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, 135 (2), 660-70

Inhibition of Integrin alphavbeta6 on Cholangiocytes Blocks Transforming Growth Factor-Beta Activation and Retards Biliary Fibrosis Progression

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Inhibition of Integrin alphavbeta6 on Cholangiocytes Blocks Transforming Growth Factor-Beta Activation and Retards Biliary Fibrosis Progression

Eleonora Patsenker et al. Gastroenterology.

Abstract

Background & aims: Integrin alphavbeta6 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)-beta1. Because its role in liver fibrosis is unknown, we studied alphavbeta6 function in vitro and explored the antifibrotic potential of the specific alphavbeta6 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 alphavbeta6 and fibrosis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. alphavbeta6-expressing cells, bile duct proliferation, and apoptosis were assessed histologically. The effect of EMD527040 on cholangiocyte adhesion, proliferation, apoptosis, and TGF-beta1 activation was studied in vitro.

Results: alphavbeta6 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 alphavbeta6 inhibition reduced activated cholangiocyte proliferation, their adhesion to fibronectin, and endogenous activation of TGF-beta1 by 50% but did not affect bile duct apoptosis.

Conclusions: Integrin alphavbeta6 is strongly up-regulated in proliferating bile duct epithelia and drives fibrogenesis via adhesion to fibronectin and auto/paracrine TGF-beta1 activation. Pharmacologic inhibition of alphavbeta6 potently inhibits the progression of primary and secondary biliary fibrosis.

Conflict of interest statement

Conflict of interest/financial disclosures: none to be declared

Figures

Figure 1
Figure 1. αvβ6 integrin expression in livers of normal and fibrotic rats and mice, and in various liver cells
Rate limiting β6 transcript levels as quantified by real time PCR in: (A) Rat livers after 6 weeks of BDL and livers of 8 wk old MDR2-/- mice, both normalized to their age-matched sham-operated and wildtype controls, respectively; data in the MDR2-/-mice are from ref.29; (B) normal rat hepatocytes (Hep), rat hepatic stellate cells (HSC), human bile duct epithelial cells (BDEC), normal human (MMNK-1) or mouse (603B) cholangiocyte cell lines, and activated tumor-derived TFK-1 cholangiocytes, normalized to β2MG mRNA (x-fold increase relative to normal human BDEC). Means±SD; *p<0.05 vs. the corresponding control group. αvβ6 immunostaining of liver sections from sham-operated rats (C), rats with BDL (D) or of normal mouse (603B) (E) and activated human (TFK-1) (F) cholangiocytes. αvβ6 protein is absent from normal livers and 603B cholangiocytes, but highly expressed on proliferating bile duct epithelial cells (red) and TFK-1 cholangiocytes (brown). Shown are representative images (magnification 40×).
Figure 2
Figure 2. Effect of αvβ6 inhibition on liver histology in rodents with biliary fibrosis
Liver sections from rodents were formalin-fixed and stained for collagen with Sirius red. A: sham-operated rats; B: rats with BDL 5 weeks + vehicle for 4 weeks; C: BDL 5 weeks + EMD527040 20mg/kg/day for 4 weeks; D: BDL 5 weeks + EMD527040 60mg/kg/day for 4 weeks (magnification 40x). E: MDR2-/- mice + vehicle; F: MDR2-/- mice + EMD527040 20mg/kg/day for 4 weeks (magnification 10×). Shown are representative images.
Figure 3
Figure 3. Effect of αvβ6 inhibition on numbers of bile duct epithelial cells in rat biliary fibrosis
Livers were harvested 5 weeks after BDL and bile duct epithelia were stained with CK19. (A) sham-operated; (B) BDL 5 weeks + vehicle for 4 weeks; (C) BDL 5 weeks + EMD527040 20mg/kg/day for 4 weeks; (D) BDL 5 weeks + EMD527040 60mg/kg/day for 4 weeks. E: Quantification of bile duct epithelial cell number was performed by a point counting method (M&M). Data are expressed as percent of CK19 positive cells per liver section (magnification 10×), (means±SD), *p<0.05 vs. BDL alone.
Figure 4
Figure 4. Effect of αvβ6 inhibition on cholangiocyte proliferation in vivo and in vitro
Double staining for CK19 and Ki-67 was performed and CK19 and Ki-67 positive cells appear in brown and dark grey, respectively. (A) BDL 5 weeks + vehicle for 4 weeks; (B) BDL 5 weeks + EMD527040 20mg/kg/day for 4 weeks; (C) BDL 5 weeks + EMD527040 60mg/kg/day for 4 weeks. (D) The percentage of proliferating bile epithelia was assessed as the number of Ki-67 positive cells divided by the number of bile duct epithelia (CK19 positive) and is expressed as per field (arbitrary units). 4 fields were assessed per each liver section (magnification 40×), (means±SD). *p<0.05 vs. BDL alone. Proliferation of TFK-1 (E), primary HSC and HepG2 (F) cells was assessed by BrdU incorporation w/wt incubation with 10-7 or 10-6M EMD527040 for 24h. Means±SD. *p<0.05 vs 10%FBS control, #p<0.05 vs 0% FBS control.
Figure 5
Figure 5. Fibrosis-related gene expression in rats with secondary biliary fibrosis treated with EMD527040
Transcript levels were measured in rats with sham-operation (n=4), or with BDL for 5 weeks, treated either with vehicle (n=8), EMD527040 at 20mg/kg/day (n=7), or EMD527040 at 60mg/kg/day for 4 weeks (n=8). Results were obtained by quantitative real time PCR, normalized to β2MG mRNA and are expressed as x-fold increase vs. the sham-operated group (means±SDs). *p<0.05 vs. BDL alone.
Figure 6
Figure 6. αvβ6 integrin inhibition blocks adhesion of human cholangiocytes to fibronectin and reduces activation of endogenous TGFβ1
(A) TFK-1 cholangiocarcinoma cells were allowed to adhere to fibronectin-coated wells for 30min in the presence or absence of EMD527040 and adherent cells were measured by calcein AM fluorescence at 520 nm. Supernatants were collected and endogenously activated TGFβ1 was measured by an ELISA for active human TGFβ1 (B), or total TGFβ1 was measured after chemical activation by 1 N HCl (C). The ratio of spontaneously active to total TGFβ1 reflects the percentage of endogenous TGFβ1 activation (n=6 per group, means±SD); *p<0.05 vs. untreated controls.

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