Inhibition of Integrin αvβ6 on Cholangiocytes Blocks Transforming Growth Factor-β Activation and Retards Biliary Fibrosis Progression

doi:10.1053/j.gastro.2008.04.009

Gastroenterology

Volume 135, Issue 2, August 2008, Pages 660-670

https://doi.org/10.1053/j.gastro.2008.04.009 Get rights and content

Background & Aims: Integrin αvβ6 is highly expressed on certain activated epithelia, where it mediates attachment to fibronectin and serves as coreceptor for the activation of latent transforming growth factor (TGF)-β1. Because its role in liver fibrosis is unknown, we studied αvβ6 function in vitro and explored the antifibrotic potential of the specific αvβ6 antagonist EMD527040. Methods: Experimental liver fibrosis was studied in rats after bile duct ligation (BDL) and in Mdr2(abcb4)^−/− mice. Different doses of EMD527040 were given to rats from week 2 to 6 after BDL and to Mdr2^−/− mice from week 4 to 8. Liver collagen was quantified, and expression of αvβ6 and fibrosis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. αvβ6-expressing cells, bile duct proliferation, and apoptosis were assessed histologically. The effect of EMD527040 on cholangiocyte adhesion, proliferation, apoptosis, and TGF-β1 activation was studied in vitro. Results: αvβ6 was highly expressed on proliferating bile duct epithelia in fibrosis, with 100-fold increased transcript levels in advanced fibrosis. EMD527040 attenuated bile ductular proliferation and peribiliary collagen deposition by 40%–50%, induced down-regulation of fibrogenic and up-regulation of fibrolytic genes, and improved liver architecture and function. In vitro αvβ6 inhibition reduced activated cholangiocyte proliferation, their adhesion to fibronectin, and endogenous activation of TGF-β1 by 50% but did not affect bile duct apoptosis. Conclusions: Integrin αvβ6 is strongly up-regulated in proliferating bile duct epithelia and drives fibrogenesis via adhesion to fibronectin and auto/paracrine TGF-β1 activation. Pharmacologic inhibition of αvβ6 potently inhibits the progression of primary and secondary biliary fibrosis.

Section snippets

Materials

All chemicals were obtained from Sigma-Aldrich (Basel, Switzerland), if not mentioned otherwise, and of the highest purity available. The nonpeptide αvβ6 integrin antagonist (3-{(S)-3-Benzyloxy-2-[5-(pyridin-2-ylamino)-pentanoylamino]-propionylamino}-3-(3,5-dichloro-phenyl)-propionic acid, Mr 587.5) (EMD527040) was developed and synthesized by Merck AG (Darmstadt, Germany).27, 28, 29 The compound is not yet commercially available. It inhibits binding of recombinant αvβ6 to fibronectin at 6

Statistical Analysis

Statistical analysis was performed using Microsoft Excel software (Microsoft Corp., Redmond, WA). Data are expressed as means ± SD. The statistical significance of differences was evaluated using the unpaired, nonparametric Student t test.

αvβ6 Integrin Is Highly Up-Regulated in Biliary Fibrosis and Expressed in Proliferating Biliary Epithelia

In rats with BDL and in Mdr2^−/− mice, β6 transcripts, which are rate limiting for αvβ6 integrin dimer formation, were up-regulated up to 100-fold, whereas normal rodents showed almost undetectable levels, confirming our prior findings in the Mdr2^−/− model²⁹ (Figure 1A). Normal liver cells and near normal immortalized bile duct epithelia showed no or very low expression of β6 when compared with highly proliferative tumor-derived TFK-1 cholangiocytes (Figure 1B). Immunohistochemistry demonstrated

Discussion

Integrin αvβ6 is expressed on certain activated epithelia and is highly up-regulated after tissue injury, in wound healing,16, 19, 20, 24 and in some types of epithelial cancers.⁴² Mice lacking this integrin are resistant to induction of pulmonary fibrosis.²⁴ This suggested an important role of αvβ6 in chronic wound healing, progression of pulmonary fibrosis, and recently liver fibrosis.29, 43 Thus, we described a significant induction of β6 integrin in human and murine liver fibrosis and

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Citation Excerpt :
Three hours following an intraperitoneal dose of EMD527040 had led to the upregulation of profibrolytic mRNA such as MMP-8 and MMP-9. In another study, EMD527040 was also shown to significantly reduce profibrogenic genes such as procollagen αI(I), connective tissue growth factor (CTGF) and α-smooth muscle actin (Patsenker et al., 2008). Despite these promising in vivo findings of EMD527040, it has not progressed into clinical trials.
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: Supported in part by grant Schu 646/14-1 from the German Research Association (DFG) and grant NIH U19 AI066313 project 4 (to DS), a scholarship from the DFG graduate college (GRK-750) and the Swiss National Fund (SNF 320000-117725/1) (to EP), and a Sheila Sherlock fellowship by the European Association for the Study of the Liver (EASL) (to Y.P.).

: Conflicts of interest/financial disclosures: None to be declared.

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Basic—Liver, Pancreas, and Biliary TractInhibition of Integrin αvβ6 on Cholangiocytes Blocks Transforming Growth Factor-β Activation and Retards Biliary Fibrosis Progression

Section snippets

Materials

Statistical Analysis

αvβ6 Integrin Is Highly Up-Regulated in Biliary Fibrosis and Expressed in Proliferating Biliary Epithelia

Discussion

J Biol Chem

Am J Pathol

Am J Pathol

Curr Opin Cell Biol

J Biol Chem

J Biol Chem

J Pediatr Surg

Am J Pathol

J Biol Chem

Exp Cell Res

Cell

Am J Pathol

J Hepatol

Hepatology

Cell

J Hepatol

J Hepatol

J Biol Chem

Gastroenterology

J Invest Dermatol

J Surg Res

J Biol Chem

J Biol Chem

Liver fibrosis

J Clin Invest

Mechanisms of disease: mechanisms of hepatic fibrosis and therapeutic implications

Nat Clin Pract Gastroenterol Hepatol

Matrix as a modulator of hepatic fibrogenesis

Semin Liver Dis

Basic—Liver, Pancreas, and Biliary Tract
Inhibition of Integrin αvβ6 on Cholangiocytes Blocks Transforming Growth Factor-β Activation and Retards Biliary Fibrosis Progression