RESUMO
BACKGROUND AND AIMS: Chronic alcohol drinking is a major risk factor for alcohol-associated liver disease (ALD). FK506-binding protein 51 (FKBP5), a cochaperone protein, is involved in many key regulatory pathways. It is known to be involved in stress-related disorders, but there are no reports regarding its role in ALD. This present study aimed to examine the molecular mechanism of FKBP5 in ALD. APPROACH AND RESULTS: We found a significant increase in hepatic FKBP5 transcripts and protein expression in patients with ALD and mice fed with chronic-plus-single binge ethanol. Loss of Fkbp5 in mice protected against alcohol-induced hepatic steatosis and inflammation. Transcriptomic analysis revealed a significant reduction of Transcriptional enhancer factor TEF-1 (TEA) domain transcription factor 1 (Tead1) and chemokine (C-X-C motif) ligand 1 (Cxcl1) mRNA in ethanol-fed Fkbp5-/- mice. Ethanol-induced Fkbp5 expression was secondary to down-regulation of methylation level at its 5' untranslated promoter region. The increase in Fkbp5 expression led to induction in transcription factor TEAD1 through Hippo signaling pathway. Fkbp5 can interact with yes-associated protein (YAP) upstream kinase, mammalian Ste20-like kinase 1 (MST1), affecting its ability to phosphorylate YAP and the inhibitory effect of hepatic YAP phosphorylation by ethanol leading to YAP nuclear translocation and TEAD1 activation. Activation of TEAD1 led to increased expression of its target, CXCL1, a chemokine-mediated neutrophil recruitment, causing hepatic inflammation and neutrophil infiltration in our mouse model. CONCLUSIONS: We identified an FKBP5-YAP-TEAD1-CXCL1 axis in the pathogenesis of ALD. Loss of FKBP5 ameliorates alcohol-induced liver injury through the Hippo pathway and CXCL1 signaling, suggesting its potential role as a target for the treatment of ALD.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Quimiocina CXCL1/metabolismo , Etanol/farmacologia , Via de Sinalização Hippo/genética , Hepatopatias Alcoólicas/genética , Proteínas de Ligação a Tacrolimo/genética , Animais , Metilação de DNA , Perfilação da Expressão Gênica , Humanos , Inflamação , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas de Sinalização YAP/metabolismoRESUMO
BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.
Assuntos
Ductos Biliares/patologia , Colestase/patologia , Cirrose Hepática/etiologia , Monoaminoxidase/fisiologia , Receptores de Serotonina/fisiologia , Serotonina/fisiologia , Triptofano Hidroxilase/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Animais , Proliferação de Células , Colangite Esclerosante/etiologia , Humanos , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2A de Serotonina/fisiologia , Receptor 5-HT2B de Serotonina/fisiologia , Receptor 5-HT2C de Serotonina/fisiologia , Serotonina/sangue , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Methionine adenosyltransferase α1 (MATα1, encoded by MAT1A) is responsible for hepatic biosynthesis of S-adenosyl methionine, the principal methyl donor. MATα1 also act as a transcriptional cofactor by interacting and influencing the activity of several transcription factors. Mat1a knockout (KO) mice have increased levels of cytochrome P450 2E1 (CYP2E1), but the underlying mechanisms are unknown. The aims of the current study were to identify binding partners of MATα1 and elucidate how MATα1 regulates CYP2E1 expression. We identified binding partners of MATα1 by coimmunoprecipitation (co-IP) and mass spectrometry. Interacting proteins were confirmed using co-IP using recombinant proteins, liver lysates, and mitochondria. Alcoholic liver disease (ALD) samples were used to confirm relevance of our findings. We found that MATα1 negatively regulates CYP2E1 at mRNA and protein levels, with the latter being the dominant mechanism. MATα1 interacts with many proteins but with a predominance of mitochondrial proteins including CYP2E1. We found that MATα1 is present in the mitochondrial matrix of hepatocytes using immunogold electron microscopy. Mat1a KO hepatocytes had reduced mitochondrial membrane potential and higher mitochondrial reactive oxygen species, both of which were normalized when MAT1A was overexpressed. In addition, KO hepatocytes were sensitized to ethanol and tumor necrosis factor α-induced mitochondrial dysfunction. Interaction of MATα1 with CYP2E1 was direct, and this facilitated CYP2E1 methylation at R379, leading to its degradation through the proteasomal pathway. Mat1a KO livers have a reduced methylated/total CYP2E1 ratio. MATα1's influence on mitochondrial function is largely mediated by its effect on CYP2E1 expression. Patients with ALD have reduced MATα1 levels and a decrease in methylated/total CYP2E1 ratio. Conclusion: Our findings highlight a critical role of MATα1 in regulating mitochondrial function by suppressing CYP2E1 expression at multiple levels.
Assuntos
Citocromo P-450 CYP2E1/genética , Metionina Adenosiltransferase/fisiologia , Mitocôndrias Hepáticas/fisiologia , Animais , Feminino , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Hepatopatias Alcoólicas/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Metilação , Camundongos , Proteínas Mitocondriais/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The Galα(1,3)Gal epitope (α-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig-to-primate and pig-to-human xenotransplantation. In response, GGTA1 gene-deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1(-/-) pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an α-GAL-terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α-GAL epitopes in GGTA1(-/-) animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross-reactivity by creating and comparing GGTA1(-/-) pigs to GGTA1(-/-) - and A3GalT2(-/-) -double-knockout pigs. METHODS: We used the CRISPR/Cas 9 system to target the GGTA1 and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single- and double-gene-knockout piglets in a single pregnancy. Once grown to maturity, the glycosphingolipid profile (including iGb3) was assayed in renal tissue by normal-phase liquid chromatography. In addition, peripheral blood mononuclear cells (PBMCs) were subjected to (i) comparative cross-match cytotoxicity analysis against human and baboon serum and (ii) IB4 staining for α-GAL/iGb3. RESULTS: Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and α-GAL/iGb3 staining were unchanged by iGb3s silencing. CONCLUSIONS: Our data suggest that iGb3s is not a contributor to antibody-mediated rejection in pig-to-primate or pig-to-human xenotransplantation. Although iGb3s gene silencing significantly changed the renal glycosphingolipid profile, the effect on Galα3Gal levels, antibody binding, and cytotoxic profiles of baboon and human sera on porcine PBMCs was neutral.
Assuntos
Galactose/metabolismo , Galactosiltransferases/genética , Rejeição de Enxerto/genética , Xenoenxertos/imunologia , Transplante Heterólogo , Doença Aguda , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Galactosiltransferases/metabolismo , Técnicas de Inativação de Genes/métodos , Humanos , Leucócitos Mononucleares/imunologia , Papio , Suínos , Transplante Heterólogo/métodosRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) remains a global health problem with unique diagnostic and therapeutic challenges, including difficulties in identifying the highest risk patients. Previous work from our lab has established the murine multidrug resistance-2 mouse (MDR2) model of HCC as a reasonable preclinical model that parallels the changes seen in human inflammatory associated HCC. The purpose of this study is to evaluate modalities of PET/CT in MDR2(-/-) mice in order to facilitate therapeutic translational studies from bench to bedside. METHODS: 18F-FDG and 11C-acetate PET/CT was performed on 12 m MDR2(-/-) mice (n = 3/tracer) with HCC and 12 m MDR2(-/+) control mice (n = 3/tracer) without HCC. To compare PET/CT to biological markers of HCC and cellular function, serum alpha-fetoprotein (AFP), lysophosphatidic acid (LPA), cAMP and hepatic tumor necrosis factor α (TNFα) were quantified in 3-12 m MDR2(-/-) (n = 10) mice using commercially available ELISA analysis. To translate results in mice to patients 11C-acetate PET/CT was also performed in 8 patents suspected of HCC recurrence following treatment and currently on the liver transplant wait list. RESULTS: Hepatic18F-FDG metabolism was not significantly increased in MDR2(-/-) mice. In contrast, hepatic 11C-acetate metabolism was significantly elevated in MDR2(-/-) mice when compared to MDR2(-/+) controls. Serum AFP and LPA levels increased in MDR2(-/-) mice contemporaneous with the emergence of HCC. This was accompanied by a significant decrease in serum cAMP levels and an increase in hepatic TNFα. In patients suspected of HCC recurrence there were 5 true positives, 2 true negatives and 1 suspected false 11C-acetate negative. CONCLUSIONS: Hepatic 11C-acetate PET/CT tracks well with HCC in MDR2(-/-) mice and patients with underlying liver disease. Consequently 11C-acetate PET/CT is well suited to study (1) HCC emergence/progression in patients and (2) reduce animal numbers required to study new chemotherapeutics in murine models of HCC.
Assuntos
Acetatos , Carbono , Carcinoma Hepatocelular/diagnóstico , Modelos Animais de Doenças , Fluordesoxiglucose F18 , Neoplasias Hepáticas/diagnóstico , Tomografia por Emissão de Pósitrons/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Camundongos , Camundongos Knockout , Imagem Multimodal/métodos , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X/métodos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND: The U.S. incidence of hepatocellular carcinoma (HCC) is increasing and is linked to hepatitis C (HepC) infection, alcohol toxicity, and obesity. This manuscript examines lysophosphatidic acid (LPA) variant biosynthesis as a biomarker and potential therapeutic target for HCC. METHODS: Serum LPA variant levels were determined in patients with HepC ± HCC, alcoholic cirrhosis ± HCC, or nonalcoholic steatohepatitis ± HCC by mass spectroscopy. To clarify the relationship between cancer and LPA variant profiles, LPA variants were evaluated in HepC + HCC patients before and after liver transplantation. Moreover, LPA variant modification of gene expression was also determined in vitro by real-time polymerase chain reaction. RESULTS: In patients diagnosed with HCC, 18:2 LPA biosynthesis was decreased, whereas 20:4 LPA biosynthesis and 20:4 LPA:18:2 LPA ratio were increased. Three days after liver transplantation, serum LPA levels and 18:2 LPA:20:4 LPA ratio were significantly reduced in patients with cancer. The 20:4 LPA selectively stimulated LPA receptor and tumor necrosis factor α expression in Hep3B cells, whereas 18:2 LPA did not. CONCLUSIONS: Serum LPA variant profiles are unique in patients with HCC allowing for the stratification of patients. Moreover, LPA variants impart individual mitogenic properties associated with tumorigenesis that may provide a potential therapeutic target. We envision that LPA profiling may accelerate diagnosis, help stratify patients at high risk of developing cancer, and provide potential targets for chemoprevention.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Lisofosfolipídeos/sangue , Fígado Gorduroso/metabolismo , Hepatite C/metabolismo , Humanos , Transplante de Fígado , Lisofosfolipídeos/biossíntese , Espectrometria de Massas , Hepatopatia Gordurosa não Alcoólica , RNA Mensageiro/análise , Receptores de Ácidos Lisofosfatídicos/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Ca2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in hepatic GRP75-mediated mitochondria-associated ER membrane (MAM) Ca2+-channeling (MCC) complex formation promotes mitochondrial dysfunction in vitro and in male mouse model of ALD. Unbiased transcriptomic analysis reveals PDK4 as a prominently inducible MAM kinase in ALD. Analysis of human ALD cohorts further corroborate these findings. Additional mass spectrometry analysis unveils GRP75 as a downstream phosphorylation target of PDK4. Conversely, non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced MCC complex formation and subsequent mitochondrial Ca2+ accumulation and dysfunction. Finally, ectopic induction of MAM formation reverses the protective effect of PDK4 deficiency in alcohol-induced liver injury. Together, our study defines a mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.
Assuntos
Retículo Endoplasmático , Hepatopatias , Camundongos , Animais , Masculino , Humanos , Retículo Endoplasmático/metabolismo , Mitocôndrias , Hepatopatias/metabolismoRESUMO
The intact telomere structure is essential for the prevention of the chromosome end-to-end fusions and maintaining genomic integrity. The maintenance of telomere length is critical for cellular homeostasis. The shortening of telomeres has been reported in patients with chronic liver diseases. The telomere length has not been systemically studied in patients with alcohol-associated liver disease (ALD) at different stages, such as alcoholic hepatitis and alcoholic cirrhosis. In this brief report, we observed evidence of telomere shortening without changes in the telomerase activity in the liver of patients with alcoholic hepatitis and alcoholic cirrhosis when compared with controls. The alterations in the genes associated with telomere binding proteins were only observed in patients with alcoholic cirrhosis. Future studies are required to determine the mechanism of how alcohol affects the length of the telomere and if the shortening impacts the disease progression in ALD.
Assuntos
Hepatite Alcoólica , Hepatopatias Alcoólicas , Telomerase , Humanos , Cirrose Hepática Alcoólica/genética , Hepatopatias Alcoólicas/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Encurtamento do TelômeroRESUMO
Alcohol-associated liver disease is the leading cause of chronic liver disease. We hypothesized that the expression of specific coding genes is critical for the progression of alcoholic cirrhosis (AC) from compensated to decompensated states. For the discovery phase, we performed RNA sequencing analysis of 16 peripheral blood RNA samples, 4 healthy controls (HCs) and 12 patients with AC. The DEGs from the discovery cohort were validated by quantitative polymerase chain reaction in a separate cohort of 17 HCs and 48 patients with AC (17 Child-Pugh A, 16 Child-Pugh B, and 15 Child-Pugh C). We observed that the numbers of differentially expressed messenger RNAs (mRNAs) were more pronounced with worsening disease severity. Pathway analysis for differentially expressed genes for patients with Child-Pugh A demonstrated genes involved innate immune responses; those in Child-Pugh B belonged to genes related to oxidation and alternative splicing; those in Child-Pugh C related to methylation, acetylation, and alternative splicing. We found significant differences in the expression of heme oxygenase 1 (HMOX1) and ribonucleoprotein, PTB binding 1 (RAVER1) in peripheral blood of those who died during the follow-up when compared to those who survived. Conclusion: Unique mRNAs that may implicate disease progression in patients with AC were identified by using a transcriptomic approach. Future studies to confirm our results are needed, and comprehensive mechanistic studies on the implications of these genes in AC pathogenesis and progression should be further explored.
Assuntos
Cirrose Hepática Alcoólica , Transcriptoma , Perfilação da Expressão Gênica , Humanos , Cirrose Hepática Alcoólica/genética , RNA Mensageiro/genética , Índice de Gravidade de Doença , Transcriptoma/genéticaRESUMO
Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp-/- mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp-/- mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα-/- hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.
Assuntos
Citocromo P-450 CYP4A/metabolismo , Ácidos Graxos/metabolismo , Hepatopatias Alcoólicas/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Amidinas , Animais , Citocromo P-450 CYP4A/antagonistas & inibidores , Modelos Animais de Doenças , Etanol/administração & dosagem , Etanol/efeitos adversos , Hepatócitos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipidômica , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Cultura Primária de Células , Pirrolidinas/administração & dosagem , RNA-Seq , Receptor EphB2 , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , Tiofenos/administração & dosagem , Regulação para CimaRESUMO
BACKGROUND: Autotaxin (ATX) is an extracellular lysophospholipase D that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been shown to be involved in many cancers. However, the functional role of ATX and the regulation of ATX expression in human hepatocellular carcinoma (HCC) remain elusive. RESULTS: In this study, ATX expression was evaluated in tissues from 38 human HCC and 10 normal control subjects. ATX was detected mainly in tumor cells within tissue sections and its over-expression in HCC was specifically correlated with inflammation and liver cirrhosis. In addition, ATX expression was examined in normal human hepatocytes and liver cancer cell lines. Hepatoma Hep3B and Huh7 cells displayed stronger ATX expression than hepatoblastoma HepG2 cells and normal hepatocytes did. Proinflammtory cytokine tumor necrosis factor alpha (TNF-alpha) promoted ATX expression and secretion selectively in Hep3B and Huh7 cells, which led to a corresponding increase in lysophospholipase-D activity. Moreover, we explored the mechanism governing the expression of ATX in hepatoma cells and established a critical role of nuclear factor-kappa B (NF-kappaB) in basal and TNF-alpha induced ATX expression. Further study showed that secreted enzymatically active ATX stimulated Hep3B cell invasion. CONCLUSIONS: This report highlights for the first time the clinical and biological evidence for the involvement of ATX in human HCC. Our observation that links the TNF-alpha/NF-kappaB axis and the ATX-LPA signaling pathway suggests that ATX is likely playing an important role in inflammation related liver tumorigenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Complexos Multienzimáticos/biossíntese , NF-kappa B/metabolismo , Fosfodiesterase I/biossíntese , Pirofosfatases/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Células Hep G2 , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Complexos Multienzimáticos/genética , NF-kappa B/genética , Fosfodiesterase I/genética , Diester Fosfórico Hidrolases , Pirofosfatases/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Transfecção , Fator de Necrose Tumoral alfa/genéticaRESUMO
OBJECTIVES: Lipids are linked to many pathological processes including hepatic steatosis and liver malignancy. This study aimed to explore lipid metabolism in hepatitis C virus (HCV) and HCV-related hepatocellular carcinoma (HCC). METHODS: Serum lipids were measured in normal, HCV and HCV-HCC patients. Whole-genome microarray was performed to identify potential signature genes involved in lipid metabolism characterizing normal vs. HCV vs. HCV-HCC conditions. RESULTS: Serum cholesterol was significantly reduced in HCV and HCV-HCC patients compared with normal controls, whereas there was no difference in glucose and triglycerides. Microarray analysis identified 224 probe sets with known functional roles in lipid metabolism (anova, 1.5-fold, P ≤ 0.001). Gene-mediated fatty acid (FA) de novo synthesis and uptake were upregulated in HCV and this upregulation was further enhanced in HCC. Genes involved in FA oxidation were downregulated in both the HCV and HCC groups. The abnormality of cholesterol metabolism in HCV was associated with downregulation of genes involved in cholesterol biosynthesis, absorption and transportation and bile acid synthesis; this abnormality was further intensified in HCC. CONCLUSIONS: Our data support the notion that HCV-related lipid metabolic abnormalities may contribute to hepatic steatosis and the development of cancer. Identification of these aberrations would stratify patients and improve treatment algorithms.
Assuntos
Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/metabolismo , Hepatite C/metabolismo , Metabolismo dos Lipídeos , Neoplasias Hepáticas/metabolismo , Análise de Variância , Glicemia/metabolismo , Índice de Massa Corporal , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Colesterol/sangue , Análise por Conglomerados , Ácidos Graxos/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/virologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hepatite C/complicações , Hepatite C/genética , Humanos , Indiana , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/sangueRESUMO
Portal hypertension (PHT) is a complication of liver cirrhosis and directly increases mortality and morbidity by increasing the propensity of venous hemorrhage. There are two main underlying causations for PHT, increased hepatic resistance and systemic hyperdynamic circulation. Both are related to localized aberrations in endothelial nitric oxide synthase (eNOS) function and NO biosynthesis. This study investigates the importance of eNOS and systemic hyperdynamic-associated hyperemia to better understand the pathophysiology of PHT. Wild-type and eNOS(-/-) mice were given the hepatotoxin CCl(4) for 4-12 wk. Hepatic fibrosis was determined histologically following collagen staining. Portal venous pressure, hepatic resistance, and hyperemia were determined by measuring splenic pulp pressure (SPP), hepatic portal-venous perfusion pressure (HPVPP), abdominal aortic flow (Qao), and portal venous flow (Qpv). Hepatic fibrosis developed equally in wild-type and eNOS(-/-) CCl(4)-exposed mice. SPP, Qao, and Qpv increased rapidly in wild-type CCl(4)-exposed mice, but HPVPP did not. In eNOS(-/-) CCl(4) mice, Qao was not increased, SPP was partially increased, and HPVPP and Qpv were increased nonsignificantly. We concluded that the systemic hyperemia component of hyperdynamic circulation is eNOS dependent and precedes increased changes in hepatic resistance. Alternative mechanisms, possibly involving cyclooxygenase, may contribute. eNOS maintains normal hepatic resistance following CCl(4)-induced fibrosis. Consequently, increased portal pressure following chronic CCl(4) exposure is linked to hyperdynamic circulation in wild-type mice and increased hepatic resistance in eNOS(-/-) mice.
Assuntos
Hipertensão Portal/enzimologia , Cirrose Hepática Experimental/enzimologia , Fígado/irrigação sanguínea , Fígado/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Pressão na Veia Porta , Sistema Porta/fisiopatologia , Alanina Transaminase/sangue , Animais , Aorta Abdominal/fisiopatologia , Tetracloreto de Carbono , Genótipo , Hiperemia/enzimologia , Hiperemia/fisiopatologia , Hipertensão Portal/induzido quimicamente , Hipertensão Portal/genética , Hipertensão Portal/fisiopatologia , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Fenótipo , Fluxo Sanguíneo Regional , Índice de Gravidade de Doença , Fatores de Tempo , Resistência VascularRESUMO
Rabbit antithymocyte globulin (rATG) use for immunosuppression induction is widespread but is contraindicated by the presence of anti-rATG antibodies. This study reports the incidence of positive anti-rATG antibody titers in patients before and after renal transplant and evaluates associated outcomes and costs. In addition, it will correlate CD40L and interleukin (IL)-21 with anti-rATG antibody titers. METHODS: Clinical and billing records from the Indiana University Transplant Laboratory were reviewed for positive versus negative anti-rATG antibody titers, graft survival, and 7-day readmission costs between 2004 and 2018. Serum from patients with positive and negative rATG antibody titers were quantitated for CD40L and IL-21 by enzyme-linked immunosorbent assay. RESULTS: On average, between 2004 and May 2018, 163 kidney transplants per year were performed. Anti-rATG antibody titers were ordered for 17 patients/year, of which 18.2% were positive at 1:100 titer either pre- or post-transplant. Time to graft loss correlated with a positive rATG titer at time of readmission. Moreover, second kidney transplant increased the anti-rATG positive rate. A weak correlation was observed between anti-rATG titer and recipient age. Seven-day readmission treatment costs were significantly lower in patients with positive anti-rATG titer. IL-21 and CD40L were significantly greater in patients with positive anti-rATG titers after transplant when compared with negative anti rATG patients. CONCLUSIONS: Positive anti-rATG antibody titer is associated with a significant negative impact on outcomes. Monitoring of anti-rATG antibody titer is recommended to optimize treatment options in patients, especially in the setting of second transplants. Elucidation of the mechanisms associated with positive anti-rATG antibody is required. IL-21 and CD40L are potential targets for future study.
RESUMO
BACKGROUND: Liver transplant and liver resection are surgical treatments for hepatocellular carcinoma (HCC) performed with curative intent. While liver transplant provides longer survival when compared to resection, the financial burden on patients and payors is significantly greater. With the increase in health care costs and the emergence of high deductible insurance policies that increase out of pocket deductibles for patients, assessment of value-based treatment is warranted. METHODS: We compiled total billable events from diagnosis of HCC through resection (N = 20) or transplant (N = 24) to death or last reported encounter from January 2011 to December 2012. RESULTS: Patients with HCC receiving resection had a model of end stage liver disease of 10.2 ± 1.2, survival 652 days (3-1, 167 days), and billable encounters of $316,873 ($2904/day). HCC patients receiving a liver transplant had a greater liver injury (model of end stage liver disease of 19.2 ± 3.7), longer survival (1579 days), and higher billable encounters, $740,714 ($2889/day). The surgical procedure represented the largest cost category (28% and 26% resection vs transplant, respectively). The cost effectiveness of treatment was directly proportional to length of survival. In resection, patients who survived >30 days (85%) cost per day dropped to $432. Transplant patients who survived >2 years (75%) saw the cost per day drop to $462. CONCLUSION: The relative financial burdens of liver resection vs liver transplant for treating HCC are comparable in patients who survive beyond a certain threshold. Transplant patients survived longer, and survival beyond 2 years makes this approach cost effective. In a health care climate aiming to contain costs and evaluate value-based treatment paradigms, expected survival and financial burden should be included in the treatment decision analysis.
Assuntos
Carcinoma Hepatocelular/economia , Custos de Cuidados de Saúde/estatística & dados numéricos , Hepatectomia/economia , Neoplasias Hepáticas/economia , Transplante de Fígado/economia , Carcinoma Hepatocelular/cirurgia , Efeitos Psicossociais da Doença , Análise Custo-Benefício , Técnicas de Apoio para a Decisão , Feminino , Humanos , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND Acute liver rejection (ALR), a significant complication of liver transplantation, burdens patients, healthcare payers, and the healthcare providers due to an increase in morbidity, cost, and resources. Despite clinical resolution, ALR is associated with an increased risk of graft loss. A unique protocol of delayed immunosuppression used in our institute provided a model to characterize metabolomic profiles in human ALR. MATERIAL AND METHODS Twenty liver allograft biopsies obtained 48 hours after liver transplantation in the absence of immunosuppression were studied. Hepatic metabolites were quantitated in these biopsies by liquid chromatography and mass spectroscopy (LC/MS). Metabolite profiles were compared among: 1) biopsies with reperfusion injury but no histological evidence of rejection (n=7), 2) biopsies with histological evidence of moderate or severe rejection (n=5), and 3) biopsies with histological evidence of mild rejection (n=8). RESULTS There were 133 metabolites consistently detected by LC/MS and these were prioritized using variable importance to projection (VIP) analysis, comparing moderate or severe rejection vs. no rejection or mild rejection using partial least squares discriminant statistical analysis (PLS-DA). Twenty metabolites were identified as progressively different. Further PLS-DA using these metabolites identified 3 metabolites (linoleic acid, γ-linolenic acid, and citrulline) which are associated with either cyclooxygenase or nitric oxide synthase functionality. CONCLUSIONS Hepatic metabolic aberrancies associated with cyclooxygenase and nitric oxide synthase function occur contemporaneous with ALR. Additional studies are required to better characterize the role of these metabolic pathways to enhance utility of the metabolomics approach in diagnosis and outcomes of ALR.
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Doença Hepática Terminal/cirurgia , Rejeição de Enxerto/metabolismo , Transplante de Fígado/efeitos adversos , Fígado/metabolismo , Metabolômica , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Idoso , Doença Hepática Terminal/metabolismo , Doença Hepática Terminal/patologia , Feminino , Rejeição de Enxerto/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Cholangiocytes are the target cells of cholangiopathies including primary sclerosing cholangitis (PSC). Vimentin is an intermediate filament protein that has been found in various types of mesenchymal cells. The aim of this study is to evaluate the role of vimentin in the progression of biliary damage/liver fibrosis and whether there is a mesenchymal phenotype of cholangiocytes in the Mdr2-/- model of PSC. METHODS: In vivo studies were performed in 12 wk. Mdr2-/- male mice with or without vimentin Vivo-Morpholino treatment and their corresponding control groups. Liver specimens from human PSC patients, human intrahepatic biliary epithelial cells (HIBEpiC) and human hepatic stellate cell lines (HHSteCs) were used to measure changes in epithelial-to-mesenchymal transition (EMT). FINDINGS: There was increased mesenchymal phenotype of cholangiocytes in Mdr2-/- mice, which was reduced by treatment of vimentin Vivo-Morpholino. Concomitant with reduced vimentin expression, there was decreased liver damage, ductular reaction, biliary senescence, liver fibrosis and TGF-ß1 secretion in Mdr2-/- mice treated with vimentin Vivo-Morpholino. Human PSC patients and derived cell lines had increased expression of vimentin and other mesenchymal markers compared to healthy controls and HIBEpiC, respectively. In vitro silencing of vimentin in HIBEpiC suppressed TGF-ß1-induced EMT and fibrotic reaction. HHSteCs had decreased fibrotic reaction and increased cellular senescence after stimulation with cholangiocyte supernatant with reduced vimentin levels. INTERPRETATION: Our study demonstrated that knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes, which leads to decreased biliary senescence and liver fibrosis. Inhibition of vimentin may be a key therapeutic target in the treatment of cholangiopathies including PSC. FUND: National Institutes of Health (NIH) awards, VA Merit awards.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Colangite Esclerosante/genética , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Vimentina/genética , Animais , Biomarcadores , Linhagem Celular , Colangite Esclerosante/diagnóstico , Colangite Esclerosante/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Fibrose , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
AIM: Portal hypertension is a common complication of liver cirrhosis and significantly increases mortality and morbidity. Previous reports have suggested that the compound thalidomide attenuates portal hypertension (PHT). However, the mechanism for this action is not fully elucidated. One hypothesis is that thalidomide destabilizes tumor necrosis factor α (TNFα) mRNA and therefore diminishes TNFα induction of nitric oxide synthase (NOS) and the production of nitric oxide (NO). To examine this hypothesis, we utilized the murine partial portal vein ligation (PVL) PHT model in combination with endothelial or inducible NOS isoform gene knockout mice. METHODS: Wild type, inducible nitric oxide synthase (iNOS)(-/-) and endothelial nitric oxide synthase (eNOS)(-/-) mice received either PVL or sham surgery and were given either thalidomide or vehicle. Serum nitrate (total nitrate, NOx) was measured daily for 7 d as a surrogate of NO synthesis. Serum TNFα level was quantified by enzyme-linked immunosorbent assay. TNFα mRNA was quantified in liver and aorta tissue by reverse transcription-polymerase chain reaction. PHT was determined by recording splenic pulp pressure (SPP) and abdominal aortic flow after 0-7 d. Response to thalidomide was determined by measurement of SPP and mean arterial pressure (MAP). RESULTS: SPP, abdominal aortic flow (Qao) and plasma NOx were increased in wild type and iNOS(-/-) PVL mice when compared to sham operated control mice. In contrast, SPP, Qao and plasma NOx were not increased in eNOS(-/-) PVL mice when compared to sham controls. Serum TNFα level in both sham and PVL mice was below the detection limit of the commercial ELISA used. Therefore, the effect of thalidomide on serum TNFα levels was undetermined in wild type, eNOS(-/-) or iNOS(-/-) mice. Thalidomide acutely increased plasma NOx in wild type and eNOS(-/-) mice but not iNOS(-/-) mice. Moreover, thalidomide temporarily (0-90 min) decreased mean arterial pressure, SPP and Qao in wild type, eNOS(-/-) and iNOS(-/-) PVL mice, after which time levels returned to the respective baseline. CONCLUSION: Thalidomide does not reduce portal pressure in the murine PVL model by modulation of NO biosynthesis. Rather, thalidomide reduces PHT by decreasing MAP by an undetermined mechanism.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Arterial/efeitos dos fármacos , Hipertensão Portal/tratamento farmacológico , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Pressão na Veia Porta/efeitos dos fármacos , Talidomida/farmacologia , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/fisiopatologia , Biomarcadores/sangue , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Hipertensão Portal/enzimologia , Hipertensão Portal/genética , Hipertensão Portal/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitratos/sangue , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Células RAW 264.7 , Fluxo Sanguíneo Regional , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangueRESUMO
The cytokine tumor necrosis factor alpha (TNFa) has previously been identified in the development of portal hypertension (PHT) by facilitating portal venous and systemic hyperemia. TNFa is reported to contribute to hyperemia via endothelial nitric oxide synthase (eNOS) induction and nitric oxide (NO) production. This study examines this hypothesis by utilizing TNFa receptor knockout mice and a murine model of pre-hepatic PHT. Plasma TNFa and NOx and tissue TNFa mRNA levels were determined in wild-type mice 0-7d post induction of pre-hepatic PHT by partial portal vein ligation (PVL). TNFa receptor knockout mice also received PVL or sham surgery and splenic pulp pressure, abdominal aortic flow and portal-systemic shunting were recorded 7d following. Portal pressure and systemic hyperemia developed rapidly following PVL. Plasma NOx was increased temporarily 2-3 days following PVL and returned to baseline by day 7. Circulating TNFa was below detectable limits of the ELISA used, as such no increase was observed. Hepatic and vascular TNFa mRNA levels were transiently changed after PVL otherwise there was no significant change. TNFa receptor targeted gene deletion did not ameliorate plasma NOx following PVL and had no effect on the development of PHT. TNFa receptor signaling plays no detectable role in the development of systemic hyperemia in the murine model of pre-hepatic PHT. Consequently, increased TNFa observed in intra-hepatic inflammatory models (CCl(4)) and in patients is probably related to inflammation associated with intra-hepatic pathology. Alternatively, TNFa may be signaling via a TNFa receptor independent mechanism.
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Apoptosis has been proposed to play an important role in the progression of renal scarring. The mechanisms that determine whether a cell enters the apoptotic program are complex. Bax and Bcl-2 are recognized modulators of this event; their relative levels determine the fate of cells. A role for apoptosis in the progression of renal scarring in the remnant kidneys of rats submitted to subtotal nephrectomy (SNx) has been described. This study investigated the expression (protein and mRNA) of Bax and Bcl-2 in remnant kidneys between day 7 and day 120 post-SNx. Northern blot analysis showed that bax mRNA was increased in remnant kidneys from day 7 (150% of control; P: < 0.05), whereas bcl-2 mRNA was decreased from day 15 (23% of control; P: < 0.05) resulting in a 14-fold increase in the ratio of bax to bcl-2 mRNA by day 120. Western blot analysis showed similar changes in Bax and Bcl-2 protein in remnant kidneys, resulting in a 147-fold increase in the ratio of Bax to Bcl-2 on day 120. Immunohistochemistry showed increases in Bax to be located predominantly in tubules in SNx kidneys. Interestingly, Bcl-2 immunostaining increased in some epithelial cells within atrophic tubules despite the overall decrease in Bcl-2 protein and mRNA. The overall renal apoptotic cells correlated closely with the ratio of bax to bcl-2 at both the mRNA and protein levels (r = 0.594 and 0.308, respectively; P: < 0.05). Furthermore, tubular apoptosis correlated positively with the mRNA level of bax (r = 0.471; P: < 0.01) and negatively with the mRNA and protein levels of bcl-2 (r = -0.443 and -0.607, respectively; P: < 0.01). The increase in the ratio of the death inducer (Bax) to the death repressor (Bcl-2) at the mRNA and protein levels may control the apoptosis associated with the progression of tubular atrophy and chronic renal fibrosis within remnant rat kidneys. These observations may have prognostic and therapeutic implications in chronic renal failure.