Yury Popov

Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies

We have made striking progress in our understanding of the biochemistry and cell biology that underlies liver fibrosis and cirrhosis, including the development of strategies and agents to prevent and reverse fibrosis. However, translation of this knowledge into clinical practice has been hampered by (1) the limitation of many in vitro and in vivo models to confirm mechanisms and to test antifibrotic agents, and (2) the lack of sensitive methodologies to quantify the degree of liver fibrosis and the dynamics of fibrosis progression or reversal in patients. Furthermore, whereas cirrhosis and subsequent decompensation are accepted hard clinical endpoints, fibrosis and fibrosis progression alone are merely plausible surrogates for future clinical deterioration. In this review we focus on an optimized strategy for preclinical antifibrotic drug development and highlight the current and future techniques that permit noninvasive assessment and quantification of liver fibrosis and fibrogenesis. The availability of such noninvasive methodologies will serve as the pacemaker for the clinical development and validation of potent antifibrotic agents. (HEPATOLOGY 2009.)

Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression.

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.

Integrin αvβ6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies

BACKGROUND/AIMS The integrin alphavbeta6 promotes proliferation of specialized epithelia and acts as a receptor for the activation of latent TGFbeta1. We studied alphavbeta6 expression in experimental and human liver fibrosis and the potential of its pharmacological inhibition for treatment of hepatic fibrosis. METHODS alphavbeta6 expression was studied by quantitative PCR and immunohistochemistry in rats with cirrhosis due to bile duct ligation (BDL), administration of thioacetamide (TAA), in Mdr2(Abcb4)(-/-) mice with spontaneous biliary fibrosis, and in livers of patients with chronic hepatitis C (n=79) and end-stage liver disease due to various etiologies (n=18). The effect of a selective alphavbeta6 inhibitor was evaluated in Mdr2(Abcb4)(-/-) mice with ongoing fibrogenesis. RESULTS Integrin beta6 mRNA increased with fibrosis stage in hepatitis C and was upregulated between 25- and 100-fold in TAA- and BDL-induced cirrhosis, in Mdr2(Abcb4)(-/-) mice and in human end-stage liver disease. alphavbeta6 protein was absent in normal livers and expressed de novo on (activated) bile duct epithelia and transitional hepatocytes. A single dose of the alphavbeta6 inhibitor injected into Mdr2(Abcb4)(-/-) mice significantly induced profibrolytic matrix metalloproteinases (MMP)-8 and -9 after 3 h, with a corresponding increase in extracellular matrix-degrading activities. In parallel profibrogenic transcripts (procollagen alpha1(I), TGFbeta2, and MMP-2) showed a trend of downregulation. CONCLUSIONS (1) Integrin alphavbeta6 is induced de novo in rodent and human liver fibrosis, where it is expressed on activated bile duct epithelia and (transitional) hepatocytes during fibrosis progression. (2) In vivo a single dose of a small molecule alphavbeta6 inhibitor induced antifibrogenic and profibrolytic genes and activities, suggesting alphavbeta6 is a unique target for treatment of liver fibrosis.

Pharmacological Inhibition of Integrin αvβ3 Aggravates Experimental Liver Fibrosis and Suppresses Hepatic Angiogenesis

The vitronectin receptor integrin αvβ3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of αvβ3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific αvβ3 (αvβ5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia‐inducible factor‐1α immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up‐regulation of profibrogenic genes and matrix metalloproteinase‐13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. αvβ3 inhibition resulted in mild liver hypoxia, as evidenced by up‐regulation of hypoxia‐inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor α, interleukin‐18, and cyclooxygenase‐2 messenger RNA indicated modest macrophage activation. Conclusion: Specific inhibition of integrin αvβ3 (αvβ5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis. (HEPATOLOGY 2009.)

Vascular endothelial growth factor promotes fibrosis resolution and repair in mice.

BACKGROUND & AIMS Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored. METHODS We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays. RESULTS VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9. CONCLUSIONS In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function.

The hedgehog pathway regulates remodelling responses to biliary obstruction in rats

Background: Chronic biliary obstruction provokes fibrosis and accumulation of immature ductular cells. This fibroductular reaction resolves following biliary decompression, suggesting that it may also be involved in the repair of biliary damage. The hedgehog (Hh) pathway becomes activated in liver after bile duct ligation (BDL), and might modulate hepatic remodelling because Hh ligands are potent morphogens. Objective: To study the induction of the Hh pathway during progression and resolution of biliary fibrosis, and to clarify whether Hh signalling regulates accumulation of bile duct progenitor cells. Design and main outcome measures: Livers from rats with BDL were examined by quantitative real-time polymerase chain reaction analysis and immunohistochemistry to identify factors that might stimulate Hh signalling. BDL rats were subjected to Roux-en-Y hepaticojejunostomy (R-Y) to relieve biliary obstruction in order to determine whether these factors and Hh signalling declined as ductular populations and concomitant fibrosis regressed. Cultures of immature ductular cells were treated with putative Hh inducers and Hh ligands to confirm their functional relevance. Results: BDL increased expression of platelet-derived growth factor-BB (PDGF-BB) and sonic hedgehog (Shh), downregulated hedgehog-interacting protein (Hip), activated Hh signalling, and expanded populations of Hh-responsive ductular cells that expressed pancyotkeratin, a liver progenitor cell marker. After R-Y, Hip remained suppressed, expression of PDGF-BB and Shh gradually declined, and populations of hedgehog-responsive ductular cells regressed. In cultured ductular cells, PDGF-BB treatment induced Shh expression, and incubation with Shh inhibited apoptotic activity. Conclusions: These results identify a mechanism for activation of the Hh pathway during cholestasis and suggest that Hh signalling regulates ductular cell accumulation after biliary injury.

Halofuginone Induces Matrix Metalloproteinases in Rat Hepatic Stellate Cells via Activation of p38 and NFκB

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen α1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor κB(NFκB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFκB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFκB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.

Fibroblast Growth Factor 21 Limits Lipotoxicity by Promoting Hepatic Fatty Acid Activation in Mice on Methionine and Choline-Deficient Diets

BACKGROUND & AIMS Nonalcoholic fatty liver disease is a common consequence of human and rodent obesity. Disruptions in lipid metabolism lead to accumulation of triglycerides and fatty acids, which can promote inflammation and fibrosis and lead to nonalcoholic steatohepatitis. Circulating levels of fibroblast growth factor (FGF)21 increase in patients with nonalcoholic fatty liver disease or nonalcoholic steatohepatitis; therefore, we assessed the role of FGF21 in the progression of murine fatty liver disease, independent of obesity, caused by methionine and choline deficiency. METHODS C57BL/6 wild-type and FGF21-knockout (FGF21-KO) mice were placed on methionine- and choline-deficient (MCD), high-fat, or control diets for 8-16 weeks. Mice were weighed, and serum and liver tissues were collected and analyzed for histology, levels of malondialdehyde and liver enzymes, gene expression, and lipid content. RESULTS The MCD diet increased hepatic levels of FGF21 messenger RNA more than 50-fold and serum levels 16-fold, compared with the control diet. FGF21-KO mice had more severe steatosis, fibrosis, inflammation, and peroxidative damage than wild-type C57BL/6 mice. FGF21-KO mice had reduced hepatic fatty acid activation and β-oxidation, resulting in increased levels of free fatty acid. FGF21-KO mice given continuous subcutaneous infusions of FGF21 for 4 weeks while on an MCD diet had reduced steatosis and peroxidative damage, compared with mice not receiving FGF21. The expression of genes that regulate inflammation and fibrosis were reduced in FGF21-KO mice given FGF21, similar to those of wild-type mice. CONCLUSIONS FGF21 regulates fatty acid activation and oxidation in livers of mice. In the absence of FGF21, accumulation of inactivated fatty acids results in lipotoxic damage and increased steatosis.

Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.