. (H) In some Hnf6 2 /2 livers, as illustrated at P10, the hepatic artery is absent and only a few SMApositive cells are found in the vicinity of the ductal plate malformation. (J) In Hnf1b 2 /2 livers at P8, the biliary epithelial cells form clusters around the portal tract. An excess of arteries (arrows), as well as SMApositive cells (arrowheads), are found close to these clusters. In contrast, in the corresponding control mice (I), the bile ducts are incorporated in the portal mesenchyme and one artery is found close to each duct. a, artery; bd, bile duct; pv, portal vein. Scale bar is 50 mm.
HNF-6 is expressed in the BEC, but not in the hepatic artery [16]. Therefore, any anomaly of the hepatic artery branches in Hnf6 2 /2 mice should result from a primary
690
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
defect in the BEC and not from an intrinsic arterial defect. We compared the formation of the arteries in control and in Hnf6 2 /2 livers. To our knowledge, the time-course of hepatic artery formation in the mouse had not been described. Therefore, we first analyzed the development of the hepatic arteries in wild-type mice. No artery formation was observed before birth (not shown). At P1, a very weak SMA staining (inset in Fig. 3A) was observed in the vicinity of bile ducts (Fig. 3A). An artery was detected from P4 onwards, appearing as SMA-positive cells surrounding an endothelium around a lumen (Fig. 3B). The intensity of the SMA staining and the thickness of the arterial wall progressively increased until P10 (Fig. 3C,D). In each portal tract the number of bile ducts was equal to that of the arteries (one or two), and the arteries were always located close to the bile ducts (Fig. 3B – D). The formation of the hepatic artery branches, like that of bile ducts, occurred according to a gradient from the hilum to the periphery. Consequently, in a given liver, portal tracts located close to the hilum contained arteries at various stages of development, while those located at a distance from the hilum were still devoid of arteries. In Hnf6 2 /2 mice the development of the hepatic arteries was severely perturbed. In four mice out of six, aged P4 – P10, an excessive number of arteries was observed near the DPM (illustrated at P7 in Fig. 3G). These individual artery branches were very similar in size and structure to those observed in the controls. The formation of these arteries started at P4 (data not shown), like in the controls. In addition, we found irregularly organized cells expressing SMA close to BEC that were either in ductal plate conformation or delineating cystic dilations (illustrated at P1 and P7, arrowheads in Fig 3E,G, respectively). Excess of arteries was observed in portal tracts close to the hilum. Peripheral portal tracts lacked arteries, as a result of immaturity or of arterial agenesis. Indeed, a distinct phenotype was observed in two out of six Hnf6 2 /2 animals. In these mice no artery was detectable (illustrated at P4 in Fig. 3F and P10 in Fig. 3H) in any portal tract. The transcription factor HNF-1b is, like HNF-6, expressed in the BEC and not in the hepatic artery [19]. We had previously shown that HNF-6 controls expression of HNF-1b in the BEC and that inactivation of the Hnf1b gene in liver induces defects in the development of the biliary tract similar to those of Hnf6 2 /2 mice [14,16]. Therefore, we investigated whether inactivation of the Hnf1b gene in the liver also induced abnormal development of the hepatic arteries. Out of three animals at P8, two showed an excessive number of arteries and expression of SMA in irregularly organized cells close to the BEC (Fig. 3J). Like in some Hnf6 2 /2 mice, one Hnf1b 2 /2 liver showed no detectable hepatic artery branch. Thus, the defects in hepatic artery development were identical in Hnf6 2 /2 and in Hnf1b 2 /2 livers. We concluded, first, that in normal mice the arteries develop after the bile ducts and that development of
the arteries is closely linked in space with that of the bile ducts. Second, in Hnf6 2 /2 and in Hnf1b 2 /2 livers, abnormal formation of the bile ducts and persistence of an excess of BEC in ductal plate conformation correlates with abnormal formation of the hepatic arteries. Since HNF-6 and HNF-1b are expressed in BEC and not in arteries, a primary defect in the BEC can induce hepatic artery malformations.
4. Discussion In some human diseases hepatic artery anomalies are associated with DPM. It is not known whether these arterial anomalies are caused by an intrinsic arterial defect or result from the biliary anomaly. We show here that in Jeune syndrome and in Meckel syndrome, two diseases with DPM, hepatic artery branch anomalies also occur and they appear after the biliary anomalies. This would favor a model in which arterial anomalies are induced by the biliary defects. Consistent with this, we find that the Hnf6 2 /2 and Hnf1b 2 /2 livers, which have a primary defect in the BEC, exhibit similar hepatic artery branch anomalies, and that these are preceded by DPM. Our data suggest that a signal originating from the bile ducts induces hepatic artery branch development. Since the arteries are separated from the ducts by mesenchymal cells, this signal most likely consists of secreted proteins. Candidate proteins are the VEGFs, the FGFs, the angiopoietins, TGF-b, the PDGFs and HGF (reviewed in [20]). Such proteins are expressed in the liver [4,21 –28]. Arterial development in humans occurs close to the BEC, but, unlike in mice, this starts at a stage when the BEC are still in ductal plate conformation. The mechanism that triggers artery formation may therefore differ in mice and humans, and this would explain why the initial formation of the artery branches is normal in human syndromes, while it is perturbed in the Hnf6 2 /2 or Hnf1b 2 /2 livers. Still, despite the different timing of artery formation in mice and humans, arterial hyperplasia occurs consecutively to the DPM. It is unlikely that the arterial anomalies are secondary to the developing fibrosis, since an increase in the number of hepatic arterial branches is not a general feature of biliary fibrosis or cirrhosis [29,30]. Also, in contrast to Blankenberg et al. [31] who analyzed fetuses with Meckel syndrome, we found no anomalies of the portal veins, neither in the human syndromes, nor in our transgenic mice (data not shown). It is therefore unlikely that perturbed portal blood flow causes the hepatic artery anomalies. Thus, we speculate that a signal inducing hepatic artery branch development is amplified in DPM by the excess of persistent biliary cells, resulting in the arterial anomaly. Our model is supported by the observations that pharmacologically-induced proliferation of the bile ducts induces proliferation of the hepatic arteries in the rat [32]. However, our transgenic mice more closely reflect the human diseases with DPM, in which cholangiocytes do not
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
proliferate but are in excess as a result from reduced apoptosis [33]. An additional finding of our work was the presence of SMA-positive cells along DPM in the liver of mutant mice, as well as in human patients. SMA-positive cells are normally found around the incorporated bile ducts, but not along the ductal plate (data not shown and [15]). In hepatic fibrosis, an increase in SMA expression has been described in myofibroblast-like cells [34 – 36]. These cells most probably arise from mesenchymal or stellate cells [37]. As Hnf6 and Hnf1b are expressed in the BEC, and not in mesenchymal or in stellate cells [16,38], abnormal SMA expression is not cell-autonomous, but is secondary to the biliary defect. We therefore propose that the persistence of an excess of BEC induces the phenotypic transition of unknown progenitors to portal myofibroblast-like cells. In Hnf1b 2 /2 livers the excess of BEC and of arteries is transient, since at 2 months of age the liver is characterized by bile duct paucity and absence of hepatic artery branches [14]. This suggests that the BEC are required to maintain the arteries. However, we can not formally exclude that the lack of arteries and ducts at 2 months of age may be secondary to the cholestatic syndrome. As the Hnf6 2 /2 mice die at P10, they are not suitable for long-term studies. The presence of hepatic artery malformations should be investigated in other animal models of congenital biliary diseases with DPM. These include the Inv-mouse [39], the cpk mouse [40,41], and the PCK rat [42]. This could provide additional models to study bile duct and hepatic artery development under normal and pathological conditions.
Acknowledgements The authors thank P. Aertsen and M.-A. Gueuning for technical help, C. Coffinier for the floxed Hnf1b mice, and M. Pontoglio, the members of the HORM unit and Prof. V. Desmet for discussions. This work was supported by grants from the Belgian State Program on Interuniversity Poles of Attraction from the D.G. Higher Education and Scientific Research of the French Community of Belgium, from the Fund for Scientific Medical Research (Belgium), and from the Human Frontier Science Program (grant no. RG0303/2000-M to F.L.). L.G. was supported by a fellowship from the French Ministry of Education and Research.
References [1] Crawford AR, Lin X-Z, Crawford JM. The normal adult human liver biopsy: a quantitative reference standard. Hepatology 1998;28: 323–331. [2] Desmet VJ, Roskams T. Cholestatic syndromes of infancy and childhood. In: Zakim D, Boyer TD, editors. Hepatology, a textbook of liver disease, 4th ed. Philadelphia: Saunders; 2002. p. 1481–1539.
691
[3] Crawford JM. Development of the intrahepatic biliary tree. Semin Liver Dis 2002;22:213– 226. [4] Gouysse G, Couvelard A, Frachon S, Bouvier R, Nejjari M, Dauge MC, et al. Relationship between vascular development and vascular differentiation during liver organogenesis in humans. J Hepatol 2002; 37:730–740. [5] Desmet VJ. Congenital diseases of intrahepatic bile ducts: variations on the theme ‘ductal plate malformation’. Hepatology 1992;16: 1069– 1083. [6] Stowens D. Congenital biliary atresia. Am J Gastroenterol 1959;32: 577–590. [7] Brough AJ, Bernstein J. Liver biopsy in the diagnosis of infantile obstructive jaundice. Pediatrics 1969;43:519 –526. [8] Ho CW, Shioda K, Shirasaki K, Takahashi S, Tokimatsu S, Maeda K. The pathogenesis of biliary atresia: a morphological study of the hepatobiliary system and the hepatic artery. J Pediatr Gastroenterol Nutr 1993;16:53–60. [9] Jorgensen MJ. The ductal plate malformation. Acta Pathol Microbiol Scand 1977;1–87. [10] Optiz JM, Howe JJ. The Meckel syndrome (dysencephalia splanchnocystica, the Gruber syndrome). Birth Defects 1969;2:167–179. [11] Salonen R, Paavola P. Meckel syndrome. J Med Genet 1998;35: 497–501. [12] Jeune M, Beraud C, Carron R. Dystrophie thoracique asphyxiante de caracte`re familial. Arch Fr Pediatr 1955;12:886–891. [13] Jacquemin P, Durviaux S, Jensen J, Godfraind C, Gradwohl G, Guillemot F, et al. The transcription factor HNF-6 regulates pancreatic endocrine cell differentiation and controls the expression of the pro-endocrine gene ngn-3. Mol Cell Biol 2000;20:4445–4454. [14] Coffinier C, Gresh L, Fiette L, Tronche F, Schutz G, Babinet C, et al. Bile system morphogenesis defects and liver dysfunction upon targeted deletion of Hnf1beta. Development 2002;129: 1829– 1838. [15] Libbrecht L, Cassiman D, Desmet V, Roskams T. The correlation between portal myofibroblasts and development of intrahepatic bile ducts and arterial branches in human liver. Liver 2002;22:252–258. [16] Clotman F, Lannoy VJ, Reber M, Cereghini S, Cassiman D, Jacquemin P, et al. The onecut transcription factor Hnf6 is required for normal development of the biliary tract. Development 2002;129: 1819–1828. [17] Sergi C, Adam S, Kahl P, Otto HF. Study of the malformation of ductal plate of the liver in Meckel syndrome and review of other syndromes presenting with this anomaly. Pediatr Dev Pathol 2000;3: 568–583. [18] Van Eyken P, Sciot R, Callea F, Van der Steen K, Moerman P, Desmet VJ. The development of the intrahepatic bile ducts in man: a keratinimmunohistochemical study. Hepatology 1988;8:1586–1595. [19] Coffinier C, Thepot D, Babinet C, Yaniv M, Barra J. Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development 1999;126:4785–4794. [20] Conway EM, Collen D, Carmeliet P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2001;49:507–521. [21] Rosmorduc O, Wendum D, Corpechot C, Galy B, Sebbagh N, Raleigh J, et al. Hepatocellular hypoxia-induced vascular endothelial growth factor expression and angiogenesis in experimental biliary cirrhosis. Am J Pathol 1999;155:1065–1073. [22] Sato T, El-Assal ON, Ono T, Yamanoi A, Dhar DK, Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver. J Hepatol 2001;34: 690–698. [23] Lau AL, Kumar TR, Nishimori K, Bonadio J, Matzuk MM. Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration. Mol Cell Biol 2000;20: 6127– 6137. [24] Ishikawa KS, Masui T, Ishikawa K, Shiojiri N. Immunolocalization of hepatocyte growth factor and its receptor (c-Met) during mouse liver development. Histochem Cell Biol 2001;116:453–462.
692
F. Clotman et al. / Journal of Hepatology 39 (2003) 686–692
[25] Malizia G, Brunt EM, Peters MG, Rizzo A, Broekelmann TJ, McDonald JA. Growth factor and procollagen type I gene expression in human liver disease. Gastroenterology 1995;108:145 –156. [26] Grappone C, Pinzani M, Parola M, Pellegrini G, Caligiuri A, DeFranco R, et al. Expression of platelet-derived growth factor in newly formed cholangiocytes during experimental biliary fibrosis in rats. J Hepatol 1999;31:100–109. [27] Faiz Kabir Uddin Ahmed A, Ohtani H, Nio M, Funaki N, Iwami D, Kumagai S. In situ expression of fibrogenic growth factors and their receptors in biliary atresia: comparison between early and late stages. J Pathol 2000;192:73–80. [28] Chow NH, Hsu PI, Lin XZ, Yang HB, Chan SH, Cheng KS, et al. Expression of vascular endothelial growth factor in normal liver and hepatocellular carcinoma: an immunohistochemical study. Hum Pathol 1997;28(6):698 –703. [29] Wanless IR. Vascular disorders. In: MacSween RNM, Burt AD, Portmann BC, Ishak KG, Scheuer PJ, Anthony PP, editors. Pathology of the liver. London: Churchill Livingstone; 2002. p. 539– 574. [30] Crawford JM. Liver cirrhosis. In: MacSween RNM, Burt AD, Portmann BC, Ishak KG, Scheuer PJ, Anthony PP, editors. Pathology of the liver. London: Churchill Livingstone; 2002. p. 575 –620. [31] Blankenberg TA, Ruebner BH, Ellis WG, Bernstein J, Dimmick JE. Pathology of renal and hepatic anomalies in Meckel syndrome. Am J Med Genet Suppl. 1987;3:395–410. [32] Masyuk TV, Ritman EL, LaRusso NF. Hepatic artery and portal vein remodelling in rat liver: vascular response to selective cholangiocyte proliferation. Am J Pathol 2003;162:1175–1182. [33] Sergi C, Kahl P, Otto HF. Contribution of apoptosis and apoptosisrelated proteins to the malformation of the primitive intrahepatic biliary system in Meckel syndrome. Am J Pathol 2000;156: 1589–1598.
[34] Nouchi T, Tanaka Y, Tsukada T, Sato C, Marumo F. Appearance of alpha-smooth-muscle-actin-positive cells in hepatic fibrosis. Liver 1991;11:100–105. [35] Seifert WF, Roholl PJ, Blauw B, van der Ham F, van Thiel-De Ruiter CF, Seifert-Bock I, et al. Fat-storing cells and myofibroblasts are involved in the initial phase of carbon tetrachloride-induced hepatic fibrosis in BN/BiRij rats. Int J Exp Pathol 1994;75: 131 –146. [36] Cassiman D, Libbrecht L, Desmet V, Denef C, Roskams T. Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers. J Hepatol 2002;36:200– 209. [37] Cassiman D, Roskams T. Beauty is in the eye of the beholder: emerging concepts and pitfalls in hepatic stellate cell research. J Hepatol 2002;37:527. [38] Coffinier C, Barra J, Babinet C, Yaniv M. Expression of the vHNF1/ HNF1beta homeoprotein gene during mouse organogenesis. Mech Dev 1999;89:211 –213. [39] Mazziotti MV, Willis LK, Heuckeroth RO, LaRegina MC, Swanson PE, Overbeek PA, et al. Anomalous development of the hepatobiliary system in the Inv mouse. Hepatology 1999;30:372–378. [40] Guay-Woodford LM, Green WJ, Lindsey JR, Beier DR. Germline and somatic loss of function of the mouse cpk gene causes biliary ductal pathology that is genetically modulated. Hum Mol Genet 2000;9: 769 –778. [41] Hou X, Mrug M, Yoder BK, Lefkowitz EJ, Kremmidiotis G, D’Eustachio P, et al. Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease. J Clin Invest 2002;109:533– 540. [42] Sanzen T, Harada K, Yasoshima M, Kawamura Y, Ishibashi M, Nakanuma Y. Polycystic kidney rat is a novel animal model of Caroli’s disease associated with congenital hepatic fibrosis. Am J Pathol 2001;158:1605– 1612.