Integrin αvβ6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies☆
Introduction
Liver fibrosis and cirrhosis are the consequences of many forms of chronic liver diseases and the major determinants of morbidity and mortality in liver patients. Therefore, there is an urgent need for antifibrotic treatments which can prevent, halt or reverse hepatic fibrosis. Significant progress has been made in our understanding of hepatic fibrosis as a dynamic process, resulting from excess extracellular matrix (ECM) production, i.e., fibrogenesis, over its degradation, i.e., fibrolysis [1], [2]. Thus several studies suggest that even advanced experimental and human liver fibrosis can reverse once pathogenic triggers are eliminated or fibrolytic stimuli prevail [3], [4], [5], [6], [7].
In hepatic fibrosis the excessive ECM is produced by activated mesenchymal cells which resemble myofibroblasts. They derive from quiescent hepatic stellate cells (HSC) and periportal or perivenular fibroblasts [8], [9]. These cells have been the major target for antifibrotic therapy [10], [11], [12], although other cells, e.g., activated Kupffer cells and bile duct epithelia, also contribute to their fibrogenic activation [13], [14], [15].
Integrins constitute a family of transmembrane heterodimeric cellular receptors that mediate cell/cell and cell/matrix interactions. The β6 integrin chain forms a heterodimer only with the αv subunit and is restricted exclusively to certain epithelial cells [16]. While the αvβ6 integrin is undetectable in most differentiated tissues, it is expressed during embryogenesis, tumorigenesis, and tissue injury [17], suggesting a role for αvβ6 in development, neoplasia and tissue repair. Interestingly, mice lacking this integrin are resistant to pulmonary fibrosis [18]. This has been explained by the role of αvβ6 in binding the TGF-β1 precursor, i.e., TGF-β1 latency-associated protein (LAP), to facilitate its proteolytic activation to biologically active TGF-β1 on the (epithelial) cell surface [18], [19]. In addition, αvβ6 is an adhesion receptor for fibronectin and tenascin-C, both central matrix molecules of granulation tissues [20], [21]. Moreover, activation of αvβ6 via adhesion to these substrates can augment epithelial cell activation and proliferation through a mitogen activated kinase (ERK) binding motif in its cytoplasmic domain [22], [23], [24].
Proliferation of bile duct epithelial cells is an almost universal finding in hepatic fibrosis [25]. Activated proliferating bile duct epithelia produce basement membrane proteins [26], [27] and release important fibrogenic growth factors for hepatic stellate cells and myofibroblasts, such as TGF-β1/2 [28], platelet derived growth factor [29] and connective tissue growth factor [13]. This ductular reaction is developmentally meaningful, since it endows the branching vascular and biliary structures of the liver with the necessary structural support provided by the induced and de novo formed ECM [30]. However, in liver fibrosis it runs out of control. It is likely that certain integrins are involved in the fibrogenic ductular reaction, and αvβ6 is a primary candidate.
Here we show that αvβ6 integrin, which is virtually absent from normal livers, is markedly upregulated on activated bile duct epithelial cells and hepatocytes with features of ductular transformation, correlating with fibrosis stage, both in rodent models of fibrosis and in patients with chronic liver disease. Furthermore, in Mdr2(Abcb4)−/− mice with spontaneously progressive liver fibrosis, a single dose of a selective small molecule inhibitor of αvβ6 caused favourable changes in hepatic fibrosis-related transcripts and upregulated activities of key fibrolytic enzymes, suggesting αvβ6 integrin as a functionally relevant and promising target in hepatic fibrosis.
Section snippets
Materials
All reagents were from Sigma (St. Louis, MO.) unless stated otherwise and of the highest purity available. The selective αvβ6 integrin antagonist EMD527040 (3-{(S)-3-Benzyloxy-2-[5-(pyridin-2-ylamino)-pentanoylamino]- propionylamino}-3-(3,5-dichloro-phenyl)-propionic acid, Mr 587.5, Fig. 4A) was developed and synthesized by Merck Pharma as previously described [31], [32] and kindly provided by S. Goodman, Merck AG, Darmstadt, Germany. This highly selective peptidomimetic antagonist was obtained
Hepatic αvβ6 integrin is virtually absent in normal liver but highly upregulated in rodent models of liver fibrosis
The β6 integrin chain forms a unique heterodimer with αv, while αv forms heterodimers with the β1,β3,β5 and β6 and β8 chain. We quantified αv, β3 and β6 transcripts at the peak of TAA- and during BDL-induced liver fibrosis, which represent models of panlobular and periportal fibrosis, respectively. Since β6 integrin forms heterodimers only with the constitutively expressed αv chain, transcript levels of the β6 chain reflect upregulation of the dimeric αvβ6 integrin [18]. While αv and β3
Discussion
In the present study we have shown that the expression of integrin αvβ6 is markedly upregulated in three experimental models of liver fibrosis and human liver fibrosis of different stage and etiology, correlating with fibrogenic activity and fibrosis stage. Interestingly, we found that αvβ6 is expressed (exclusively) by activated bile duct epithelial cells and transitional hepatocytes, cells that are prominent in (portal) fibrosis. Further, we provided evidence that αvβ6 is functionally
Acknowledgements
We thank Dr. Michael Bauer (University of Erlangen) for designing some of the primers used in the study and expert advice in real time PCR. This work was supported by Grant Schu 646/14-1 by the German Research Council (D.F.G.), and an appointment Grant to D.S. by the Beth Israel Deaconess Medical Center. Y.P. was recipient of a Sheila Sherlock fellowship of the European Association for the Study of the Liver and E.P. of a scholarship by the graduate college GRK 750.
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The authors declare that they do not have anything to disclose regarding funding from industries or conflict of interest with respect to this manuscript. Data in part were presented by us in abstract form at the annual meeting of the American Association for the Study of the Liver in 2004 (linking integrin avb6 expression to progression of liver fibrosis) and in 2006 (presenting first evidence of its functional role in experimental liver fibrosis) in Boston.