Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment
© Spee et al; licensee BioMed Central Ltd. 2005
Received: 10 February 2005
Accepted: 07 December 2005
Published: 07 December 2005
We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls.
We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor β1 (TGF-β1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-β1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-β1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-β1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis.
The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic inflammatory diseases and cirrhosis.
Chronic liver disease is characterized by decreased regeneration of hepatocytes and increased formation of fibrous tissue. These characteristics may be the sequel of various chronic processes such as cholestasis, viral infections, toxin exposure, and metabolic disorders. Dogs have complex liver diseases such as hepatitis and cirrhosis which are highly comparable with the human counterparts. Moreover, coding sequences of dogs proved highly homologous to the human sequences , especially compared to the rodent genome. Thus, dogs may fulfill a role as a spontaneous animal model in between toxin-induced or surgical models in rodents, and spontaneous diseases in man. The complex interplay of many factors active in chronic liver disease makes it difficult to unravel the roles of different individual pathogenetic pathways. Dogs display liver diseases, which are potentially valuable models to compare complex with simple pathologic entities.
We have chosen these two congenital dog diseases for comparative analysis of liver growth/regeneration, fibrosis, and hepatic homeostasis: congenital portosystemic shunt (CPSS) and primary portal vein hypoplasia (PPVH). CPSS is characterized by an abnormal single large communication between the portal vein and a major systemic vein (cava or azygos). This results in the virtual absence of portal vein perfusion to the liver from birth onwards. Liver growth remains nearly absent but there is essentially no liver pathology [2, 3]. PPVH is a developmental abnormality in which the terminal vein branches are not or only partially present and, in most cases, in combination with congenital portal fibrosis, but without inflammation . PPVH is associated with portal hypertension and reduced liver growth. Thus, these two congenital diseases represent relatively simple models for reduced liver growth associated with fibrosis (PPVH) or without fibrosis (CPSS). Both diseases have a decrease in liver growth due to differences in portal perfusion which results in a massive reduction of liver size.
Because hepatocyte growth factor (HGF) is one of the most important genes involved in liver growth/regeneration [5–7], abnormal expression of HGF could play a major role in the decreased liver size in CPSS or PPVH. Therefore, treatment of dogs with HGF could be a possible therapeutic approach. A pre-requisite for treatment is that HGF signaling components are unaffected in those dogs. Consequently, we focused on measuring gene products involved in signaling of HGF and counteracting transforming growth factor β1 (TGF-β1). All biological responses induced by HGF are elicited by binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-MET). The signaling cascade triggered by HGF begins with phosphorylation of the receptor and is mediated by concomitant activation of different cytoplasmic effectors that bind to the same multifunctional binding site. The c-MET mediated response includes two key pathways involved in cell survival and mitogenesis . The first; protein kinase B (PKB/Akt) is activated by phosphoinositide 3-kinase (PI3K) and elicits cell survival [9, 10]. The second; ERK1/2 (also known as p42/44 MAPK), a member of the mitogen-activated protein (MAP) kinase family, is activated by the RAS-RAF-MEK pathway and is responsible for mitogenesis . A third response of HGF is the branching morphogenesis which next to the PKB and ERK pathways requires involvement of the signal transducer and activator of transcription (STAT) 3 pathway .
It is well established that an increase of TGF-β1 in liver promotes the formation of extracellular matrix (ECM) components and suppresses hepatocyte proliferation [13, 14]. Prolonged overexpression of TGF-β1 in non-parenchymal cells causes hepatic fibrosis in humans and experimental animals. In several fibrosis models, fibrotic lesions are associated with an increase in collagens and TGF-β1 mRNAs . The intracellular pathway that is activated by TGF-β1 receptors is mediated by Smads. Smad2 is activated via carboxy-terminal phosphorylation by TGF-β1 type I receptor kinases. When bound with co-Smads, they act as TGF-β1-induced transcriptional activators of target genes .
Cell homeostasis is the result of balance between cell death, cell proliferation, and growth-arrest. Therefore we investigated expression levels of pro-apoptotic Fas ligand and caspase-3, anti-apoptotic Bcl-2 , cell-cycle stimulating TGFα, and cell-cycle inhibitor p27kip. All of these gene-products are regulated directly or indirectly by PKB .
The present study was designed to describe the differential gene-expression of the above indicated crucial pathways involved in growth/regeneration, fibrosis, and cellular homeostasis in liver tissues of dogs with CPSS (reduced growth/regeneration without fibrosis) and PPVH (reduced growth/regeneration and fibrosis) in comparison with healthy animals. These simple congenital dog models may be used to unravel the roles of different gene products in those pathways. These well-defined large animal models are intended to serve as the first spontaneous liver diseases to investigate novel regenerative/anti-fibrotic therapies, such as HGF treatment. This study may also serve as a basis for future comparison with more complex diseases like chronic hepatitis and cirrhosis.
Histological grading of fibrosis
HGF/c-MET signaling pathway involved in regeneration and growth
TGF-β1 cascade signaling pathway involved in fibrosis
Gene-expression of apoptosis-related signaling proteins and hypoxia induced factor
Gene-expression of extracellular matrix gene products
Western blot analysis of HGF, c-MET, PKB, STAT3, ERK, TGF-β1, Smad2, Collagen I, and Caspase-3
In order to analyze the possibility of growth factor therapy, two congenital canine liver diseases were molecularly dissected. The expression of a total of 17 gene products involved in liver growth/regeneration, fibrosis, ECM, and cellular homeostasis was measured and normalized to the average amount of two reference genes (Q-PCR). Western blot analysis confirmed the quantitative mRNA results and, furthermore, showed activated pathways. These two independent techniques provided insight into the effects of portal venous hypoperfusion in two canine hepatic diseases; congenital portosystemic shunt (CPSS) without fibrosis and primary portal vein hypoplasia (PPVH) with fibrosis. Taken together, the obtained data provided insights in the feasibility for HGF-treatment.
The normalization performed in this study was obtained by averaging the amount of two different reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase (HPRT). No samples were more than 5 percent apart from the individual measured reference genes levels (data not shown). This normalization strategy, using the average amount of two reference genes, is taken as a prerequisite for accurate Q-PCR expression profiling which enables us to measure small expression differences and allows the study of their biological relevance .
It is well known that HGF plays an essential role in development  and regeneration of the liver, and increases hepatocyte viability. The found decrease in gene-expression of both HGF and its receptor agrees with the reduced liver size in these canine disorders. However, and in contrast to the c-MET levels which correlate nicely with the found protein levels, the amount of HGF mRNA does not seem to reflect protein levels. This can be contributed to HGF which can be a paracrine but also an endocrine factor. Extra-hepatic HGF could have been present in the pancreas or intestinal tract .
Although HGF and c-MET mRNA levels were decreased, downstream targets of this tyrosine cascade signaling pathway were still active. Downstream targets, such as Fas ligand and p27kip, were chosen as direct or indirect targets of the HGF-cMET-PI3K-PKB axis. Fas ligand transcription is regulated by FOXO (forkhead box, sub-group "O" transcription factors). Therefore, the decrease in Fas ligand can be explained by an active PKB which directly phosphorylates FOXO . A similar result can be seen in the reduced levels of p27kip mRNA, as this is down-regulated at the gene-transcription level by active PKB . Combined, this indicates that PKB is active in both diseases, which was confirmed by Western blot analysis. It remains to be seen whether other receptor tyrosine kinases (e.g., EGF receptor or insulin receptor) activate this pathway in these dogs . Next to the activated PKB pathway, we have analyzed other c-MET mediated responses in CPSS and PPVH. ERK1/2 showed to be activated in both diseases to a similar level as the healthy controls. The significance of the slightly reduced phosphorylated STAT3 in PPVH, which is phosphorylated by HGF on serine 727 , needs to be further investigated. Taken together, the pathways which elicit all major biological functions of c-MET showed to be active in CPSS and PPVH.
Prolonged or overexpression of TGF-β1 acts to suppress cell proliferation, and induces a deposition of ECM proteins, resulting in fibrosis in major organs such as liver [26, 27]. We showed that in PPVH the TGF-β1 pathway through Smad2 is activated, consistent with the fibrosis seen in PPVH. Measurements on fibrosis related gene products revealed no elevated activity of the TGF-β1 pathway in CPSS. Gene expression levels related to the TGF-β1 pathway, including its receptors, and the proteolytic activator of TGF-β1 (uPA) were elevated in PPVH, thus indicating an active Smad pathway that could subsequently lead to fibrosis. Western blot analysis confirmed found TGF-β1 levels. Measurements on collagen gene-expression, especially collagen I, confirm the current paradigm of TGF-β1 signaling in fibrous tissues like PPVH . Contrary, non-fibrotic CPSS did not show any alterations in collagen expression. The observation of phosphorylated Smad2 in healthy liver tissue showed that the phosphorylation of Smad2 is a dynamic process and has already been described in other publications [29, 30].
The expressions of the pro-apoptotic genes Fas ligand and caspase-3 were clearly decreased. Bcl-2 gene-expression was elevated two times in PPVH; but not in CPSS (Figure 4). Western blot analysis showed that the unprocessed form of caspase-3 was present in lesser amount in CPSS and PPVH; however, the amount of processed or active bands compared to healthy control was higher in the diseases compared to healthy controls. This indicates that although the total amount of caspase-3 is lower, there is more cleavage of the caspase-3 to its active forms in the diseases, possibly leading to an increase in apoptosis.
Both HGF and TGF-β1 need extracellular processing to become biologically active. The serine protease HGF activator is responsible for activation of proHGF . Our studies revealed that HGF activator gene-expression was doubled in dogs with CPSS and halved in case of PPVH. This indicated an increased HGF activation in CPSS. Although levels of HGF activator were reduced in PPVH, this does not necessarily indicate a lack of extracellular processing of HGF. Interestingly uPA, the activator of TGF-β1, was expressed at an increased level in dogs with PPVH. This may, via active TGF-β1-receptor interaction, indicate an activation of Smads and thus the formation of collagens.
Differential gene expression measurements on hepatic diseases have been performed in the past; nevertheless, little is known about levels of genes that play an important role in fibrosis. There have been measurements on cirrhosis in man and rat that indicate an up or down-regulated expression of several proteins . Although these results might be significant in severe forms of fibrosis, these data depict an end-point of the disease whereas earlier stages may be more informative.
Regeneration with recombinant HGF has been achieved in rodent models of liver failure [33, 34]. Moreover, besides its regenerative capacity, HGF is known to have an antifibrogenic effect [35, 36] and thus reduces or prevent fibrosis in PPVH. TGF-β1 intervention to halt the progression of liver fibrosis and positively effect regeneration, has been applied successfully  even in cirrhosis . The measured gene products involved in fibrosis in PPVH make it a good spontaneous animal model to investigate new therapeutic strategies to influence the HGF and/or TGF-β1 pathways in vivo. Furthermore, most fibrogenic models are induced by toxins such as dimethylnitrosamine (DMN), CCl4, or thioacetamide . The canine PPVH model is not drug-induced; therefore, may be better to compare with human diseases and thus fill the gap between induced rodent models and human diseases.
This study is the first to measure expression profiles of crucial pathways of liver growth/regeneration, fibrosis, and hepatic homeostasis in spontaneous canine liver diseases. The present findings in two diseases with relatively simple pathogenesis may also serve as basis for evaluation of more complex diseases like hepatitis and cirrhosis. Evaluation of such complex diseases in dogs is highly suitable for comparative studies on the roles of different pathways in the pathogenesis of liver diseases in man. Two further conclusions can be deduced from the data presented here. First, the pathophysiological differences between CPSS and PPVH can nicely be explained by the Q-PCR measurements and Western blots. Second, although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational background to design controlled studies for HGF-supplementation in CPSS and PPVH.
All samples are obtained from different dog breeds appearing in the clinic with spontaneous diseases. Samples were randomly chosen and aimed to encompass different dog-breeds and both sexes in each group. The procedures were approved by the Ethical Committee as required under Dutch legislation.
The congenital portosystemic shunt (n = 11 dogs) and primary portal vein hypoplasia group (n = 8 dogs) were compared with a group of healthy dogs (n = 11 dogs). The inclusion criteria for CPSS were increased fasting plasma ammonia concentration, abnormal ammonia tolerance test (peak ammonia ≥ 150 μmol/l plasma) and ultrasonographic visualization of a small liver and a congenital portosystemic shunt with a diameter as wide as the portal vein trunk. The presence of the shunt was further confirmed with surgery, during which a wedge liver biopsy was taken and immediately put in liquid nitrogen and stored at -70°C, until analysis. In CPSS there is no portal hypertension. The inclusion criteria for PPVH were the visualization of a small liver with ultrasonography, presence of multiple small acquired portosystemic collaterals due to portal hypertension, and an abnormal ammonia tolerance test (peak ammonia ≥ 150 μmol/l plasma). Liver tissue of dogs with PPVH was obtained under local anaesthesia by ultrasound-guided biopsy with a true cut 16G biopsy needle. Two biopsies were immediately immersed in liquid nitrogen, and stored at -70°C, until analysis. The healthy control dogs were age-matched, and had AP, ALT, and fasting bile acids in plasma within the reference range. Ultrasonographically the control dog livers had a normal size, shape, and structure, and there were no histological abnormalities in stained histological sections.
Histological grading of fibrosis
Liver samples were fixed in 10% buffered formalin and routinely embedded in paraffin. Sections (4 μm) were stained with haematoxylin-eosin, the Van Gieson stain, and the reticulin stain according to Gordon and Sweet. Histologically, the presence of fibrosis was evaluated semi-quantitatively (absent, slight, moderate, or marked) as well as with respect to its localization. Fibrosis scoring was performed according to Scheuer, a defined scoring method for fibrosis in hepatitis. The slides were independently examined by one certified veterinary pathologist.
RNA isolation and reverse-transcription polymerase chain reaction
Total cellular RNA was isolated from each frozen canine liver tissue in duplicate, using the RNeasy Mini Kit (Qiagen, Leusden, The Netherlands) according to the manufacturer's instructions. The RNA samples were treated with Dnase-I (Qiagen Rnase-free DNase kit). In total 3 μg of RNA was incubated with poly(dT) primers at 42°C for 45 min, in a 60 μl reaction volume, using the Reverse Transcription System from Promega (Promega Benelux, Leiden, The Netherlands).
Quantitative measurements of the mRNA levels of HGF, TGF-β1, and other related signaling molecules
Nucleotide Sequences of Dog-Specific Primers for Real-Time Q-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
CCG CCT TGG TGG TGG TCT CC
AGG GCG CTG GGC TTC TCG T
ACA CAG ACG TTT GGC ATC GAG AAG TAT
AAA CTG GAG CGG ATG GCA CAG
CGG AGG GAC GCC AAA CAG G
GTC CCG GGT CAA CTC TTC GTG
CAA GGA TCT GGG CTG GAA GTG GA
CCA GGA CCT TGC TGT ACT GCG TGT
TGF-β1 R I
CAG TCA CCG AGA CCA CAG ACA AAG T
TGA AGA TGG TGC ACA AAC AAA TGG
TGF-β1 R II
GAC CTG CTG CCT GTG TGA CTT TG
GGA CTT CGG GAG CCA TGT ATC TTG
CTG GGG AGA TGA AGT TTG AGG TGG
TGG AAC GGA TCT TCA GCA AGG C
TGG AGA GCG TCA ACC GGG AGA TGT
AGG TGT GCA GAT GCC GGT TCA GGT
GGG GTC AGT CCT GCA ACA ACA A
ATC TTC CCC TCC ATC AGC ATC AG
ATC ACT GAA GAT GGA TGG GTT GGT
GAA AGG AGC ATG TTC TGA AGT AGC ACT
TTA CGT TCC TTC GAT CAG TTG TCA
GAG GAG GTT CTT GCA TTG GAG TC
GTG TGT ACA GAA CGG CCT CA
TCG CAA ATC ACG TCA TCG
ATA GAG GCT TTG ATG GAC GAA
CCT CGC TCA CCA GGA GC
CAC AGC CAG ACA ACA GAT GC
GCA TGG TAC TGA AGC GAC G
AGG TTG TTA CCA TGG GCA
GCA TAA TGG GAA ACC GTG TAG
A Kolmogorov-Smirnov test was performed to establish a normal distribution and a Levene's test for the homogeneity of variances. All samples included in this study were normally distributed and homogeneous in variance. The statistical significance of differences between diseased and control animals was determined by using the Student's t-test. A p-value < 0.05 was considered statistically significant. Analysis was performed using SPSS software (SPSS Benelux BV, Gorinchem, The Netherlands).
Western blot analysis
Used antibodies in Western blot experiments.
Product Size (kDa)
p-c-MET (Tyr 1230/1234/1235)
p-PKB (Thr 308)
p-STAT3 (Ser 727)
p-Erk1/2 (Thr 202/Tyr 204)
p-Smad2 (Ser 465/467)
Beta-actin (pan Ab-5)
The authors are indebted to Dr. Alexandra Pietersen, Dr. Bernard Roelen, and Dr. Peter ten Dijke for their invaluable advice.
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