Thiago A. Pereira

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

BACKGROUND & AIMS Repair 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. METHODS Immature 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. RESULTS In 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. CONCLUSIONS Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

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)

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.

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.

Macrophage‐derived hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni

Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury.

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

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. HEPATOLOGY 2010

Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease.

BACKGROUND & AIMS Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem cell associated factor, controls liver growth in healthy adult mice. Increasing nuclear localization of YAP triggers accumulation of reactive-appearing ductular cells (YAP+RDC) with liver progenitor capabilities. The significance of YAP activation, and mechanisms involved, are unknown in diseased livers. We evaluated the hypothesis that YAP is more activated in injured livers that are scarring than in those that are regenerating effectively. METHODS Immunohistochemistry and qRT-PCR analysis were used to localize and quantify changes in YAP and RDC in 52 patients with non-alcoholic fatty liver disease (NAFLD) and two mouse models of diet-induced non-alcoholic steatohepatitis (NASH). Results were correlated with liver disease severity, metabolic risk factors, and factors proven to control NAFLD progression. RESULTS YAP increased in NAFLD where it mainly localized in nuclei of RDC that expressed progenitor markers. Accumulation of YAP+RDC paralleled the severity of hepatocyte injury and accumulation of Sonic hedgehog, but not steatosis or metabolic risk factors. YAP+RDC expressed osteopontin, a Shh-regulated fibrogenic factor. Myofibroblast accumulation, fibrosis, and numbers of YAP+RDC strongly correlated. In murine NASH models, atrophic fibrotic livers contained significantly more YAP+RDC than livers with less severe NASH. CONCLUSION YAP+RDC promote scarring, rather than effective regeneration, during NASH.

Genetic differences in oxidative stress and inflammatory responses to diet-induced obesity do not alter liver fibrosis in mice.

Objective: To determine how genetic factors might influence the progression of nonalcoholic fatty liver disease (NAFLD).