Thiago De Almeida Pereira

All Publications

• Serum osteopontin is a biomarker of severe fibrosis and portal hypertension in human and murine schistosomiasis mansoni INTERNATIONAL JOURNAL FOR PARASITOLOGY Pereira, T. A., Syn, W., Pereira, F. E., Lambertucci, J. R., Secor, W. E., Diehl, A. M. 2016; 46 (13-14): 829-832

  Abstract

  Schistosomiasis is a major cause of fibrosis and portal hypertension. The reason 4-10% of infected subjects develops hepatosplenic schistosomiasis remains unclear. Chronically infected male CBA/J mice reproduce the dichotomic forms of human schistosomiasis. Most mice (80%) develop moderate splenomegaly syndrome (similar to hepatointestinal disease in humans) and 20% present severe hypersplenomegaly syndrome (analogous to human hepatosplenic disease). We demonstrated that the profibrogenic molecule osteopontin discriminates between mice with severe and mild disease and could be a novel morbidity biomarker in murine and human schistosomiasis. Failure to downregulate osteopontin during the chronic phase may explain why hepatosplenic subjects develop severe fibrosis.

  View details for DOI 10.1016/j.ijpara.2016.08.004

  View details for Web of Science ID 000390726800001

  View details for PubMedID 27729270

  View details for PubMedCentralID PMC5584370

• Osteopontin Is Upregulated in Human and Murine Acute Schistosomiasis Mansoni PLOS NEGLECTED TROPICAL DISEASES Pereira, T. A., Syn, W., Amancio, F. F., Diniz Cunha, P. H., Morais Caporali, J. F., de Melo Trindade, G. V., Santos, E. T., Souza, M. M., Andrade, Z. A., Witek, R. P., Secor, W. E., Lima Pereira, F. E., Lambertucci, J. R., Diehl, A. M. 2016; 10 (10)

  Abstract

  Symptomatic acute schistosomiasis mansoni is a systemic hypersensitivity reaction against the migrating schistosomula and mature eggs after a primary infection. The mechanisms involved in the pathogenesis of acute schistosomiasis are not fully elucidated. Osteopontin has been implicated in granulomatous reactions and in acute hepatic injury. Our aims were to evaluate if osteopontin plays a role in acute Schistosoma mansoni infection in both human and experimentally infected mice and if circulating OPN levels could be a novel biomarker of this infection.Serum/plasma osteopontin levels were measured by ELISA in patients with acute (n = 28), hepatointestinal (n = 26), hepatosplenic (n = 39) schistosomiasis and in uninfected controls (n = 21). Liver osteopontin was assessed by immunohistochemistry in needle biopsies of 5 patients. Sera and hepatic osteopontin were quantified in the murine model of schistosomiasis mansoni during acute (7 and 8 weeks post infection, n = 10) and chronic (30 weeks post infection, n = 8) phase. Circulating osteopontin levels are increased in patients with acute schistosomiasis (p = 0.0001). The highest levels of OPN were observed during the peak of clinical symptoms (7-11 weeks post infection), returning to baseline level once the granulomas were modulated (>12 weeks post infection). The plasma levels in acute schistosomiasis were even higher than in hepatosplenic patients. The murine model mirrored the human disease. Macrophages were the major source of OPN in human and murine acute schistosomiasis, while the ductular reaction maintains OPN production in hepatosplenic disease. Soluble egg antigens from S. mansoni induced OPN expression in primary human kupffer cells.S. mansoni egg antigens induce the production of OPN by macrophages in the necrotic-exudative granulomas characteristic of acute schistosomiasis mansoni. Circulating OPN levels are upregulated in human and murine acute schistosomiasis and could be a non-invasive biomarker of this form of disease.

  View details for DOI 10.1371/journal.pntd.0005057

  View details for Web of Science ID 000386676200038

  View details for PubMedID 27755536

  View details for PubMedCentralID PMC5068698

• Macrophages and endothelial cells orchestrate tumor-associated angiogenesis in oral cancer via hedgehog pathway activation TUMOR BIOLOGY Valverde, L. d., Pereira, T. d., Dias, R. B., Nazare Guimaraes, V. S., Goncalves Ramos, E. A., Santos, J. N., Gurgel Rocha, C. A. 2016; 37 (7): 9233-9241

  Abstract

  The present study aimed to evaluate the role of Hedgehog (Hh) molecule expression in association with the clinical aspects of oral squamous cell carcinoma (OSCC), as well as angiogenesis and CD163+ macrophages. Twenty-eight cases of OSCC, nine cases of tumor-free resection margins (TM), and four cases of non-neoplastic oral mucosa (NNM) were submitted to immunohistochemistry to detect proteins Sonic Hedgehog (SHH), Indian Hedgehog (IHH), GLI1, CD163, and CD105. Protein colocalization with respect to SHH/CD163, IHH/CD163, GLI1/CD163, and GLI1/CD105 was assessed by immunohistochemical double staining. In tumor parenchyma, SHH and IHH were present in the cytoplasm of neoplastic cells, while GLI1 was observed in cytoplasm and nucleus. Endothelial cells were found to express SHH, IHH, and GLI1 within CD105+ vessels, and a positive correlation between infiltrating macrophage density (IMD) and microvascular density (MVD) was observed in cases of OSCC and TM. When compared to TM and NNM, the OSCC cases demonstrated higher immunoreactivity for SHH (p = 0.01), IHH (p = 0.39), GLI1 (p = 0.03), IMD (p = 0.0002), and MVD (p = 0.0002). Our results suggest the participation of the Hh pathway in OSCC by way of autocrine and paracrine signaling, in addition to the participation of both SHH and IHH ligands. Endothelial cells were also found to exhibit positivity with respect to Hh pathway components and we surmise that these molecules may play a role in tumor angiogenesis. CD163+ macrophages were also observed to express IHH, a ligand of this pathway, in addition to being associated with tumor neovascularization.

  View details for DOI 10.1007/s13277-015-4763-6

  View details for Web of Science ID 000382174500074

  View details for PubMedID 26768620

• Schistosome-induced cholangiocyte proliferation and osteopontin secretion correlate with fibrosis and portal hypertension in human and murine schistosomiasis mansoni CLINICAL SCIENCE Pereira, T. A., Syn, W., Machado, M. V., Vidigal, P. V., Resende, V., Voieta, I., Xie, G., Otoni, A., Souza, M. M., Santos, E. T., Chan, I. S., Trindade, G. V., Choi, S. S., Witek, R. P., Pereira, F. E., Secor, W. E., Andrade, Z. A., Lambertucci, J. R., Diehl, A. M. 2015; 129 (10): 875-883

  Abstract

  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.

  View details for DOI 10.1042/CS20150117

  View details for Web of Science ID 000361048900004

  View details for PubMedID 26201095

  View details for PubMedCentralID PMC4558314

• Macrophage-derived hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni LIVER INTERNATIONAL Pereira, T. A., Xie, G., Choi, S. S., Syn, W., Voieta, I., Lu, J., Chan, I. S., Swiderska, M., Amaral, K. B., Antunes, C. M., Secor, W. E., Witek, R. P., Lambertucci, J. R., Pereira, F. L., Diehl, A. M. 2013; 33 (1): 149-161

  Abstract

  Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury.Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved.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.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.SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.

  View details for DOI 10.1111/liv.12016

  View details for Web of Science ID 000312302800020

  View details for PubMedID 23121638

  View details for PubMedCentralID PMC3518740

• Viral factors induce Hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma LABORATORY INVESTIGATION Pereira, T. d., Witek, R. P., Syn, W., Choi, S. S., Bradrick, S., Karaca, G. F., Agboola, K. M., Jung, Y., Omenetti, A., Moylan, C. A., Yang, L., Fernandez-Zapico, M. E., Jhaveri, R., Shah, V. H., Pereira, F. E., Diehl, A. M. 2010; 90 (12): 1690-1703

  Abstract

  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.

  View details for DOI 10.1038/labinvest.2010.147

  View details for Web of Science ID 000284751600001

  View details for PubMedID 20697376

  View details for PubMedCentralID PMC2980808

• Anti-IL-13Rα2 therapy promotes recovery in a murine model of inflammatory bowel disease Mucosal Immunology Karmele, E., Pasricha, T., Ramalingam, T., Thompson, R., Gieseck, R., Knilans, K., Hegen, M., Farmer, M., Jin, F., Kleinman, A., Hinds, D., The 23andMe Research Team , Pereira, T. A., Prado, R. Q., Bing, N., Tchistiakova, L., Kasaian, M., Wynn, T. A., Vannella, K. M. 2019

  View details for DOI 10.1038/s41385-019-0189-6

• TARGETING THE HEDGEHOG PATHWAY IS A NOVEL THERAPEUTIC STRATEGY TO TREAT SCHISTOSOMIASIS FIBROSIS AND PORTAL HYPERTENSION Pereira, T., Vidigal, P., Voieta, I., Resende, V., Witek, R., Jegga, A., Arron, J., Madala, S., Lambertucci, J., Diehl, A., Wynn, T., Beachy, P. AMER SOC TROP MED & HYGIENE. 2019: 10–11

  View details for Web of Science ID 000507364502033

• Sensitivity and specificity of the circulating cathodic antigen rapid urine test in the diagnosis of Schistosomiasis mansoni infection and evaluation of morbidity in a low- endemic area in Brazil. Revista da Sociedade Brasileira de Medicina Tropical Ferreira, F. T., Fidelis, T. A., Pereira, T. A., Otoni, A., Queiroz, L. C., Amâncio, F. F., Antunes, C. M., Lambertucci, J. R. ; 50 (3): 358–64

  Abstract

  The Kato-Katz technique is the standard diagnostic test for Schistosoma mansoni infection in rural areas. However, the utility of this method is severely limited by the day-to-day variability in host egg excretion in the stool. In high-transmission areas, the point-of-care circulating cathodic antigen (POC-CCA) urine assay has proven to be a reliable test. However, investigations of the reliability of the POC-CCA assay in low-transmission regions are under way. This study aimed to evaluate the sensitivity and specificity of the POC-CCA assay and the morbidity of schistosomiasis in a low-endemic area in Brazil.Pains City is a low-transmission zone for schistosomiasis. A total of 300 subjects aged 7-76 years were randomly selected for the POC-CCA cassette test. For S. mansoni diagnosis, three stool samples on six slides were compared with one urine sample for each subject. The sensitivity and specificity in the absence of a gold standard were calculated using latent class analysis. Clinical examinations and abdominal ultrasounds were performed in 181 volunteers to evaluate morbidity associated with schistosomiasis.The sensitivity and specificity of the Kato-Katz technique were 25.6% and 94.6%, respectively. By contrast, the sensitivity and specificity of the POC-CCA assay were 68.1% and 72.8%, respectively. Hepatosplenic schistosomiasis was diagnosed in two patients (1.1%).Overall, the POC-CCA urine assay proved to be a useful test for diagnosing S. mansoni in a low-endemic area in Brazil. Severe clinical forms of schistosomiasis can be present even in such low-endemic areas.

  View details for DOI 10.1590/0037-8682-0423-2016

  View details for PubMedID 28700054

• Emerging Role of HMGB1 in the Pathogenesis of Schistosomiasis Liver Fibrosis. Frontiers in immunology Vicentino, A. R., Carneiro, V. C., Allonso, D., Guilherme, R. d., Benjamim, C. F., Dos Santos, H. A., Xavier, F., Pyrrho, A. D., Gomes, J. d., Fonseca, M. d., de Oliveira, R. C., Pereira, T. A., Ladislau, L., Lambertucci, J. R., Fantappié, M. R. 2018; 9: 1979

  Abstract

  In chronic schistosomiasis, liver fibrosis is linked to portal hypertension, which is a condition associated with high mortality and morbidity. High mobility group box 1 (HMGB1) was originally described as a nuclear protein that functions as a structural co-factor in transcriptional regulation. However, HMGB1 can also be secreted into the extracellular milieu under appropriate signal stimulation. Extracellular HMGB1 acts as a multifunctional cytokine that contributes to infection, injury, inflammation, and immune responses by binding to specific cell-surface receptors. HMGB1 is involved in fibrotic diseases. From a clinical perspective, HMGB1 inhibition may represent a promising therapeutic approach for treating tissue fibrosis. In this study, we demonstrate elevated levels of HMGB1 in the sera in experimental mice or in patients with schistosomiasis. Using immunohistochemistry, we demonstrated that HMGB1 trafficking in the hepatocytes of mice suffering from acute schistosomiasis was inhibited by Glycyrrhizin, a well-known HMGB1 direct inhibitor, as well as by DIC, a novel and potential anti-HMGB1 compound. HMGB1 inhibition led to significant downregulation of IL-6, IL4, IL-5, IL-13, IL-17A, which are involved in the exacerbation of the immune response and liver fibrogenesis. Importantly, infected mice that were treated with DIC or GZR to inhibit HMGB1 pro-inflammatory activity showed a significant increase in survival and a reduction of over 50% in the area of liver fibrosis. Taken together, our findings indicate that HMGB1 is a key mediator of schistosomotic granuloma formation and liver fibrosis and may represent an outstanding target for the treatment of schistosomiasis.

  View details for PubMedID 30258438

  View details for PubMedCentralID PMC6143665

• Bone marrow-derived monocyte infusion improves hepatic fibrosis by decreasing osteopontin, TGF-β1, IL-13 and oxidative stress. World journal of gastroenterology de Souza, V. C., Pereira, T. A., Teixeira, V. W., Carvalho, H., de Castro, M. C., D'assunção, C. G., de Barros, A. F., Carvalho, C. L., de Lorena, V. M., Costa, V. M., Teixeira, Á. A., Figueiredo, R. C., de Oliveira, S. A. 2017; 23 (28): 5146–57

  Abstract

  To evaluate the therapeutic effects of bone marrow-derived CD11b+CD14+monocytes in a murine model of chronic liver damage.Chronic liver damage was induced in C57BL/6 mice by administration of carbon tetrachloride and ethanol for 6 mo. Bone marrow-derived monocytes isolated by immunomagnetic separation were used for therapy. The cell transplantation effects were evaluated by morphometry, biochemical assessment, immunohistochemistry and enzyme-linked immunosorbent assay.CD11b+CD14+monocyte therapy significantly reduced liver fibrosis and increased hepatic glutathione levels. Levels of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6 and IL-1β, in addition to pro-fibrotic factors, such as IL-13, transforming growth factor-β1 and tissue inhibitor of metalloproteinase-1 also decreased, while IL-10 and matrix metalloproteinase-9 increased in the monocyte-treated group. CD11b+CD14+monocyte transplantation caused significant changes in the hepatic expression of α-smooth muscle actin and osteopontin.Monocyte therapy is capable of bringing about improvement of liver fibrosis by reducing oxidative stress and inflammation, as well as increasing anti-fibrogenic factors.

  View details for DOI 10.3748/wjg.v23.i28.5146

  View details for PubMedID 28811709

  View details for PubMedCentralID PMC5537181

• Thrombocytopenia as a marker of liver steatosis in a low-endemic area for schistosomiasis mansoni. Revista da Associacao Medica Brasileira (1992) Otoni, A., Antunes, C. a., Tavares, F. F., Araújo, D. H., Pereira, T. d., Queiroz, L. C., Amâncio, F. F., Lambertucci, J. R. 2017; 63 (6): 532–37

  Abstract

  Thrombocytopenia is commonly found in patients living in highly endemic areas for Schistosoma mansoni. Recently, different degrees of liver steatosis have also been associated with low platelet counts worldwide. We investigated the association of platelet counts with hepatosplenic schistosomiasis and with liver steatosis in an area of low prevalence of schistosomiasis in Brazil.Pains, a city in the state of Minas Gerais, Brazil, had a population of 8,307 inhabitants and a schistosomiasis prevalence of 8%. Four micro-areas comprising 1,045 inhabitants were selected for this study. Blood sample was collected and a complete blood count (CBC) was performed. Eighty-seven (87) patients had low platelet counts (group 1 - 8.3%) and 94 volunteers presenting normal CBC were randomized (group 2 - 8.9%). They underwent clinical and ultrasound examinations. Liver steatosis was determined as either present or absent using abdominal ultrasound. A spleen > 12 cm in length, measured by ultrasound (US), was considered to be increased. Data collected were analyzed using SPSS software version 19.0.Twenty-two patients (22/25.3%) in group 1 had liver steatosis compared with 11 volunteers (11.7%) in group 2 (p=0.02). Hepatosplenic schistosomiasis was diagnosed in two patients (p>0.05).Thrombocytopenia was not a good marker of hepatosplenic schistosomiasis mansoni in a low prevalence area in Brazil. Liver steatosis was associated with thrombocytopenia in our study.

  View details for DOI 10.1590/1806-9282.63.06.532

  View details for PubMedID 28876430

• MCM3: A Novel Proliferation Marker in Oral Squamous Cell Carcinoma. Applied immunohistochemistry & molecular morphology : AIMM Valverde, L. d., de Freitas, R. D., Pereira, T. d., de Resende, M. F., Agra, I. M., dos Santos, J. N., Dos Reis, M. G., Sales, C. B., Gurgel Rocha, C. A. 2016: -?

  Abstract

  The present study sought to evaluate and compare the immunoexpression of proteins minichromosome maintenance (MCM) 3 and Ki-67 in oral squamous cell carcinoma (OSCC) to assess the potential of these proteins as markers of cellular proliferation. Twenty-eight cases of OSCC, 9 of tumor-free resection margins (TM), and 4 of non-neoplastic oral mucosa (NNM) were subjected to immunohistochemistry to detect the expression of proteins MCM3 and Ki-67. All OSCCs demonstrated positivity for both proteins. In these tumors, greater MCM3 immunoreactivity was observed in comparison with Ki-67, whereas TMs and NNMs exhibited greater Ki-67 expression compared with MCM3. The immunoexpression of Ki-67 seemed to be influenced by the inflammatory process, particularly in TM and NNM. Our findings indicate that although both MCM3 and Ki-67 represent reliable markers of cellular proliferation in OSCC, as MCM3 expression does not appear to be influenced by external factors, this protein may emerge as a novel marker of cellular proliferation in these types of tumors.

  View details for PubMedID 27258565

• Correction: Vestibular Evoked Myogenic Potential (VEMP) Triggered by Galvanic Vestibular Stimulation (GVS): A Promising Tool to Assess Spinal Cord Function in Schistosomal Myeloradiculopathy. PLoS neglected tropical diseases Caporali, J. F., Gonçalves, D. U., Labanca, L., de Oliveira, L. D., Trindade, G. V., Pereira, T. d., Cunha, P. H., Mourão, M. S., Lambertucci, J. R. 2016; 10 (5)

  Abstract

  [This corrects the article DOI: 10.1371/journal.pntd.0004672.].

  View details for DOI 10.1371/journal.pntd.0004763

  View details for PubMedID 27232501

  View details for PubMedCentralID PMC4884095

• Caspase-2 promotes obesity, the metabolic syndrome and nonalcoholic fatty liver disease CELL DEATH & DISEASE Machado, M. V., Michelotti, G. A., Jewell, M. L., Pereira, T. A., Xie, G., Premont, R. T., Diehl, A. M. 2016; 7

  Abstract

  Obesity and its resulting metabolic disturbances are major health threats. In response to energy surplus, overtaxed adipocytes release fatty acids and pro-inflammatory factors into the circulation, promoting organ fat accumulation (including nonalcoholic fatty liver disease), insulin resistance and the metabolic syndrome. Recently, caspase-2 was linked to lipoapoptosis, so we hypothesized that caspase-2 might be a critical determinant of metabolic syndrome pathogenesis. Caspase-2-deficient and wild-type mice were fed a Western diet (high-fat diet, enriched with saturated fatty acids and 0.2% cholesterol, supplemented with fructose and glucose in the drinking water) for 16 weeks. Metabolic and hepatic outcomes were evaluated. In vitro studies assessed the role of caspase-2 in adipose tissue proliferative properties and susceptibility for lipoapoptosis. Caspase-2-deficient mice fed a Western diet were protected from abdominal fat deposition, diabetes mellitus, dyslipidemia and hepatic steatosis. Adipose tissue in caspase-2-deficient mice was more proliferative, upregulated mitochondrial uncoupling proteins consistent with browning, and was resistant to cell hypertrophy and cell death. The liver was protected from steatohepatitis through a decrease in circulating fatty acids and more efficient hepatic fat metabolism, and from fibrosis as a consequence of reduced fibrogenic stimuli from fewer lipotoxic hepatocytes. Caspase-2 deficiency protected mice from diet-induced obesity, metabolic syndrome and nonalcoholic fatty liver disease. Further studies are necessary to assess caspase-2 as a therapeutic target for those conditions.

  View details for DOI 10.1038/cddis.2016.19

  View details for Web of Science ID 000379791300008

  View details for PubMedID 26890135

  View details for PubMedCentralID PMC5399190

• Vitamin B5 and N-Acetylcysteine in Nonalcoholic Steatohepatitis: A Preclinical Study in a Dietary Mouse Model DIGESTIVE DISEASES AND SCIENCES Machado, M. V., Kruger, L., Jewell, M. L., Michelotti, G. A., Pereira, T. d., Xie, G., Moylan, C. A., Diehl, A. M. 2016; 61 (1): 137-148

  Abstract

  Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation.We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH.CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH.Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity and correlated with worse liver cell apoptosis, inflammation, and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice.In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH.

  View details for DOI 10.1007/s10620-015-3871-x

  View details for Web of Science ID 000367609300020

  View details for PubMedID 26403427

  View details for PubMedCentralID PMC4703517

• In Vivo MRI Assessment of Experimental Schistosomiasis TRENDS IN PARASITOLOGY Lambertucci, J. R., Mamede, M., Pereira, T. A. 2016; 32 (1): 3-5

  Abstract

  A new study using magnetic resonance imaging (MRI) evaluated successfully the transverse relaxation time T2 as a non-invasive imaging biomarker for monitoring hepatic fibrogenesis in schistosomiasis. However, there are some drawbacks that need special attention. This preliminary data opens new opportunities to understand and monitor liver fibrosis in schistosomiasis and other fibrogenic diseases.

  View details for DOI 10.1016/j.pt.2015.11.003

  View details for Web of Science ID 000368206900003

  View details for PubMedID 26615712

• Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease JOURNAL OF HEPATOLOGY Machado, M. V., Michelotti, G. A., Pereira, T. A., Xie, G., Premont, R., Cortez-Pinto, H., Diehl, A. M. 2015; 63 (4): 962-970

  Abstract

  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.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.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.YAP+RDC promote scarring, rather than effective regeneration, during NASH.

  View details for Web of Science ID 000361729900025

  View details for PubMedID 26070409

  View details for PubMedCentralID PMC4575842

• Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis GUT Machado, M. V., Michelotti, G. A., Pereira, T. d., Boursier, J., Kruger, L., Swiderska-Syn, M., Karaca, G., Xie, G., Guy, C. D., Bohinc, B., Lindblom, K. R., JOHNSON, E., Kornbluth, S., Diehl, A. M. 2015; 64 (7): 1148-U226

  Abstract

  Caspase-2 is an initiator caspase involved in multiple apoptotic pathways, particularly in response to specific intracellular stressors (eg, DNA damage, ER stress). We recently reported that caspase-2 was pivotal for the induction of cell death triggered by excessive intracellular accumulation of long-chain fatty acids, a response known as lipoapoptosis. The liver is particularly susceptible to lipid-induced damage, explaining the pandemic status of non-alcoholic fatty liver disease (NAFLD). Progression from NAFLD to non-alcoholic steatohepatitis (NASH) results, in part, from hepatocyte apoptosis and consequential paracrine-mediated fibrogenesis. We evaluated the hypothesis that caspase-2 promotes NASH-related cirrhosis.Caspase-2 was localised in liver biopsies from patients with NASH. Its expression was evaluated in different mouse models of NASH, and outcomes of diet-induced NASH were compared in wild-type (WT) and caspase-2-deficient mice. Lipotoxicity was modelled in vitro using hepatocytes derived from WT and caspase-2-deficient mice.We showed that caspase-2 is integral to the pathogenesis of NASH-related cirrhosis. Caspase-2 is localised in injured hepatocytes and its expression was markedly upregulated in patients and animal models of NASH. During lipotoxic stress, caspase-2 deficiency reduced apoptosis, inhibited induction of profibrogenic hedgehog target genes in mice and blocked production of hedgehog ligands in cultured hepatocytes.These data point to a critical role for caspase-2 in lipid-induced hepatocyte apoptosis in vivo for the production of apoptosis-associated fibrogenic factors and in the progression of lipid-induced liver fibrosis. This raises the intriguing possibility that caspase-2 may be a promising therapeutic target to prevent progression to NASH.

  View details for DOI 10.1136/gutjnl-2014-307362

  View details for Web of Science ID 000356022700019

  View details for PubMedID 25053716

  View details for PubMedCentralID PMC4303564

• Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease PLOS ONE Machado, M. V., Michelotti, G. A., Xie, G., de Almeida, T. P., Boursier, J., Bohnic, B., Guy, C. D., Diehl, A. M. 2015; 10 (5)

  Abstract

  Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our time. Although there are several animal models of NASH, consensus regarding the optimal model is lacking. We aimed to compare features of NASH in the two most widely-used mouse models: methionine-choline deficient (MCD) diet and Western diet.Mice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared.The metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation.Feeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH.

  View details for DOI 10.1371/journal.pone.0127991

  View details for Web of Science ID 000355185600105

  View details for PubMedID 26017539

  View details for PubMedCentralID PMC4446215

• Repair-Related Activation of Hedgehog Signaling in Stromal Cells Promotes Intrahepatic Hypothyroidism ENDOCRINOLOGY Bohinc, B. N., Michelotti, G., Xie, G., Pang, H., Suzuki, A., Guy, C. D., Piercy, D., Kruger, L., Swiderska-Syn, M., Machado, M., Pereira, T., Zavacki, A. M., Abdelmalek, M., Diehl, A. M. 2014; 155 (11): 4591-4601

  Abstract

  Thyroid hormone (TH) is important for tissue repair because it regulates cellular differentiation. Intrahepatic TH activity is controlled by both serum TH levels and hepatic deiodinases. TH substrate (T4) is converted into active hormone (T3) by deiodinase 1 (D1) but into inactive hormone (rT3) by deiodinase 3 (D3). Although the relative expressions of D1 and D3 are known to change during liver injury, the cell types and signaling mechanisms involved are unclear. We evaluated the hypothesis that changes in hepatic deiodinases result from repair-related activation of the Hedgehog pathway in stromal cells. We localized deiodinase expression, assessed changes during injury, and determined how targeted manipulation of Hedgehog signaling in stromal cells impacted hepatic deiodinase expression, TH content, and TH action in rodents. Humans with chronic liver disease were also studied. In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, whereas stromal expression of D3 increased, particularly in myofibroblasts. Conditionally disrupting Hedgehog signaling in myofibroblasts normalized deiodinase expression. Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression. In patients, this was reflected by increased serum rT3. Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4. In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

  View details for DOI 10.1210/en.2014-1302

  View details for Web of Science ID 000343422800045

  View details for PubMedID 25121996

  View details for PubMedCentralID PMC4256825

• Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication. Clinical science Choi, S. S., Claridge, L. C., Jhaveri, R., Swiderska-Syn, M., Clark, P., Suzuki, A., Pereira, T. A., Mi, Z., Kuo, P. C., Guy, C. D., Pereira, F. E., Diehl, A. M., Patel, K., Syn, W. 2014; 126 (12): 845-855

  Abstract

  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.

  View details for DOI 10.1042/CS20130473

  View details for PubMedID 24438228

  View details for PubMedCentralID PMC4055032

• Alcohol activates the hedgehog pathway and induces related procarcinogenic processes in the alcohol-preferring rat model of hepatocarcinogenesis. Alcoholism, clinical and experimental research Chan, I. S., Guy, C. D., Machado, M. V., Wank, A., Kadiyala, V., Michelotti, G., Choi, S., Swiderska-Syn, M., Karaca, G., Pereira, T. A., Yip-Schneider, M. T., Max Schmidt, C., Diehl, A. M. 2014; 38 (3): 787-800

  Abstract

  Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared with only 5% of water-drinking controls.Herein, we used quantitative real-time PCR and quantitative immunocytochemistry to compare liver tissues from alcohol-consuming P rats and water-fed P rat controls after 6, 12, or 18 months of drinking. We aimed to identify potential mechanisms that might underlie the differences in liver cancer formation and hypothesized that chronic alcohol ingestion would activate Hedgehog (HH), a regenerative signaling pathway that is overactivated in HCC.Chronic alcohol ingestion amplified age-related degenerative changes in hepatocytes, but did not cause appreciable liver inflammation or fibrosis even after 18 months of heavy drinking. HH signaling was also enhanced by alcohol exposure, as evidenced by increased levels of mRNAs encoding HH ligands, HH-regulated transcription factors, and HH target genes. Immunocytochemistry confirmed increased alcohol-related accumulation of HH ligand-producing cells and HH-responsive target cells. HH-related regenerative responses were also induced in alcohol-exposed rats. Three of these processes (i.e., deregulated progenitor expansion, the reverse Warburg effect, and epithelial-to-mesenchymal transitions) are known to promote cancer growth in other tissues.Alcohol-related changes in Hedgehog signaling and resultant deregulation of liver cell replacement might promote hepatocarcinogenesis.

  View details for DOI 10.1111/acer.12279

  View details for PubMedID 24164383

  View details for PubMedCentralID PMC4054878

• Brain schistosomiasis in mice experimentally infected with Schistosoma mansoni. Revista da Sociedade Brasileira de Medicina Tropical Lambertucci, J. R., Fidelis, T. A., Pereira, T. A., Coelho, P. M., Araujo, N., Souza, M. M., Brasileiro Filho, G., Pereira, F. E., Antunes, C. M. 2014; 47 (2): 251-253

  Abstract

  Human neuroschistosomiasis has been reported in the literature, but the possibility of modeling neuroschistosomiasis in mice is controversial.In two research laboratories in Brazil that maintain the Schistosoma mansoni life cycle in rodents, two mice developed signs of brain disease (hemiplegia and spinning), and both were autopsied.S. mansoni eggs, both with and without granuloma formation, were observed in the brain and meninges of both mice by optical microscopy.This is the first description of eggs in the brains of symptomatic mice that were experimentally infected with S. mansoni. An investigation of experimental neuroschistosomiasis is now feasible.

  View details for PubMedID 24861305

• Hepatocellular carcinoma and liver cirrhosis TP53 mutation analysis reflects a moderate dietary exposure to aflatoxins in Espirito Santo State, Brazil MOLECULAR BIOLOGY REPORTS de Carvalho, F. M., Pereira, T. d., Goncalves, P. L., Jarske, R. D., Lima Pereira, F. E., Louro, I. D. 2013; 40 (8): 4883-4887

  Abstract

  The close relationship between aflatoxins and 249ser TP53 gene mutation (AGG to AGT, Arg to Ser) in hepatocellular carcinoma (HCC) makes this mutation an indirect indicator of dietary contamination with this toxin. We have examined the prevalence of codon 249 TP53 mutation in 41 HCC and 74 liver cirrhosis (without HCC) cases diagnosed at the HUCAM University Hospital in Vitoria, Espírito Santo State, Brazil. DNA was extracted from paraffin sections and from plasma. The mutation was detected by DNA amplification, followed by restriction endonuclease digestion and confirmed by direct sequencing. DNA restriction showed 249ser mutation in 16 HCC and 13 liver cirrhosis, but sequencing confirmed mutations in only 6 HCC and 1 liver cirrhosis. In addition, sequencing revealed 4 patients with mutations at codon 250 (250ser and 250leu) in HCC cases. The prevalence of TP53 mutation was 10/41 (24.3%) in HCC and 1/74 (1.4%) in liver cirrhosis. No relationship between the presence of mutations and the etiology of HCC was observed. TP53 exon 7 mutations, which are related to aflatoxins exposure, were found at 14.6% (249ser), 7.3% (250leu) and 2.4% (250ser) in 41 cases of HCC and 1.4% in 74 liver cirrhosis (without HCC) cases, suggesting a moderate dietary exposure to aflatoxins in the Espírito Santo State, Brazil.

  View details for DOI 10.1007/s11033-013-2587-2

  View details for Web of Science ID 000322392300025

  View details for PubMedID 23649769

• Paracrine Hedgehog Signaling Drives Metabolic Changes in Hepatocellular Carcinoma CANCER RESEARCH Chan, I. S., Guy, C. D., Chen, Y., Lu, J., Swiderska-Syn, M., Michelotti, G. A., Karaca, G., Xie, G., Krueger, L., Syn, W., Anderson, B. R., Pereira, T. A., Choi, S. S., Baldwin, A. S., Diehl, A. M. 2012; 72 (24): 6344-6350

  Abstract

  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.

  View details for DOI 10.1158/0008-5472.CAN-12-1068

  View details for Web of Science ID 000312591900005

  View details for PubMedID 23066040

  View details for PubMedCentralID PMC3525764

• NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease GUT Syn, W., Agboola, K. M., Swiderska, M., Michelotti, G. A., Liaskou, E., Pang, H., Xie, G., Philips, G., Chan, I. S., Karaca, G. F., Pereira, T. d., Chen, Y., Mi, Z., Kuo, P. C., Choi, S. S., Guy, C. D., Abdelmalek, M. F., Diehl, A. M. 2012; 61 (9): 1323-1329

  Abstract

  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.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.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.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.

  View details for DOI 10.1136/gutjnl-2011-301857

  View details for Web of Science ID 000307809900016

  View details for PubMedID 22427237

  View details for PubMedCentralID PMC3578424

• Osteopontin is Induced by Hedgehog Pathway Activation and Promotes Fibrosis Progression in Nonalcoholic Steatohepatitis HEPATOLOGY Syn, W., Choi, S. S., Liaskou, E., Karaca, G. F., Agboola, K. M., Oo, Y. H., Mi, Z., Pereira, T. A., Zdanowicz, M., Malladi, P., Chen, Y., Moylan, C., Jung, Y., Bhattacharya, S. D., Teaberry, V., Omenetti, A., Abdelmalek, M. F., Guy, C. D., Adams, D. H., Kuo, P. C., Michelotti, G. A., Whitington, P. F., Diehl, A. M. 2011; 53 (1): 106-115

  Abstract

  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).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.

  View details for DOI 10.1002/hep.23998

  View details for Web of Science ID 000286406300013

  View details for PubMedID 20967826

  View details for PubMedCentralID PMC3025083

• Activation of Rac1 Promotes Hedgehog-Mediated Acquisition of the Myofibroblastic Phenotype in Rat and Human Hepatic Stellate Cells HEPATOLOGY Choi, S. S., Witek, R. P., Yang, L., Omenetti, A., Syn, W., Moylan, C. A., Jung, Y., Karaca, G. F., Teaberry, V. S., Pereira, T. A., Wang, J., Ren, X., Diehl, A. M. 2010; 52 (1): 278-290

  Abstract

  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.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.

  View details for DOI 10.1002/hep.23649

  View details for Web of Science ID 000279409200030

  View details for PubMedID 20578145

  View details for PubMedCentralID PMC2920128

• Accumulation of Natural Killer T Cells in Progressive Nonalcoholic Fatty Liver Disease HEPATOLOGY Syn, W., Oo, Y. H., Pereira, T. A., Karaca, G. F., Jung, Y., Omenetti, A., Witek, R. P., Choi, S. S., Guy, C. D., Fearing, C. M., Teaberry, V., Pereira, F. E., Adams, D. H., Diehl, A. M. 2010; 51 (6): 1998-2007

  Abstract

  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.Hh pathway activation leads to hepatic enrichment with NKT cells that contribute to fibrosis progression in NASH.

  View details for DOI 10.1002/hep.23599

  View details for Web of Science ID 000278261600017

  View details for PubMedID 20512988

  View details for PubMedCentralID PMC2920131

• Pan-Caspase Inhibitor VX-166 Reduces Fibrosis in an Animal Model of Nonalcoholic Steatohepatitis HEPATOLOGY Witek, R. P., Stone, W. C., Karaca, F. G., Syn, W., Pereira, T. A., Agboola, K. M., Omenetti, A., Jung, Y., Teaberry, V., Choi, S. S., Guy, C. D., Pollard, J., Charlton, P., Diehl, A. M. 2009; 50 (5): 1421-1430

  Abstract

  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 >or=6). Nevertheless, VX-166-treated MCD-fed mice demonstrated decreased alpha-smooth muscle actin expression (4 weeks, P < 0.05; 8 weeks, P < 0.005) and had reduced hepatic levels of collagen 1alpha1 messenger RNA (8 weeks, P < 0.05). Hydroxyproline content and Sirius red staining of VX-166-treated livers confirmed decreases in fibrosis.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.

  View details for DOI 10.1002/hep.23167

  View details for Web of Science ID 000271565600014

  View details for PubMedID 19676126

• Hedgehog-Mediated Epithelial-to-Mesenchymal Transition and Fibrogenic Repair in Nonalcoholic Fatty Liver Disease GASTROENTEROLOGY Syn, W., Jung, Y., Omenetti, A., Abdelmalek, M., Guy, C. D., Yang, L., Wang, J., Witek, R. P., Fearing, C. M., Pereira, T. A., Teaberry, V., Choi, S. S., Conde-Vancells, J., Karaca, G. F., Diehl, A. M. 2009; 137 (4): 1478-1488

  Abstract

  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.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.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.Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

  View details for DOI 10.1053/j.gastro.2009.06.051

  View details for Web of Science ID 000270255200039

  View details for PubMedID 19577569

  View details for PubMedCentralID PMC2757536

• Genetic differences in oxidative stress and inflammatory responses to diet-induced obesity do not alter liver fibrosis in mice LIVER INTERNATIONAL Syn, W., Yang, L., Chiang, D. J., Qian, Y., Jung, Y., Karaca, G., Choi, S. S., Witek, R. P., Omenetti, A., Pereira, T. A., Diehl, A. M. 2009; 29 (8): 1262-1272

  Abstract

  To determine how genetic factors might influence the progression of nonalcoholic fatty liver disease (NAFLD).Beginning in adolescence, male C57BL6 (BL6) and 129/SVJ mice were fed control (n=15/group) or high-fat (HF) diets (n=30/group) for 6 months.Assessed were body weight, insulin resistance, hepatic production of free radicals, expression of cytokines and fibrosis-related genes and severity of hepatic steatosis, injury and fibrosis.High-fat diets induced comparable obesity, hepatic steatosis and insulin resistance in the two strains. Compared with BL6 mice, 129/SVJ mice had impaired induction of antioxidant genes, generated three- to four-fold more free radicals and exhibited two-fold greater induction of profibrogenic cytokines (interleukin-4 and transforming growth factor-beta1) and fibrosis-related genes (fibronectin and tissue inhibitor of metalloproteinase-1) (all P<0.05 for 129 vs BL6). Surprisingly, however, induction of collagen I alpha1 mRNA and accumulation of Sirius red-stained fibrils and hepatic hydroxyproline were similar in BL6 and 129/SVJ mice, and although patchy sinusoidal fibrosis emerged in both strains, neither developed bridging fibrosis.Although BL6 and 129/SVJ mice with diet-induced obesity, insulin resistance and steatosis differed with respect to several factors that are thought to influence human NAFLD progression, they developed comparable liver fibrosis. Moreover, none of the risk factors for NAFLD-related cirrhosis in humans, including obesity, insulin resistance, chronic inflammatory and oxidant stress, steatohepatitis or activation of fibrogenic genes, proved to be sufficient to cause cirrhosis in these mice, even when exposure to one or more of these insults was very prolonged.

  View details for DOI 10.1111/j.1478-3231.2009.02036.x

  View details for Web of Science ID 000268653200020

  View details for PubMedID 19490416

  View details for PubMedCentralID PMC3610179