Hedgehog-YAP Signaling Pathway Regulates Glutaminolysis to Control Activation of Hepatic Stellate Cells.

BACKGROUND & AIMS: Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP. METHODS: Primary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis. RESULTS: Expression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression. CONCLUSIONS: Glutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.

The role of external beam radiotherapy in the treatment of hepatocellular cancer.

Hepatocellular carcinoma (HCC) is increasing in incidence and mortality. Although the prognosis remains poor, long-term survival has improved from 3% in 1970 to an 18% 5-year survival rate today. This is likely because of the introduction of well tolerated, oral antiviral therapies for hepatitis C. Curative options for patients with HCC are often limited by underlying liver dysfunction/cirrhosis and medical comorbidities. Less than one-third of patients are candidates for surgery, which is the current gold standard for cure. Nonsurgical treatments include embolotherapies, percutaneous ablation, and ablative radiation. Technological advances in radiation delivery in the past several decades now allow for safe and effective ablative doses to the liver. Conformal techniques allow for both dose escalation to target volumes and normal tissue sparing. Multiple retrospective and prospective studies have demonstrated that hypofractionated image-guided radiation therapy, used as monotherapy or in combination with other liver-directed therapies, can provide excellent local control that is cost effective. Therefore, as the HCC treatment paradigm continues to evolve, ablative radiation treatment has moved from a palliative treatment to both a "bridge to transplant" and a definitive treatment.

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis.

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.

OBJECTIVE: The ductular reaction (DR) involves mobilisation of reactive-appearing duct-like cells (RDC) along canals of Hering, and myofibroblastic (MF) differentiation of hepatic stellate cells (HSC) in the space of Disse. Perivascular cells in stem cell niches produce pleiotrophin (PTN) to inactivate the PTN receptor, protein tyrosine phosphatase receptor zeta-1 (PTPRZ1), thereby augmenting phosphoprotein-dependent signalling. We hypothesised that the DR is regulated by PTN/PTPRZ1 signalling. DESIGN: PTN-GFP, PTN-knockout (KO), PTPRZ1-KO, and wild type (WT) mice were examined before and after bile duct ligation (BDL) for PTN, PTPRZ1 and the DR. RDC and HSC from WT, PTN-KO, and PTPRZ1-KO mice were also treated with PTN to determine effects on downstream signaling phosphoproteins, gene expression, growth, and migration. Liver biopsies from patients with DRs were also interrogated. RESULTS: Although quiescent HSC and RDC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was demonstrated in healthy liver. BDL induced PTN in MF-HSC and increased PTPRZ1 in MF-HSC and RDC. In WT mice, BDL triggered a DR characterised by periportal accumulation of collagen, RDC and MF-HSC. All aspects of this DR were increased in PTN-KO mice and suppressed in PTPRZ1-KO mice. In vitro studies revealed PTN-dependent accumulation of phosphoproteins that control cell-cell adhesion and migration, with resultant inhibition of cell migration. PTPRZ1-positive cells were prominent in the DRs of patients with ductal plate defects and adult cholestatic diseases. CONCLUSIONS: PTN, and its receptor, PTPRZ1, regulate the DR to liver injury by controlling the migration of resident cells in adult liver progenitor niches.

Role of Fn14 in acute alcoholic steatohepatitis in mice.

TNF-like weak inducer of apoptosis (TWEAK) is a growth factor for bipotent liver progenitors that express its receptor, fibroblast growth factor-inducible 14 (Fn14), a TNF receptor superfamily member. Accumulation of Fn14(+) progenitors occurs in severe acute alcoholic steatohepatitis (ASH) and correlates with acute mortality. In patients with severe ASH, inhibition of TNF-α increases acute mortality. The aim of this study was to determine whether deletion of Fn14 improves the outcome of liver injury in alcohol-consuming mice. Wild-type (WT) and Fn14 knockout (KO) mice were fed control high-fat Lieber deCarli diet or high-fat Lieber deCarli diet with 2% alcohol (ETOH) and injected intraperitoneally with CCl4 for 2 wk to induce liver injury. Mice were euthanized 3 or 10 days after CCl4 treatment. Survival was assessed. Liver tissues were analyzed for cell death, inflammation, proliferation, progenitor accumulation, and fibrosis by quantitative RT-PCR, immunoblot, hydroxyproline content, and quantitative immunohistochemistry. During liver injury, Fn14 expression, apoptosis, inflammation, hepatocyte replication, progenitor and myofibroblast accumulation, and fibrosis increased in WT mice fed either diet. Mice fed either diet expressed similar TWEAK/Fn14 levels, but ETOH-fed mice had higher TNF-α expression. The ETOH-fed group developed more apoptosis, inflammation, fibrosis, and regenerative responses. Fn14 deletion did not reduce hepatic TNF-α expression but improved all injury parameters in mice fed the control diet. In ETOH-fed mice, Fn14 deletion inhibited TNF-α induction and increased acute mortality, despite improvement in liver injury. Fn14 mediates wound-healing responses that are necessary to survive acute liver injury during alcohol exposure.

Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease.

UNLABELLED: Clinicians rely upon the severity of liver fibrosis to segregate patients with well-compensated nonalcoholic fatty liver disease (NAFLD) into subpopulations at high- versus low-risk for eventual liver-related morbidity and mortality. We compared hepatic gene expression profiles in high- and low-risk NAFLD patients to identify processes that distinguish the two groups and hence might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low-risk, "mild" NAFLD patients (fibrosis stage 0-1; n = 40) and high-risk, "severe" NAFLD patients (fibrosis stage 3-4; n = 32). Findings were validated in a second, independent cohort and confirmed by real-time polymerase chain reaction and immunohistochemistry (IHC). As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64-gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20-gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism-related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and IHC confirmed deregulation of metabolic and regenerative pathways in severe NAFLD and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer. CONCLUSION: By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD-related morbidity and mortality.

Schistosome-induced cholangiocyte proliferation and osteopontin secretion correlate with fibrosis and portal hypertension in human and murine schistosomiasis mansoni.

Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce osteopontin (OPN), a pro-fibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Cultured cholangiocytes, Kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and ELISA; cell proliferation was assessed by BrdU (5-bromo-2'-deoxyuridine). Mice were infected with Schistosoma mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry (IHC) and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n=72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. SEA-induced cholangiocyte proliferation and OPN secretion (P<0.001 compared with controls). Cholangiocytes were OPN (+) in Schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, P=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, P=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; P=0.001). S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.

Cross-talk between Notch and Hedgehog regulates hepatic stellate cell fate in mice.

UNLABELLED: Liver repair involves phenotypic changes in hepatic stellate cells (HSCs) and reactivation of morphogenic signaling pathways that modulate epithelial-to-mesenchymal/mesenchymal-to-epithelial transitions, such as Notch and Hedgehog (Hh). Hh stimulates HSCs to become myofibroblasts (MFs). Recent lineage tracing studies in adult mice with injured livers showed that some MFs became multipotent progenitors to regenerate hepatocytes, cholangiocytes, and HSCs. We studied primary HSC cultures and two different animal models of fibrosis to evaluate the hypothesis that activating the Notch pathway in HSCs stimulates them to become (and remain) MFs through a mechanism that involves an epithelial-to-mesenchymal-like transition and requires cross-talk with the canonical Hh pathway. We found that when cultured HSCs transitioned into MFs, they activated Hh signaling, underwent an epithelial-to-mesenchymal-like transition, and increased Notch signaling. Blocking Notch signaling in MFs/HSCs suppressed Hh activity and caused a mesenchymal-to-epithelial-like transition. Inhibiting the Hh pathway suppressed Notch signaling and also induced a mesenchymal-to-epithelial-like transition. Manipulating Hh and Notch signaling in a mouse multipotent progenitor cell line evoked similar responses. In mice, liver injury increased Notch activity in MFs and Hh-responsive MF progeny (i.e., HSCs and ductular cells). Conditionally disrupting Hh signaling in MFs of bile-duct-ligated mice inhibited Notch signaling and blocked accumulation of both MF and ductular cells. CONCLUSIONS: The Notch and Hedgehog pathways interact to control the fate of key cell types involved in adult liver repair by modulating epithelial-to-mesenchymal-like/mesenchymal-to-epithelial-like transitions.

Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication.

OPN (osteopontin)) is a Hh (Hedgehog)-regulated cytokine that is up-regulated during chronic liver injury and directly promotes fibrosis. We have reported that Hh signalling enhances viral permissiveness and replication in HCV (hepatitis C virus)-infected cells. Hence we hypothesized that OPN directly promotes HCV replication, and that targeting OPN could be beneficial in HCV. In the present study, we compared the expression of OPN mRNA and protein in HCV (JFH1)-infected Huh7 and Huh7.5 cells, and evaluated whether modulating OPN levels using exogenous OPN ligands (up-regulate OPN) or OPN-specific RNA-aptamers (neutralize OPN) leads to changes in HCV expression. Sera and livers from patients with chronic HCV were analysed to determine whether OPN levels were associated with disease severity or response to therapy. Compared with Huh7 cells, Huh7.5 cells support higher levels of HCV replication (15-fold) and expressed significantly more OPN mRNA (30-fold) and protein. Treating Huh7 cells with OPN ligands led to a dose-related increase in HCV (15-fold) and OPN (8-fold) mRNA. Conversely, treating Huh7.5 cells with OPN-specific RNA aptamers inhibited HCV RNA and protein by >50% and repressed OPN mRNA to basal levels. Liver OPN expression was significantly higher (3-fold) in patients with advanced fibrosis. Serum OPN positively correlated with fibrosis-stage (P=0.009), but negatively correlated with ETBCR (end-of-treatment biochemical response), ETVR (end-of-treatment virological response), SBCR (sustained biochemical response) and SVR (sustained virological response) (P=0.007). The OPN fibrosis score (serum OPN and presence of fibrosis ≥F2) may be a predictor of SVR. In conclusion, OPN is up-regulated in the liver and serum of patients with chronic hepatitis C, and supports increased viral replication. OPN neutralization may be a novel therapeutic strategy in chronic hepatitis C.

Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation.

OBJECTIVE: Vascular remodelling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signalling regulates vascular development and increases during liver injury. This study therefore examined its role in capillarisation. DESIGN: Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacological, antibody-mediated and genetic approaches were used to manipulate Hh signalling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by quantitative reverse transcription PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry. RESULTS: Freshly isolated LSEC expressed Hh ligands, Hh receptors and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and upregulated capillarisation-associated genes; whereas Hh signalling antagonist or Hh ligand neutralising antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from Smo(loxP/loxP) transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signalling, less induction of capillarisation-associated genes and retention of fenestrae. In mice with injured livers, inhibiting Hh signalling prevented capillarisation. CONCLUSIONS: LSEC produce and respond to Hh ligands, and use Hh signalling to regulate complex phenotypic changes that occur during capillarisation.

Hedgehog controls hepatic stellate cell fate by regulating metabolism.

BACKGROUND & AIMS: The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism. METHODS: Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity. RESULTS: Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh. CONCLUSIONS: Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.

Macrophage-derived Hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni.

BACKGROUND: Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. AIMS: Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. METHODS: Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated using qRT-PCR. Inhibition of the Hedgehog pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. RESULTS: Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog responsive [Gli2(+)] and accumulated in parallel with fibrosis stage (P < 0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA-stimulated macrophages to express Ihh and Shh mRNA (P < 0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (P < 0.001) and Hedgehog inhibitors blocked this effect (P < 0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh pathway inhibitors abrogated this response (P < 0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. CONCLUSION: SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. DESIGN: The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. RESULTS: When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18-/- and CD1d-/- mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. CONCLUSION: Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.

Pancreatic secretory trypsin inhibitor I reduces the severity of chronic pancreatitis in mice overexpressing interleukin-1ß in the pancreas.

IL-1ß is believed to play a pathogenic role in the development of pancreatitis. Expression of human IL-1ß in pancreatic acinar cells produces chronic pancreatitis, characterized by extensive intrapancreatic inflammation, atrophy, and fibrosis. To determine if activation of trypsinogen is important in the pathogenesis of chronic pancreatitis in this model, we crossed IL-1ß transgenic [Tg(IL1ß)] mice with mice expressing a trypsin inhibitor that is normally produced in rat pancreatic acinar cells [pancreatic secretory trypsin inhibitor (PTSI) I]. We previously demonstrated that transgenic expression of PSTI-I [Tg(Psti1)] increased pancreatic trypsin inhibitor activity by 190%. Tg(IL1ß) mice were found to have marked pancreatic inflammation, characterized by histological changes, including acinar cell loss, inflammatory cell infiltration, and fibrosis, as well as elevated myeloperoxidase activity and elevated pancreatic trypsin activity, as early as 6 wk of age. In contrast to Tg(IL1ß) mice, pancreatitis was significantly less severe in dual-transgenic [Tg(IL1ß)-Tg(Psti1)] mice expressing IL-1ß and PSTI-I in pancreatic acinar cells. These findings indicate that overexpression of PSTI-I reduces the severity of pancreatitis and that pancreatic trypsin activity contributes to the pathogenesis of an inflammatory model of chronic pancreatitis.

Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication.

UNLABELLED: Studies of the hepatitis C virus (HCV) life-cycle rely heavily on Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh-related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here we demonstrate that Huh7.5 cells, when compared with Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers, and markedly up-regulated Hh pathway activity: Shh, >100-fold, Gli1, >30-fold, Ptc, 2-fold. In Huh7.5 cells, we found that cyclopamine, an Hh pathway antagonist, reduced HCV RNA levels by 50% compared with vehicle and inactive isomer controls. Moreover, in Huh7 cells treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2-fold and 4-fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose-response manner. CONCLUSION: We have identified a relationship between HCV and Hh signaling where up-regulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high-level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.

Hedgehog activity, epithelial-mesenchymal transitions, and biliary dysmorphogenesis in biliary atresia.

UNLABELLED: Biliary atresia (BA) is notable for marked ductular reaction and rapid development of fibrosis. Activation of the Hedgehog (Hh) pathway promotes the expansion of populations of immature epithelial cells that coexpress mesenchymal markers and may be profibrogenic. We examined the hypothesis that in BA excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Livers and remnant extrahepatic ducts from BA patients were evaluated by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and immunostaining for Hh ligands, target genes, and markers of mesenchymal cells or ductular progenitors. Findings were compared to children with genetic cholestatic disease, age-matched deceased donor controls, and adult controls. Ductular cells isolated from adult rats with and without bile duct ligation were incubated with Hh ligand-enriched medium ± Hh-neutralizing antibody to determine direct effects of Hh ligands on epithelial to mesenchymal transition (EMT) marker expression. Livers from pediatric controls showed greater innate Hh activation than adult controls. In children with BA, both intra- and extrahepatic ductular cells demonstrated striking up-regulation of Hh ligand production and increased expression of Hh target genes. Excessive accumulation of Hh-producing cells and Hh-responsive cells also occurred in other infantile cholestatic diseases. Further analysis of the BA samples demonstrated that immature ductular cells with a mesenchymal phenotype were Hh-responsive. Treating immature ductular cells with Hh ligand-enriched medium induced mesenchymal genes; neutralizing Hh ligands inhibited this. CONCLUSION: BA is characterized by excessive Hh pathway activity, which stimulates biliary EMT and may contribute to biliary dysmorphogenesis. Other cholestatic diseases show similar activation, suggesting that this is a common response to cholestatic injury in infancy.

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

UNLABELLED: 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.

Leptin promotes the myofibroblastic phenotype in hepatic stellate cells by activating the hedgehog pathway.

Trans-differentiation of quiescent hepatic stellate cells (Q-HSCs), which exhibit epithelial and adipocytic features, into myofibroblastic-HSC (MF-HSCs) is a key event in liver fibrosis. Culture models demonstrated that Hedgehog (Hh) pathway activation is required for transition of epithelioid/adipocytic Q-HSCs into MF-HSCs. Hh signaling inhibits adiposity and promotes epithelial-to-mesenchymal transitions (EMTs). Leptin (anti-adipogenic, pro-EMT factor) promotes HSC trans-differentiation and liver fibrosis, suggesting that the pathways may interact to modulate cell fate. This study aimed to determine whether leptin activates Hh signaling and whether this is required for the fibrogenic effects of leptin. Cultures of primary HSCs from lean and fa/fa rats with an inherited ObRb defect were examined. Inhibitors of PI3K/Akt, JAK/STAT, and Hh signaling were used to delineate how ObRb activation influenced Hh signaling and HSC trans-differentiation. Fibrogenesis was compared in wild type and db/db mice (impaired ObRb function) to assess the profibrotic role of leptin. The results demonstrate that leptin-ObR interactions activate Hh signaling with the latter necessary to promote trans-differentiation. Leptin-related increases in Hh signaling required ObR induction of PI3K/Akt, which was sufficient for leptin to repress the epithelioid/adipocytic program. Leptin-mediated induction of JAK/STAT was required for mesenchymal gene expression. Leptin-ObRb interactions were not necessary for HSC trans-differentiation to occur in vitro or in vivo but are important because liver fibrogenesis was attenuated in db/db mice. These findings reveal that leptin activates Hh signaling to alter gene expression programs that control cell fate and have important implications for liver fibrosis and other leptin-regulated processes involving EMTs, including development, obesity, and cancer metastasis.