Elsevier

Journal of Hepatology

Volume 43, Issue 6, December 2005, Pages 1045-1054
Journal of Hepatology

Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes

https://doi.org/10.1016/j.jhep.2005.06.025Get rights and content

Background/Aims

Mdr2 (Abcb4)-/- mice develop hepatic lesions resembling primary sclerosing cholangitis. Our aim was to characterize the evolution of fibrosis in Mdr2-/- mice.

Methods

Mdr2-/-mice and their wild-type littermates were sacrificed at 2, 4 and 8 weeks after birth. Hepatic collagen was determined biochemically. Fibrosis related transcript levels were quantified from livers by real-time RT-PCR, and MMP activities determined by substrate assays. Liver histology was assessed by connective tissue staining and immunohistochemistry for α-smooth muscle actin (α-SMA).

Results

Mdr2-/- mice demonstrated a time-dependent increase of relative and total hepatic collagen (fivefold at 8 weeks, compared to wildtype controls), and maximal α-SMA immunoreactivity at 4 weeks. Compared to wildtype controls profibrogenic mRNA levels for procollagen α1(I), TGFβ1, TGFβ2, MMP-2 and -13, TIMP-1, PDGFβ receptor, and PAI-1 were upregulated up to 27-fold. Most transcripts peaked at 4 weeks, but procollagen α1(I) mRNA increased steadily, TIMP-1 mRNA was constantly elevated (20-fold), MMP-13 mRNA was suppressed and interstitial collagenase and gelatinase activities were downregulated.

Conclusions

Mdr2-/- mice spontaneously progress to severe biliary fibrosis. This is due to a characteristic temporal pattern of upregulated profibrogenic and downregulated fibrolytic genes and activities. These mice are an attractive model to test potential antifibrotics for the treatment of (biliary) liver fibrosis.

Introduction

Liver cirrhosis, the frequent consequence of chronic liver disease, is associated with a high morbidity and mortality. In chronic cholangiopathies such as PBC and PSC development of cirrhosis is frequent and often requires transplantation as the only effective treatment modality. Therapies that halt disease progression are of limited efficacy and pharmacological reversion of advanced fibrosis and cirrhosis is unavailable. In view of the growing demand for liver transplantation confronted with an increasing donor shortage, effective antifibrotic therapies are urgently needed.

Potential antifibrotic agents have to be tested in vivo. Several experimental fibrosis models have been developed in rats and mice. So far there is no suitable animal model for biliary fibrosis, since ligation of the common bile duct results in a rapidly progressive interstitial (biliary) fibrosis, associated with massive proliferation of bile ducts which is only rarely observed in man [1]. Thus, there is a need for a biliary fibrosis model, which resembles human pathology and is characterized by a progressive, homogeneous and reproducible liver fibrosis, allowing for testing of large numbers of potential drug candidates and their combinations.

Mice deficient in the canalicular phospholipid flippase (Mdr2/Abcb4-/- mice) spontaneously develop liver injury, due to the absence of phospholipid from bile [2], which leads to morphological features of primary sclerosing cholangitis (PSC) [3]. Moreover, these animals can be considered an animal model for human MDR3 deficiency ranging from progressive familial intrahepaic cholestasis type 3 to adult liver cirrhosis [4]. The hepatic pathogenesis in Mdr2-/- mice was recently characterized as a sequence of events that include disruption of tight junctions and basement membranes of bile ducts, and bile leakage to the portal tract. These features trigger a multistep process that ultimately leads to the formation of periportal biliary fibrosis [5]. However, neither the extent nor the dynamics of fibrogenesis were investigated in these mice in detail. Here we present an analysis of the evolution of fibrosis and the temporal expression of fibrogenic and fibrolytic genes in Mdr2-/- mice. Our results show that these mice may also serve as a valuable in vivo tool to test potential antifibrotic agents.

Section snippets

Animal studies

Mdr2-/- knockout and Mdr2+/+ wildtype mice were obtained from Jackson Laboratory (Jackson Laboratory, Bar Harbor, ME) and housed with a 12-h light-dark cycle with water and standard mouse pellet chow ad libitum. At age 2-, 4-, and 8-weeks mice were sacrificed by cervical dislocation under general anesthesia (400 mg avertin/kg i/p), livers and spleens were excised and weighed. Liver specimens from two lobes were either fixed in 4% buffered formalin or snap-frozen in liquid nitrogen for further

Results

Mdr2-/- mice and their wildtype littermates were sacrificed at 2, 4 and 8 weeks of age, previously established to reflect important time points for the pathogenesis of liver injury [5].

Discussion

Mdr2/Abcb4 (mouse orthologue of human MDR3/ABCB4) knockout mice (Mdr2-/-) were recently shown to develop biliary fibrosis shortly after birth [3] as a result of leakage of potentially toxic bile acids (e.g. cholic acid) into the periductal area [5]. The model resembles biliary fibrosis in humans, such as PSC or congenital MDR3 deficiency ranging from progressive familial intrahepaic cholestasis type 3 to adult liver cirrhosis [4], and should therefore be well suited to better understand the

Acknowledgements

This work was supported by the German research Council (DFG) grant 646/14-1, and grants by the German Network for Viral Hepatitis (Hepnet) and the Interdisciplinary Center for Clinical Research (IZKF) of the University of Erlangen-Nuernberg to D.S. and grant P-15502 from the Austrian Science Foundation and a GEN-AU project grant from the Austrian Ministry for Science to M.T. Y.P. was a recipient of EASL/Yamanouchi (2002) and EASL/Sheila Sherlock (2003) Fellowships, and E.P. of a DFG-Graduate

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