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.

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.

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.

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.

TWEAK/Fn14 signaling is required for liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. METHODS: To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. RESULTS: In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12-8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. CONCLUSIONS: TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.

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.

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.

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.

Paracrine Hedgehog signaling drives metabolic changes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) typically develops in cirrhosis, a condition characterized by Hedgehog (Hh) pathway activation and accumulation of Hh-responsive myofibroblasts. Although Hh signaling generally regulates stromal-epithelial interactions that support epithelial viability, the role of Hh-dependent myofibroblasts in hepatocarcinogenesis is unknown. Here, we used human HCC samples, a mouse HCC model, and hepatoma cell/myofibroblast cocultures to examine the hypothesis that Hh signaling modulates myofibroblasts' metabolism to generate fuels for neighboring malignant hepatocytes. The results identify a novel paracrine mechanism whereby malignant hepatocytes produce Hh ligands to stimulate glycolysis in neighboring myofibroblasts, resulting in release of myofibroblast-derived lactate that the malignant hepatocytes use as an energy source. This discovery reveals new diagnostic and therapeutic targets that might be exploited to improve the outcomes of cirrhotic patients with HCCs.

Hedgehog signaling blockade delays hepatocarcinogenesis induced by hepatitis B virus X protein.

The hepatitis B virus (HBV) encoded X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). Aberrant activation of the Hedgehog (Hh) pathway has been linked to many tumor types including HCC. Thus, experiments were designed to test the hypothesis that HBx promotes HCC via activation of Hh signaling. HBx expression correlated with an upregulation of Hh markers in human liver cancer cell lines, in liver samples from HBV infected patients with HCC, and in the livers of HBx transgenic mice (HBxTg) that develop hepatitis, steatosis, and dysplasia, culminating in the appearance of HCC. The findings in human samples provide clinical validation for the in vitro results and those in the HBxTg. Blockade of Hh signaling inhibited HBx stimulation of cell migration, anchorage-independent growth, tumor development in HBxTg, and xenograft growth in nude mice. Results suggest that the ability of HBx to promote cancer is at least partially dependent upon the activation of the Hh pathway. This study provides biologic evidence for the role of Hh signaling in the pathogenesis of HBV-mediated HCC and suggests cause and effect for the first time. The observation that inhibition of Hh signaling partially blocked the ability of HBx to promote growth and migration in vitro and tumorigenesis in two animal models implies that Hh signaling may represent an "oncogene addiction" pathway for HBV-associated HCC. This work could be central to designing specific treatments that target early development and progression of HBx-mediated HCC.

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.

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.

The role of Hedgehog signaling in fibrogenic liver repair.

Repair of adult liver, like many tissues, involves the coordinated response of a number of different cell types. In adult livers, fibroblastic cells, ductular cells, inflammatory cells, and progenitor cells contribute to this process. Our studies demonstrate that the fates of such cells are dictated, at least in part, by Hedgehog, a fetal morphogenic pathway that was once thought to be active mainly during embryogenesis. Studies of injured adult human and rodent livers demonstrate that injury-related activation of the Hedgehog pathway modulates several important aspects of repair, including the growth of hepatic progenitor populations, hepatic accumulation of myofibroblasts, repair-related inflammatory responses, vascular remodeling, liver fibrosis and hepatocarcinogenesis. These findings identify the Hedgehog pathway as a potentially important target for biomarker development and therapeutic manipulation, and emphasize the need for further research to advance knowledge about how this pathway is regulated by and interacts with other signals that regulate adult liver repair.

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.

Viral factors induce Hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma.

Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCCs) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCCs develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (ie, liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. In conclusion, HBV/HCV infection increases hepatocyte production of Hh ligands and expands the types of Hh-responsive cells that promote liver fibrosis and cancer.

Activation of Rac1 promotes hedgehog-mediated acquisition of the myofibroblastic phenotype in rat and human hepatic stellate cells.

UNLABELLED: Hepatic accumulation of myofibroblastic hepatic stellate cells (MF-HSCs) is pivotal in the pathogenesis of cirrhosis. Two events are necessary for MF-HSCs to accumulate in damaged livers: transition of resident, quiescent hepatic stellate cells (Q-HSCs) to MF-HSCs and expansion of MF-HSC numbers through increased proliferation and/or reduced apoptosis. In this study, we identified two novel mediators of MF-HSC accumulation: Ras-related C3 botulinum toxin substrate 1 (Rac1) and Hedgehog (Hh). It is unclear whether Rac1 and Hh interact to regulate the accumulation of MF-HSCs. We evaluated the hypothesis that Rac1 promotes activation of the Hh pathway, thereby stimulating signals that promote transition of Q-HSCs into MF-HSCs and enhance the viability of MF-HSCs. Using both in vitro and in vivo model systems, Rac1 activity was manipulated through adenoviral vector-mediated delivery of constitutively active or dominant-negative rac1. Rac1-transgenic mice with targeted myofibroblast expression of a mutated human rac1 transgene that produces constitutively active Rac1 were also examined. Results in all models demonstrated that activating Rac1 in HSC enhanced Hh signaling, promoted acquisition/maintenance of the MF-HSC phenotype, increased MF-HSC viability, and exacerbated fibrogenesis. Conversely, inhibiting Rac1 with dominant-negative rac1 reversed these effects in all systems examined. Pharmacologic manipulation of Hh signaling demonstrated that profibrogenic actions of Rac1 were mediated by its ability to activate Hh pathway-dependent mechanisms that stimulated myofibroblastic transition of HSCs and enhanced MF-HSC viability. CONCLUSION: These findings demonstrate that interactions between Rac1 and the Hh pathway control the size of MF-HSC populations and have important implications for the pathogenesis of cirrhosis.

Signals from dying hepatocytes trigger growth of liver progenitors.

OBJECTIVE: The death rate of mature hepatocytes is chronically increased in various liver diseases, triggering responses that prevent liver atrophy, but often cause fibrosis. Mice with targeted disruption of inhibitor kappa B kinase (Ikk) in hepatocytes (HEP mice) provide a model to investigate this process because inhibiting Ikk-nuclear factor-kappaB (NF-kappaB) signalling in hepatocytes increases their apoptosis. METHODS: Cell proliferation, apoptosis, progenitors, fibrosis and production of Hedgehog (Hh) ligands (progenitor and myofibroblast growth factors) were compared in HEP and control mice before and after feeding methionine choline-deficient ethionine-supplemented (MCDE) diets. Ikkbeta was deleted from primary hepatocytes to determine the effects on Hh ligand production; Hh signalling was inhibited directly in progenitors to determine the effects on viability. Liver sections from patients were examined to assess relationships between hepatocyte production of Hh ligands, accumulation of myofibroblastic cells and liver fibrosis. RESULTS: Disrupting the Ikk-NF-kappaB pathway in hepatocytes inhibited their proliferation but induced their production of Hh ligands. The latter provided viability signals for progenitors and myofibroblasts, enhancing accumulation of these cell types and causing fibrogenesis. Findings in the mouse models were recapitulated in diseased human livers. CONCLUSION: Dying mature hepatocytes produce Hh ligands which promote the compensatory outgrowth of progenitors and myofibroblasts. These results help to explain why diseases that chronically increase hepatocyte death promote cirrhosis.