Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation

Abstract

Hepatic fibrosis is a common outcome of a variety of chronic liver diseases. Here we evaluated the therapeutic efficacy of hepatocyte growth factor (HGF) on liver fibrosis induced by bile duct ligation (BDL) and investigated potential mechanisms. Mice underwent BDL, followed by intravenous injections of naked HGF expression plasmid or control vector. HGF gene therapy markedly ameliorated hepatic fibrotic lesions, as demonstrated by reduced alpha-smooth muscle actin (alphaSMA) expression, attenuated deposition of type I and type III collagen, and normalized total hydroxyproline content. HGF also suppressed transforming growth factor-beta1 (TGF-beta1) expression. Interestingly, colocalization of alphaSMA and cytokeratin-19 in bile duct epithelium was observed, suggesting the possibility of biliary epithelial to myofibroblast transition after BDL. Cells that were still positive for cytokeratin-19 but actively producing type I collagen were found in the biliary epithelia and periductal region. Laminin staining revealed an impaired basement membrane of the bile duct epithelium in diseased liver. These lesions were largely prevented by HGF administration. In vitro, treatment of human biliary epithelial cells with TGF-beta1 induced alphaSMA and fibronectin expression and suppressed cytokeratin-19. HGF abolished the phenotypic conversion of biliary epithelial cells induced by TGF-beta1. These results suggest that HGF ameliorates hepatic biliary fibrosis in part by blocking bile duct epithelial to mesenchymal transition.

Figure 1

HGF gene therapy attenuates liver fibrosis induced by BDL. Masson’s trichrome staining of liver tissue sections from pcDNA3- (A and C) and pCMV-HGF-treated (B and D) mice at 12 weeks after BDL. Extensive peribiliary (A) and interstitial collagen (C) staining was noticeable in liver sections of the mice injected with pcDNA3. Significantly less hepatic collagen staining was observed in pCMV-HGF-treated mice (B and D).

Figure 2

HGF reduces hepatic hydroxyproline content and suppresses type I and type III collagen deposition. A:Total tissue hydroxyproline content was determined by biochemical assay. Data were expressed as micrograms of hydroxyproline per milligram of tissue and presented as mean ± SEM (n = 4–6). *, P < 0.05 versus sham; **, P < 0.01 versus sham; †, P < 0.05 versus pcDNA3 control. B–E: Representative micrographs showed the deposition of type I (B and C) and type III (D and E) collagen in the portal tract region of liver sections. B and D: pcDNA3; C and E: pCMV-HGF.

Figure 3

HGF inhibits hepatic TGF-β1 expression. A: Reverse transcription-PCR showed an inhibitory effect of HGF on hepatic TGF-β1 expression induced by BDL. The mRNA levels of hepatic TGF-β1 was determined by reverse transcription-PCR at 12 weeks after BDL. The numbers 1 through 12 indicate each individual animal. B: Graphic presentation of the relative abundance of TGF-β1 after normalization with actin in various groups as indicated. *, P < 0.01 versus sham control; †, P < 0.05 versus pcDNA3 control. C–E: Representative micrographs showed the localization of TGF-β1 by immunohistochemical staining in sham control (C) and BDL mice treated with either pcDNA3 (D) or pCMV-HGF (E). Strong staining for TGF-β1 was observed in bile duct epithelium, as well as in periductal fibroblast cells in the liver at 12 weeks after BDL (D). F and G: Western blot analyses showed hepatic TGF-β1 type I receptor (TβR-I) abundance at 4 (F) and 12 weeks (G) after BDL. Liver homogenates were probed with antibodies against TβR-I and actin, respectively. H–J: Representative micrographs showed the localization of TβR-I by immunohistochemical staining in sham control (H) and BDL mice treated with either pcDNA3 (I) or pCMV-HGF (J). There was strong immunostaining for TβR-I in bile duct epithelium in all three groups. Weak staining for TβR-I was also noticeable in hepatocytes.

Figure 4

Colocalization of the bile duct epithelial marker cytokeratin-19 and myofibroblast marker αSMA. A and B: Exogenous HGF inhibited hepatic expression of αSMA after BDL. Western blot analyses showed hepatic αSMA abundance at 4 (A) and 12 weeks (B) after BDL. Liver homogenates were probed with antibodies against αSMA and actin, respectively. C–K: Double immunofluorescence staining showed the localization of cytokeratin-19 (red, left column) and αSMA (green, center column) in the liver sections at 12 weeks after BDL. Merging of cytokeratin-19 and αSMA staining is presented in right column (E, H, and K). C–E, sham control; F–H, BDL liver injected with pcDNA3; I–K, BDL liver injected with pCMV-HGF. Arrowheads denote bile duct epithelia. Arrows indicate αSMA-positive hepatic arteriole. Colocalization of cytokeratin-19 and αSMA is clearly evident in the bile duct epithelium after ligation (H). L–N: Immunohistochemical staining showed the localization of αSMA in bile duct epithelium at 4 and 12 weeks after BDL, respectively. L, 4 weeks after BDL; M, 12 weeks after BDL; N, liver injected with pCMV-HGF, 4 weeks after BDL.

Figure 5

Double immunostaining shows a transition of the bile duct epithelial cells to matrix-producing interstitial cells. Liver sections were prepared from the mice receiving pcDNA3 (A–F) or pCMV-HGF (G–I) injections at 12 weeks after BDL, and stained using antibodies against cytokeratin-19 (red, left column) and hsp47 (brown, middle column). Merging of cytokeratin-19 and hsp47 staining is presented in right column (C, F, and I). Arrowheads denote a cluster of cells that retained bile duct epithelial cell appearance, but lost cytokeratin-19, and were hsp47-positive. Arrows indicate cytokeratin-19-positive cells that localized in interstitial compartment, with strong staining for hsp47. Boxed area (F) showed a cluster of bile duct epithelial cells that they were all positive for hsp47 staining, but only some of them were still positive for cytokeratin-19.

Figure 6

Double immunostaining shows the expression of type I collagen in bile duct epithelial cells after BDL. Liver sections were prepared from the mice receiving pcDNA3 (A–D) or pCMV-HGF (E–H) injections at 12 weeks after BDL and stained using antibodies against type I collagen (A and E) and cytokeratin-19 (B and F). Nuclear staining with 4′,6-diamidino-2-phenylindole, HCl was shown (C and G). Merging of the images was presented (D and H). Arrowheads denote the bile duct epithelial cells that stained positively for type I collagen. Arrows indicate the bile duct epithelial cells that were negative for type I collagen in mice receiving pCMV-HGF injections.

Figure 7

Double immunostaining demonstrates the impairment of the bile duct basement membrane and the migration of cytokeratin-19-positive cells after BDL. Liver sections were prepared from sham control (A–C), BDL mice injected with pcDNA3 (D–F) or pCMV-HGF (G–I) and stained using antibodies against cytokeratin-19 (red, left column) and laminin (green, middle column). Merging of cytokeratin-19 and laminin staining is presented in right column (C, F, and I). The integrity of bile duct basement membrane, as shown by laminin staining (arrowheads), was clearly impaired after ligation. The cytokeratin-19-positive epithelial cells could easily migrate into interstitial compartment through broken basement membrane (arrowheads in E and F).

Figure 8

TGF-β1 induces bile duct EMT. HIBEpiCs were incubated with 2 ng/ml of TGF-β1 in serum-free medium for various periods of time as indicated. Whole cell lysates were immunoblotted with antibodies against αSMA, fibronectin, cytokeratin-19, and actin, respectively. A: Representative Western blots. B: Graphic presentation of the relative abundance of αSMA and cytokeratin 19 after normalization with actin.

Figure 9

HGF blocks TGF-β1-mediated bile duct EMT. A: HIBEpiCs were incubated without (control) or with 2 ng/ml TGF-β1, 40 ng/ml HGF, or both for 3 days. Whole cell lysates were immunoblotted with antibodies against αSMA, fibronectin, cytokeratin-19, and actin, respectively. B: Western blot demonstrates that HGF blocks TGF-β1-mediated bile duct EMT in a dose-dependent fashion. Cells were treated with a fixed dose of TGF-β1 (2 ng/ml) and an increasing amount of HGF as indicated. C–N: Representative micrographs show the immunofluorescence staining for αSMA (C–F), fibronectin (G–J), and cytokeratin 19 (K–N) in bile duct epithelial cells after various treatments. Control (C, G, and K); TGF-β1, (D, H, and L); HGF (E, I, and M); and TGF-β1 plus HGF (F, J, and N).