Major HGF-mediated regenerative pathways are similarly affected in human and canine cirrhosis
- Bart Spee†1, 2,
- Brigitte Arends†1,
- Ted SGAM van den Ingh1,
- Tania Roskams2,
- Jan Rothuizen1 and
- Louis C Penning1Email author
© Spee et al; licensee BioMed Central Ltd. 2007
Received: 19 December 2006
Accepted: 31 July 2007
Published: 31 July 2007
The availability of non-rodent animal models for human cirrhosis is limited. We investigated whether privately-owned dogs (Canis familiaris) are potential model animals for liver disease focusing on regenerative pathways. Several forms of canine hepatitis were examined: Acute Hepatitis (AH), Chronic Hepatitis (CH), Lobular Dissecting Hepatitis (LDH, a specific form of micronodulair cirrhosis), and Cirrhosis (CIRR). Canine cirrhotic samples were compared to human liver samples from cirrhotic stages of alcoholic liver disease (hALC) and chronic hepatitis C infection (hHC).
Canine specific mRNA expression of the regenerative hepatocyte growth factor (HGF) signaling pathway and relevant down-stream pathways were measured by semi-quantitative PCR and Western blot (STAT3, PKB, ERK1/2, and p38-MAPK). In all canine groups, levels of c-MET mRNA (proto-oncogenic receptor for HGF) were significantly decreased (p < 0.05). Surprisingly, ERK1/2 and p38-MAPK were increased in CH and LDH. In the human liver samples Western blotting indicated a high homology of down-stream pathways between different etiologies (hALC and hHC). Similarly activated pathways were found in CIRR, hALC, and hHC.
In canine hepatitis and cirrhosis the major regenerative downstream pathways were activated. Signaling pathways are similarly activated in human cirrhotic liver samples, irrespective of the differences in etiology in the human samples (alcohol abuse and HCV-infection). Therefore, canine hepatitis and cirrhosis could be an important clinical model to evaluate novel interventions prior to human clinical trials.
Chronic hepatitis (CH) and end-stage cirrhosis (CIRR) are an increasing medical problem, affecting over 5% of the world population [1, 2]. The best-studied animal model for these liver diseases is tetracarbon-induced fibrotic liver diseases inflicted in rats . Many more models have been devised to mimic liver diseases in man; the time-course of the development however, is not always comparable to the human situation [4–8]. Furthermore, the variability in the affected human population regarding, sex, age, social factors, eating- and drinking behavior, body weight etc. is not fully covered in standardized laboratory conditions.
Liver regeneration is a complex interplay of different factors . One of the main growth factors identified in liver regeneration is Hepatocyte Growth Factor (HGF). HGF activates the proto-oncogenic receptor tyrosine kinase c-MET and subsequent down-stream pathways, including the anti-apoptotic protein kinase-B (PKB/Akt) cascade, the proliferative MAP-kinase pathway (ERK1/2 and p38MAPK), and the STAT3 signaling (signal transducers and activators of transcription) [15–17]. At present, a comparison between these regeneration signal transduction pathways in human and canine livers is missing, hampering the application of dogs as (pre-) clinical model animals for human medicine.
We have analyzed HGF-mediated regeneration signaling in canine samples from dogs with Acute Hepatitis (AH), Chronic Hepatitis (CH), and Lobular Dissecting Hepatitis (LDH, a specific form of micronodulair cirrhosis similar to neonatal hepatitis in human hepatology) [18, 19], and CIRR. The cirrhotic samples were compared to two human cirrhotic diseases with different etiologies; HCV-induced (hHC) and alcohol-induced (hALC). This study will elucidate the potential of (non-experimental) dogs to bridge between toxin-induced rodent models and human clinical situation.
HGF/c-MET signaling pathways involved in liver regeneration in dogs with AH, CH, CIRR, and LDH
Western blot analysis on STAT3, PKB/Akt, ERK1/2, and p38MAPK in canine liver homogenates
Western blot analysis on human cirrhotic explant samples after alcohol abuse (hALC) and after hepatitis C virus infection (hHC)
To investigate if hepatic regenerative signal transduction pathways are similarly affected in liver diseases between man and dogs, the expression of HGF and c-MET was measured. Furthermore, Western blot analysis was used to show (de)activation of important signaling pathways of liver regeneration. This provided insight into major regeneration pathways in AH, CH, LDH, CIRR. A comparison between canine cirrhosis and human cirrhotic samples with different etiologies provided additional information on the suitability of the canine-model.
The increased mRNA levels of HGF and the decreased levels of c-MET mRNA in fibrotic canine diseases (CH, LDH, and CIRR), were in line with publications from human samples . Together this suggests that HGF-mediated regeneration in human cirrhosis is similarly affected in canine CH, LDH, and CIRR. The reduced c-MET protein levels indicate a utilization of c-MET which is degraded through endocytosis after phosphorylation. Intracellular degradation is dependent on the interaction with Cbl ubiquitin ligases . Semi-quantitative hepatocyte proliferation studies on canine samples with a Ki67 antibody indicate a moderate hepatocyte proliferation in CH; whereas cirrhotic samples (CIRR and LDH) as well as healthy samples are virtually negative (data not shown). Similar results have been described in human chronic and cirrhotic liver samples, irrespective of the etiology [22, 23]. This indicates the ability of hepatocytes to proliferate after c-MET activation in more chronic stages while cirrhotic samples do not complete the cell-cycle of which the cause remains to be elucidated.
To further substantiate the molecular comparison between human and canine fibrotic livers the activation status of HGF/c-MET downstream signaling components involved in regeneration was analyzed. HCV cirrhotic samples had high levels of Ser-phosphorylated STAT3 compared to alcohol induced cirrhotic samples. This HCV-induced up regulation of STAT3 phosphorylation was shown before in man and rodent models in vivo as well as in vitro studies on cell-lines [24, 25]. In other studies the levels of STAT phosphorylation indicated phosphorylated STAT3 protein in HCV affected cirrhotic livers compared to primary biliary cirrhotic samples and healthy tissue . In the canine samples including the healthy control biopsies STAT3-Ser phosphorylation was strongly present. The levels of STAT3-Tyr phosphorylation were the lowest in AH and strongest in LDH. As the amount of STAT3-Tyr phosphorylation indicates the DNA binding capacity of the protein, the pathways seems to be activated in fibrotic diseases and not in acute hepatitis.
Next to STAT3 phosphorylation, HCV induces ERK1/2 phosphorylation . In the canine samples, an up regulation of the levels of phosphorylated ERK1/2 was observed in CH and LDH. Finally, the increase in p38MAPK phosphorylation as observed in CH, LDH, and CIRR has been described in fibrotic tissues in man . In general, HGF/c-MET downstream signaling in CH, LDH, and CIRR is to a high degree comparable with the molecular data obtained from human clinical samples.
Surgical animal models for liver regeneration, such as partial hepatectomy (PH), represent an over-simplification by the absence of inflammation or overperfusion; furthermore, all hepatocytes are stimulated by PH to enter the G1 phase simultaneously. Toxic models induced by dimethylnitrosamine, CCl4, acetominophen, or thioacetamide can represent chronic as well as acute/fulminant hepatitis [29–31]. Toxic models are better clinical models as hepatotoxins can be used to selectively induce centrolobular and periportal necrotic lesions and thus mimic clinical liver diseases. However, toxin-induced models do not represent the full range of changes seen in human liver diseases .
Comparison between canine and human diseases was obtained by using cirrhotic human samples derived from alcohol abuse or hepatitis C infection, two of the most common causes of hepatitis in the Western world [33, 34]. As in humans, chronic hepatitis in dogs is associated with progressive fibrosis, reduction in liver size and regeneration, and finally disruption of the liver architecture (cirrhosis), which may cause portal hypertension, ascites, and portosystemic encephalopathy . Although histologically highly comparable to their human counterparts, the etiology of canine hepatitis is largely unknown . However, human samples (hALC and hHC) showed the same degree of activation in the signal transduction pathways, irrespective of the different underlying etiology. Therefore, despite unknown etiology in dogs the underlying mechanisms are similarly activated.
This study is the first to measure expression profiles of crucial pathways of liver regeneration in canine liver diseases in comparison with man. Previously, a high similarity of affected fibrotic pathways between human- and canine-liver diseases was found . Combining these measurements on fibrotic- and regenerative-signaling pathways, privately owned dogs may help to fill in the gap between toxin-induced rodent models and human diseases. Furthermore, this study provides the basis to analyze more acute forms of hepatitis such as (sub)acute hepatitis in dog. Taken together, these results indicated that CH, LDH, and CIRR are suitable spontaneous large animal models to evaluate the clinical application of therapies such as cell transplantation or the administration of growth factors.
All samples were obtained from privately owned canines of different breeds referred to our veterinary clinic. All procedures were approved by Utrecht University's Ethical Committee, as required under Dutch legislation. Each disease group (n = 11 dogs) was compared to age-matched healthy control dogs (n = 12), without clinical signs of hepatitis or other disease (histopathology did not reveal any abnormalities). Liver biopsies were obtained (ultrasound-guided) from all dogs under local anesthesia with a true cut 14G biopsy needle, preceded by ultrasonographic evaluation of the liver to exclude non-homogeneous hepatic changes. Two formalin-fixed biopsies were embedded in paraffin, sliced, and stained with hematoxylin and eosin-, van Gieson-, and reticulin-stain according to Gordon and Sweet. All histological examinations were performed by one experienced, certified veterinary pathologist. Two other biopsies were snap-frozen and stored at -70°C until molecular analysis.
All liver samples were obtained from surgical patients transplanted at the Department of Abdominal Transplantation in the University Hospital Leuven, Leuven, Belgium. The procedures were approved by Leuven University's Ethical Committee, as required under Belgian legislation. Human explant samples were collected directly after surgery and immediately snap-frozen. All patients, predominantly male, were presented with micronodular cirrhosis. The Alcoholic Cirrhosis (hALC) group contained five patients (n = 5) characterized by cirrhosis with neutrophil infiltrations, alcohol related morphological changes (hepatocyte ballooning, Mallory bodies, necrosis), and in some cases steatosis and increased iron deposition. The Hepatitis C (hHC) group contained four patients (n = 4) characterized by cirrhosis with neutrophil infiltrations, lymphoid follicles and aggregates. All cases were presented with hepatocyte decay, apoptosis/necrosis, regeneration, and fibrosis.
Nucleotide Sequences of Dog-Specific Primers for Real-Time Quantitative PCR
Product size (bp)
TGT CCC CAC CCC CAA TGT ATC
CTC CGA TGC CTG CTT CAC TAC CTT
AGC TTG CTG GTG AAA AGG AC
TTA TAG TCA AGG GCA TAT CC
AAA GGA GAT GAG AAA CGC AAA CAG
GGC CTA GCA AGC TTC AGT AAT ACC
TGT GCT GTG AAA TCC CTG AAT AGA AATC
CCA AGA GTG AGA GTA CGT TTG GAT GAC
Primary Antibodies used in Western blot experiments
Product size (kDa)
Goat anti-human HGF
Goat anti-human c-MET
Rabbit anti-human c-MET (Tyr1230/1234/1235)
Rabbit anti-human/dog STAT3
Rabbit anti-human phospho-STAT3 (Ser727)
Mouse anti-human phospho-STAT3 (Tyr705)
Mouse anti-human/dog PKB
Rabbit anti-human phospho-PKB (Thr308)
Rabbit anti-human Erk1/2
Rabbit anti-human phospho-ERK1/2 (Thr202/Tyr204)
Rabbit anti-human p38-MAPK
Rabbit anti-human phospho-p38-MAPK (Thr180/Tyr182)
Mouse anti-human/dog Beta-actin (pan Ab-5)
Hepatocyte Growth Factor
Lobular Dissecting Hepatitis
Mitogen activated protein kinase (MAPK) 1 and 3
cirrhotic stage of alcoholic liver disease
cirrhotic stage of chronic hepatitis C infection
Mitogen activated protein kinase (MAPK) 14
Protein Kinase B
Quantitative real-time PCR
Signal Transducer and Activator of Transcription 3.
The authors thank Dr. Libbrecht for the (description of) the human samples.
- Motola-Kuba D, Zamora-Valdes D, Uribe M, Mendez-Sanchez N: Hepatocellular carcinoma. An overview. Ann Hepatol. 2006, 5: 16-24.PubMedGoogle Scholar
- Williams R: Global challenges in liver disease. Hepatology. 2006, 44: 521-526. 10.1002/hep.21347.View ArticlePubMedGoogle Scholar
- Constandinou C, Henderson N, Iredale JP: Modeling liver fibrosis in rodents. Methods Mol Med. 2005, 117: 237-250.PubMedGoogle Scholar
- Anstee QM, Goldin RD: Mouse models in non-alcoholic fatty liver disease and steatohepatitis research. Int J Exp Pathol. 2006, 87: 1-16. 10.1111/j.0959-9673.2006.00465.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Belanger M, Butterworth RF: Acute liver failure: a critical appraisal of available animal models. Metab Brain Dis. 2005, 20: 409-423. 10.1007/s11011-005-7927-z.View ArticlePubMedGoogle Scholar
- Dandri M, Lutgehetmann M, Volz T, Petersen J: Small animal model systems for studying hepatitis B virus replication and pathogenesis. Semin Liver Dis. 2006, 26: 181-191. 10.1055/s-2006-939760.View ArticlePubMedGoogle Scholar
- Nanji AA, French SW: Animal models of alcoholic liver disease--focus on the intragastric feeding model. Alcohol Res Health. 2003, 27: 325-330.PubMedGoogle Scholar
- Siegmund SV, Haas S, Singer MV: Animal models and their results in gastrointestinal alcohol research. Dig Dis. 2005, 23: 181-194. 10.1159/000090165.View ArticlePubMedGoogle Scholar
- Ostrander EA, Wayne RK: The canine genome. Genome Res. 2005, 15: 1706-1716. 10.1101/gr.3736605.View ArticlePubMedGoogle Scholar
- Sutter NB, Ostrander EA: Dog star rising: the canine genetic system. Nat Rev Genet. 2004, 5: 900-910. 10.1038/nrg1492.View ArticlePubMedGoogle Scholar
- Spee B, Arends B, van den Ingh TSGAM, Brinkhof B, Nederbragt H, Ijzer J, Roskams T, Penning LC, Rothuizen J: Transforming growth factor beta-1 signalling in canine hepatic diseases: new models for human fibrotic liver pathologies. Liver Int. 2006, 26: 716-725. 10.1111/j.1478-3231.2006.01277.x.View ArticlePubMedGoogle Scholar
- Twedt DC, van den Ingh TSGAM, Charles JA: Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Disease. 2006, Saunders ElsevierGoogle Scholar
- Neff MW, Rine J: A fetching model organism. Cell. 2006, 124: 229-231. 10.1016/j.cell.2006.01.008.View ArticlePubMedGoogle Scholar
- Koniaris LG, McKillop IH, Schwartz SI, Zimmers TA: Liver regeneration. J Am Coll Surg. 2003, 197: 634-659. 10.1016/S1072-7515(03)00374-0.View ArticlePubMedGoogle Scholar
- Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF: Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003, 4: 915-925. 10.1038/nrm1261.View ArticlePubMedGoogle Scholar
- Hanada M, Feng J, Hemmings BA: Structure, regulation and function of PKB/AKT--a major therapeutic target. Biochim Biophys Acta. 2004, 1697: 3-16.View ArticlePubMedGoogle Scholar
- Okano J, Shiota G, Matsumoto K, Yasui S, Kurimasa A, Hisatome I, Steinberg P, Murawaki Y: Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway. Biochem Biophys Res Commun. 2003, 309: 298-304. 10.1016/j.bbrc.2003.04.002.View ArticlePubMedGoogle Scholar
- Bennett AM, Davies JD, Gaskell CJ, Lucke VM: Lobular dissecting hepatitis in the dog. Vet Pathol. 1983, 20: 179-188.View ArticlePubMedGoogle Scholar
- van den Ingh TSGAM, Rothuizen J: Lobular dissecting hepatitis in juvenile and young adult dogs. J Vet Intern Med. 1994, 8: 217-220.View ArticlePubMedGoogle Scholar
- Cramer T, Schuppan D, Bauer M, Pfander D, Neuhaus P, Herbst H: Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver Int. 2004, 24: 335-344. 10.1111/j.1478-3231.2004.0926.x.View ArticlePubMedGoogle Scholar
- Hammond DE, Carter S, McCullough J, Urbe S, Vande WG, Clague MJ: Endosomal dynamics of Met determine signaling output. Mol Biol Cell. 2003, 14: 1346-1354. 10.1091/mbc.E02-09-0578.PubMed CentralView ArticlePubMedGoogle Scholar
- Farinati F, Cardin R, D'Errico A, De MN, Naccarato R, Cecchetto A, Grigioni W: Hepatocyte proliferative activity in chronic liver damage as assessed by the monoclonal antibody MIB1 Ki67 in archival material: the role of etiology, disease activity, iron, and lipid peroxidation. Hepatology. 1996, 23: 1468-1475. 10.1002/hep.510230625.View ArticlePubMedGoogle Scholar
- Quaglia A, McStay M, Stoeber K, Loddo M, Caplin M, Fanshawe T, Williams G, Dhillon A: Novel markers of cell kinetics to evaluate progression from cirrhosis to hepatocellular carcinoma. Liver Int. 2006, 26: 424-432. 10.1111/j.1478-3231.2006.01242.x.View ArticlePubMedGoogle Scholar
- Waris G, Turkson J, Hassanein T, Siddiqui A: Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol. 2005, 79: 1569-1580. 10.1128/JVI.79.3.1569-1580.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoshida T, Hanada T, Tokuhisa T, Kosai K, Sata M, Kohara M, Yoshimura A: Activation of STAT3 by the hepatitis C virus core protein leads to cellular transformation. J Exp Med. 2002, 196: 641-653. 10.1084/jem.20012127.PubMed CentralView ArticlePubMedGoogle Scholar
- Starkel P, de Saeger C, Leclercq I, Strain A, Horsmans Y: Deficient Stat3 DNA-binding is associated with high Pias3 expression and a positive anti-apoptotic balance in human end-stage alcoholic and hepatitis C cirrhosis. J Hepatol. 2005, 43: 687-695. 10.1016/j.jhep.2005.03.024.View ArticlePubMedGoogle Scholar
- Giambartolomei S, Covone F, Levrero M, Balsano C: Sustained activation of the Raf/MEK/Erk pathway in response to EGF in stable cell lines expressing the Hepatitis C Virus (HCV) core protein. Oncogene. 2001, 20: 2606-2610. 10.1038/sj.onc.1204372.View ArticlePubMedGoogle Scholar
- Furukawa F, Matsuzaki K, Mori S, Tahashi Y, Yoshida K, Sugano Y, Yamagata H, Matsushita M, Seki T, Inagaki Y, Nishizawa M, Fujisawa J, Inoue K: p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology. 2003, 38: 879-889.View ArticlePubMedGoogle Scholar
- Gupta S, Rajvanshi P, Aragona E, Lee CD, Yerneni PR, Burk RD: Transplanted hepatocytes proliferate differently after CCl4 treatment and hepatocyte growth factor infusion. Am J Physiol. 1999, 276: G629-G638.PubMedGoogle Scholar
- Kosai K, Matsumoto K, Funakoshi H, Nakamura T: Hepatocyte growth factor prevents endotoxin-induced lethal hepatic failure in mice. Hepatology. 1999, 30: 151-159. 10.1002/hep.510300102.View ArticlePubMedGoogle Scholar
- Matsuda Y, Matsumoto K, Yamada A, Ichida T, Asakura H, Komoriya Y, Nishiyama E, Nakamura T: Preventive and therapeutic effects in rats of hepatocyte growth factor infusion on liver fibrosis/cirrhosis. Hepatology. 1997, 26: 81-89. 10.1002/hep.510260111.View ArticlePubMedGoogle Scholar
- Palmes D, Spiegel HU: Animal models of liver regeneration. Biomaterials. 2004, 25: 1601-1611. 10.1016/S0142-9612(03)00508-8.View ArticlePubMedGoogle Scholar
- Corrao G, Zambon A, Torchio P, Arico S, La Vecchia C, di Orio F: Attributable risk for symptomatic liver cirrhosis in Italy. Collaborative Groups for the Study of Liver Diseases in Italy. J Hepatol. 1998, 28: 608-614. 10.1016/S0168-8278(98)80284-5.View ArticlePubMedGoogle Scholar
- Morgan G: Beneficial effects of NSAIDs in the gastrointestinal tract. Eur J Gastroenterol Hepatol. 1999, 11: 393-400.View ArticlePubMedGoogle Scholar
- Jalan R, Hayes PC: Hepatic encephalopathy and ascites. Lancet. 1997, 350: 1309-1315. 10.1016/S0140-6736(97)07503-X.View ArticlePubMedGoogle Scholar
- Boomkens SY, Penning LC, Egberink HF, van den Ingh TSGAM, Rothuizen J: Hepatitis with special reference to dogs. A review on the pathogenesis and infectious etiologies, including unpublished results of recent own studies. Vet Q. 2004, 26: 107-114.View ArticlePubMedGoogle Scholar
- Spee B, Mandigers PJ, Arends B, Bode P, van den Ingh TSGAM, Hoffmann G, Rothuizen J, Penning LC: Differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in Doberman pinschers. Comp Hepatol. 2005, 4: 3-10.1186/1476-5926-4-3.PubMed CentralView ArticlePubMedGoogle Scholar
- Brinkhof B, Spee B, Rothuizen J, Penning LC: Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem. 2006, 356: 36-43. 10.1016/j.ab.2006.06.001.View ArticlePubMedGoogle Scholar
- Spee B, Penning LC, van den Ingh TSGAM, Arends B, Ijzer J, van Sluijs FJ, Rothuizen J: Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment. Comp Hepatol. 2005, 4: 7-10.1186/1476-5926-4-7.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.