Youngmi Jung

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.

Hedgehog-Mediated Epithelial-to-Mesenchymal Transition and Fibrogenic Repair in Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMSnRepair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors.nnnMETHODSnImmature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc(+/-)) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD.nnnRESULTSnIn cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc(+/-) mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD.nnnCONCLUSIONSnHh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis

Myofibroblastic hepatic stellate cells (MF-HSC) are derived from quiescent hepatic stellate cells (Q-HSC). Q-HSC express certain epithelial cell markers and have been reported to form junctional complexes similar to epithelial cells. We have shown that Hedgehog (Hh) signaling plays a key role in HSC growth. Because Hh ligands regulate epithelial-to-mesenchymal transition (EMT), we determined whether Q-HSC express EMT markers and then assessed whether these markers change as Q-HSC transition into MF-HSC and whether the process is modulated by Hh signaling. Q-HSC were isolated from healthy livers and cultured to promote myofibroblastic transition. Changes in mRNA and protein expression of epithelial and mesenchymal markers, Hh ligands, and target genes were monitored in HSC treated with and without cyclopamine (an Hh inhibitor). Studies were repeated in primary human HSC and clonally derived HSC from a cirrhotic rat. Q-HSC activation in vitro (culture) and in vivo (CCl(4)-induced cirrhosis) resulted in decreased expression of Hh-interacting protein (Hhip, an Hh antagonist), the EMT inhibitors bone morphogenic protein (BMP-7) and inhibitor of differentiation (Id2), the adherens junction component E-cadherin, and epithelial keratins 7 and 19 and increased expression of Gli2 (an Hh target gene) and mesenchymal markers, including the mesenchyme-associated transcription factors Lhx2 and Msx2, the myofibroblast marker alpha-smooth muscle actin, and matrix molecules such as collagen. Cyclopamine reverted myofibroblastic transition, reducing mesenchymal gene expression while increasing epithelial markers in rodent and human HSC. We conclude that Hh signaling plays a key role in transition of Q-HSC into MF-HSC. Our findings suggest that Q-HSC are capable of transitioning between epithelial and mesenchymal fates.

Fate-Mapping Evidence that Hepatic Stellate Cells are Epithelial Progenitors in Adult Mouse Livers

Liver injury activates quiescent hepatic stellate cells (Q‐HSC) to proliferative myofibroblasts. Accumulation of myofibroblastic hepatic stellate cells (MF‐HSC) sometimes causes cirrhosis and liver failure. However, MF‐HSC also promote liver regeneration by producing growth factors for oval cells, bipotent progenitors of hepatocytes and cholangiocytes. Genes that are expressed by primary hepatic stellate cell (HSC) isolates overlap those expressed by oval cells, and hepatocytic and ductular cells emerge when HSC are cultured under certain conditions. We evaluated the hypothesis that HSC are a type of oval cell and, thus, capable of generating hepatocytes to regenerate injured livers. Because Q‐HSC express glial fibrillary acidic protein (GFAP), we crossed mice in which GFAP promoter elements regulated Cre‐recombinase with ROSA‐loxP‐stop‐loxP‐green fluorescent protein (GFP) mice to generate GFAP‐Cre/GFP double‐transgenic mice. These mice were fed methionine choline‐deficient, ethionine‐supplemented diets to activate and expand HSC and oval cell populations. GFP(+) progeny of GFAP‐expressing precursors were characterized by immunohistochemistry. Basal expression of mesenchymal markers was negligible in GFAP(+)Q‐HSC. When activated by liver injury or culture, HSC downregulated expression of GFAP but remained GFP(+); they became highly proliferative and began to coexpress markers of mesenchyme and oval cells. These transitional cells disappeared as GFP‐expressing hepatocytes emerged, began to express albumin, and eventually repopulated large areas of the hepatic parenchyma. Ductular cells also expressed GFAP and GFP, but their proliferative activity did not increase in this model. These findings suggest that HSC are a type of oval cell that transitions through a mesenchymal phase before differentiating into hepatocytes during liver regeneration.

Pan‐caspase inhibitor VX‐166 reduces fibrosis in an animal model of nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is a potentially progressive liver disease that culminates in cirrhosis. Cirrhosis occurs more often in individuals with nonalcoholic steatohepatitis (NASH) than in those with steatosis (nonalcoholic fatty liver [NAFL]). The difference between NAFL and NASH is the extent of hepatocyte apoptosis, which is more extensive in NASH. Because phagocytosis of apoptotic cells activates hepatic stellate cells (HSCs), we examined the hypothesis that a pan‐caspase inhibitor, VX‐166, would reduce progression of fibrosis in a mouse model of NASH. Male db/db mice were fed methionine/choline‐deficient (MCD) diets to induce NASH and liver fibrosis. Mice were gavaged once daily with either the pan‐caspase inhibitor VX‐166 (6 mg/kg/d; Vertex, Abingdon, UK) or vehicle only and sacrificed at 4 or 8 weeks. Treatment with an MCD diet increased alanine aminotransferase (ALT), caspase‐3 activity, terminal deoxynucleotidyl transferase–mediated dUTP nick‐end labeling (TUNEL)‐positive cells, NASH, and fibrosis. Treatment of MCD‐fed mice with VX‐166 decreased active caspase‐3, TUNEL‐positive cells, and triglyceride content (P < 0.05). However, ALT levels were similar in VX‐166–treated mice and vehicle‐treated controls. Histological findings also confirmed that both groups had comparable liver injury (NAFLD activity score ≥6). Nevertheless, VX‐166–treated MCD‐fed mice demonstrated decreased α‐smooth muscle actin expression (4 weeks, P < 0.05; 8 weeks, P < 0.005) and had reduced hepatic levels of collagen 1α1 messenger RNA (8 weeks, P < 0.05). Hydroxyproline content and Sirius red staining of VX‐166–treated livers confirmed decreases in fibrosis. Conclusion: Inhibiting hepatic apoptosis suppresses the development of fibrosis in mice with NASH. Beneficial effects on liver fibrosis were associated with reductions in hepatic steatosis, but occurred without obvious improvement in liver injury. These findings are consistent with evidence that apoptosis triggers HSC activation and liver fibrosis and suggest that caspase inhibitors may be useful as an antifibrotic NASH therapy. (HEPATOLOGY 2009.)

Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease

Liver inflammation is greater in nonalcoholic steatohepatitis (NASH) than steatosis, suggesting that immune responses contribute to nonalcoholic fatty liver disease (NAFLD) progression. Livers normally contain many natural killer T (NKT) cells that produce factors that modulate inflammatory and fibrogenic responses. Such cells are relatively depleted in steatosis, but their status in more advanced NAFLD is uncertain. We hypothesized that NKT cells accumulate and promote fibrosis progression in NASH. We aimed to determine if livers become enriched with NKT cells during NASH‐related fibrosis; identify responsible mechanisms; and assess if NKT cells stimulate fibrogenesis. NKT cells were analyzed in wildtype mice and Patched‐deficient (Ptc+/−) mice with an overly active Hedgehog (Hh) pathway, before and after feeding methionine choline‐deficient (MCD) diets to induce NASH‐related fibrosis. Effects of NKT cell‐derived factors on hepatic stellate cells (HSC) were examined and fibrogenesis was evaluated in CD1d‐deficient mice that lack NKT cells. NKT cells were quantified in human cirrhotic and nondiseased livers. During NASH‐related fibrogenesis in wildtype mice, Hh pathway activation occurred, leading to induction of factors that promoted NKT cell recruitment, retention, and viability, plus liver enrichment with NKT cells. Ptc+/− mice accumulated more NKT cells and developed worse liver fibrosis; CD1d‐deficient mice that lack NKT cells were protected from fibrosis. NKT cell‐conditioned medium stimulated HSC to become myofibroblastic. Liver explants were 2‐fold enriched with NKT cells in patients with non‐NASH cirrhosis, and 4‐fold enriched in patients with NASH cirrhosis. Conclusion: Hh pathway activation leads to hepatic enrichment with NKT cells that contribute to fibrosis progression in NASH. (HEPATOLOGY 2010;)

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

Accumulation of hedgehog-responsive progenitors parallels alcoholic liver disease severity in mice and humans.

BACKGROUND & AIMSnImproving outcomes in alcoholic liver disease (ALD) necessitates better understanding of how habitual ethanol (EtOH) consumption alters normal regenerative mechanisms within the liver. Hedgehog (Hh) pathway activation promotes expansion of progenitor populations in other tissues. We evaluated the hypothesis that chronic EtOH exposure activates Hh signaling in liver.nnnMETHODSnHh signaling, liver progenitors, transforming growth factor (TGF)-beta induction, and liver damage were compared in mice fed chow, high-fat diets (HF), or HF + EtOH for 4 weeks. Susceptibility to TGF-beta-mediated apoptosis was compared in Hh-responsive liver cells (eg, immature cholangiocytes and oval cells) and mature hepatocytes (which are unresponsive to Hh). Hepatic accumulation of Hh-responsive cells were compared in controls and ALD patients and correlated with a discriminant function (DF) that predicts subacute mortality.nnnRESULTSnHh signaling and numbers of Hh-responsive cells were increased in HF mice and greatest in HF+EtOH mice. In both, progenitor and stromal cell populations harbored Hh-responsive cells. More ductular-type progenitors and fibrosis markers were noted in HF+EtOH mice than in HF mice. The former also expressed more TGF-beta-1. TGF-beta-1 treatment selectively promoted the viability of Hh-responsive immature liver cells and caused mature hepatocytes that survived to produce Hh ligands. Hh-responsive cells were increased in ALD patients. Lobular accumulation of Hh-responsive immature ductular cells was greater in those with a DF >32 than those with a DF <32.nnnCONCLUSIONSnHh signaling is increased in ALD and may influence ALD outcomes by promoting hepatic accumulation of immature ductular cells.

Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation

In bile duct-ligated (BDL) rodents, as in humans with chronic cholangiopathies, biliary obstruction triggers proliferation of bile ductular cells that are surrounded by fibrosis produced by adjacent myofibroblastic cells in the hepatic mesenchyme. The proximity of the myofibroblasts and cholangiocytes suggests that mesenchymal–epithelial crosstalk promotes the fibroproliferative response to cholestatic liver injury. Studying BDL mice, we found that bile duct obstruction induces activity of the Hedgehog (Hh) pathway, a system that regulates the viability and differentiation of various progenitors during embryogenesis. After BDL, many bile ductular cells and fibroblastic-appearing cells in the portal stroma express Hh ligands, receptor and/or target genes. Transwell cocultures of an immature cholangiocyte line that expresses the Hh receptor, Patched (Ptc), with liver myofibroblastic cells demonstrated that both cell types produced Hh ligands that enhanced each others viability and proliferation. Further support for the concept that Hh signaling modulates the response to BDL was generated by studying PtcLacZ mice, which have an impaired ability to constrain Hh signaling due to a heterozygous deficiency of Ptc. After BDL, PtcLacZ mice upregulated fibrosis gene expression earlier than wild-type controls and manifested an unusually intense ductular reaction, more expanded fibrotic portal areas, and a greater number of lobular necrotic foci. Our findings reveal that adult livers resurrect developmental signaling systems, such as the Hh pathway, to guide remodeling of the biliary epithelia and stroma after cholestatic injury.

Liver Cell–Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

BACKGROUND & AIMSnAngiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC).nnnMETHODSnMF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats.nnnRESULTSnPlatelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL.nnnCONCLUSIONSnHh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.