Background & Aims
Well established risk factors for intrahepatic cholangiocarcinoma such as biliary tract inflammation and liver flukes are not present in most patients in Western countries. Although cirrhosis and other causes of chronic liver disease have been implicated, their contribution as risk factors for cholangiocarcinoma is unclear and our aims were to analyze these emerging potential risk factors by systematic examination of case-control series from geographically diverse regions.
We performed a literature review and meta-analysis of case-control studies on intrahepatic cholangiocarcinoma and cirrhosis and related risk factors. Tests of heterogeneity, publication bias and sensitivity analyses were performed and an overall odds ratio and 95% confidence intervals calculated.
Eleven studies from both high and low prevalence regions were identified. All studies except for those evaluating cirrhosis, diabetes, and obesity exhibited significant heterogeneity. Cirrhosis was associated with a combined OR of 22.92 (95% CI = 18.24 – 28.79). Meta-analysis estimated the overall odds ratio (with 95% confidence intervals) for defined risk factors such as hepatitis B: 5.10 (2.91–8.95), hepatitis C: 4.84 (2.41–9.71), obesity: 1.56 (1.26–1.94), diabetes mellitus type II: 1.89 (1.74–2.07), smoking: 1.31 (0.95–1.82), and alcohol use: 2.81 (1.52–5.21). Sensitivity analysis did not alter the odds ratio for any risk factors except smoking and there was no evidence of publication bias.
Cirrhosis, chronic hepatitis B and C, alcohol use, diabetes, and obesity are major risk factors for intrahepatic cholangiocarcinoma. These data suggest a common pathogenesis of primary intrahepatic epithelial cancers.
cholangiocarcinoma; risk factors; biliary neoplasia
Background & Aim
The mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK) and p38 mediate liver ischemia/reperfusion (I/R) injury via cell death and inflammatory cytokine expression, respectively. Nilotinib is an orally available receptor tyrosine kinase inhibitor used for chronic myelogenous leukemia that also has in vitro activity against JNK and p38. In this study, we examine its therapeutic potential against hepatic I/R injury.
The effects of nilotinib on liver I/R injury were tested using a murine model of warm, segmental liver I/R. Serum ALT was measured and livers were analyzed by histology, RT-PCR, western blot, and flow cytometry. The in vitro effects of nilotinib on hepatocyte and nonparenchymal cell (NPC) MAPK activation and cytokine production were also tested.
Mice receiving nilotinib had markedly lower serum ALT levels and less histologic injury and apoptosis following liver I/R. Nilotinib did not inhibit its known receptor tyrosine kinases. Nilotinib lowered intrahepatic expression of IL-1β, IL-6, MCP-1, and MIP-2 and systemic levels of IL-6, MCP-1, and TNF. Nilotinib reduced NPC activation of p38 MAPK signaling and decreased the recruitment of inflammatory monocytes and their production of TNF. Nilotinib attenuated JNK phosphorylation and hepatocellular apoptosis. In vitro, nilotinib demonstrated direct inhibition of JNK activation in isolated hepatocytes cultured under hypoxic conditions, and blocked activation of p38 MAPK and cytokine production by stimulated NPCs.
Nilotinib lowers both liver JNK activation and NPC p38 MAPK activation and may be useful for ameliorating liver I/R injury in humans.
p38 MAPK; c-Jun NH2-terminal kinase; hepatocytes; nonparenchymal cells; cytokines
Background & Aims
Ethanol-inducible cytochrome P450 2E1 (CYP2E1) activity contributes to oxidative stress. However, CYP2E1 may have an important role in the pathogenesis of high-fat mediated nonalcoholic steatohepatitis (NASH). Thus, the role of CYP2E1 in high-fat mediated NASH development was evaluated.
Male wild-type (WT) and Cyp2e1-null mice were fed a low-fat diet (LFD, 10% energy-derived) or high-fat diet (HFD, 60% energy-derived) for 10 weeks. Liver histology and tissue homogenates were examined for various parameters of oxidative stress and inflammation.
Liver histology showed that only WT mice fed a HFD developed NASH despite increased steatosis in both WT and Cyp2e1-null mice fed HFD. Markers of oxidative stress such as elevated CYP2E1 activity and protein amounts, lipid peroxidation, protein carbonylation, nitration, and glycation with increased phospho-JNK were all markedly elevated only in the livers of HFD-fed WT mice. Furthermore, while the levels of inflammation markers osteopontin and F4/80 were higher in HFD-fed WT mice, TNFα and MCP-1 contents were lower compared to the corresponding LFD-fed WT. Finally, only HFD-fed WT mice exhibited increased insulin resistance and impaired glucose tolerance.
These data suggest that CYP2E1 is critically important in NASH development by promoting oxidative/nitrosative stress, protein modifications, inflammation and insulin resistance.
Liver; CYP2E1; null mice; high-fat diet; NAFLD; NASH; oxidative stress; protein modifications; inflammation; insulin resistance
Background & Aim
Steatohepatitis (SH) is associated with mitochondrial dysfunction and superoxide overgeneration, which can be converted into H2O2 by SOD2. Since mitochondrial GSH (mGSH) plays a critical role in H2O2 reduction, we explored the interplay between superoxide, H2O2 and mGSH in nutritional and genetic models of SH, which exhibit mGSH depletion.
Isolated mitochondria and primary hepatocytes, as well as in vivo SH models showing mGSH depletion to test the consequences of superoxide scavenging.
In isolated mitochondria and primary hepatocytes superoxide scavenging by SOD mimetics or purified SOD decreased superoxide and peroxynitrite generation but increased H2O2 following mGSH depletion despite mitochondrial peroxiredoxin/thioredoxin defense. Selective mGSH depletion sensitized hepatocytes to cell death induced by SOD mimetics and this was prevented by RIP1 kinase inhibition with necrostatin-1 or GSH repletion with GSH ethyl ester (GSHee). Mice fed the methinonie-choline deficient (MCD) diet or MAT1A−/− mice exhibit reduced SOD2 activity; in vivo treatment with SOD mimetics increased liver damage, inflammation, and fibrosis, despite decreased superoxide and 3-nitrotyrosine immunoreactivity, effects that were ameliorated by mGSH replenishment with GSHee but not NAC. As a proof-of-principle of the detrimental role of superoxide scavenging in the face of mGSH depletion, transgenic mice overexpressing SOD2 exhibited enhanced susceptibility to MCD-mediated SH.
These findings underscore a critical role for mGSH in the therapeutic potential of superoxide scavenging in SH, and suggest that the combined approach of superoxide scavenging with mGSH replenishment may be of significance in SH.
Background & Aims
Ballooned hepatocytes in nonalcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine if ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro.
Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using Huh-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3−/− mice.
Ballooned hepatocytes in NASH display diminished expression of the caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which via AP-1 induces SHH expression. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3−/− cells during lipotoxic stress.
Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.
caspase 3; c-Jun-N-terminal Kinase (JNK); lipoapoptosis; lysophosphatidylcholine; palmitate
Hepatocyte transplantation; Injury; Rescue; Stem cells; Tissue engineering
Purinergic signalling has been postulated as a mechanism of cellular signalling since the early 1970s. Cellular responses triggered by extracellular nucleotides and nucleosides occur by defined adenosine (P1) and ATP (P2) receptors, respectively, and play a prominent role in many aspects of health and disease, including those involving the liver. In normal physiology, extracellular nucleotides modulate many of the normal biologic and hepatic metabolic processes such as gluconeogenesis and insulin responsiveness. Further, in multiple disease states, ATP and certain nucleotides serve as danger signals and are involved in heightened purinergic receptor activation in a myriad of pathologic processes. Recently, others and we have shown the regulation of purinergic signalling by ectonucleotidases to play an important role in the acute vascular pathobiology of liver inflammation, regeneration and immunity, as in ischemia reperfusion and transplantation. Increased understanding into mechanisms of extracellular ATP metabolism by such ecto enzymes has also led to novel insights into the exquisite balance of nucleotide P2-receptor and adenosinergic P1-receptor signalling in those chronic hepatic diseases, characterized by steatosis, fibrosis and malignancy. This review will explore the developing role of purinergic signalling in the pathophysiology of liver disease and comment on potential future clinical applications.
Ectonucleotidases; P2-receptor; CD39; CD73; T lymphocytes; Liver disease
Background & Aims
Repair from biliary damages requires the biliary specification of hepatic progenitor cells and the remodeling of ductular reactive structures into branching biliary tubules. We hypothesized that the morphogenetic role of Notch signaling is maintained during the repair process and have addressed this hypothesis using pharmacologic and genetic models of defective Notch signaling.
Treatment with DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) or ANIT (alpha-naphthyl-isothiocyanate) was used to induce biliary damage in wild type mice and in mice with a liver specific defect in the Notch-2 receptor (Notch-2-cKO) or in RPB-Jk. Hepatic progenitor cells, ductular reaction, and mature ductules were quantified using K19 and SOX-9.
In DDC treated wild type mice, pharmacologic Notch inhibition with dibenzazepine decreased the number of both ductular reaction and hepatic progenitor cells. Notch-2-cKO mice treated with DDC or ANIT accumulated hepatic progenitor cells that failed to progress into mature ducts. In RBP-Jk-cKO mice, mature ducts and hepatic progenitor cells were both significantly reduced with respect to similarly treated wild type mice. The mouse progenitor cell line BMOL cultured on matrigel, formed a tubular network allowing the study of tubule formation in vitro; γ-secretase inhibitor treatment and siRNAs silencing of Notch-1, Notch-2 or Jagged-1 significantly reduced both the length and number of tubular branches.
These data demonstrate that Notch signaling plays an essential role in biliary repair. Lack of Notch-2 prevents biliary tubule formation, both in vivo and in vitro. Lack of RBP-Jk inhibits the generation of biliary-committed precursors and tubule formation.
Cholangiocytes; Alagille syndrome; Ductular reaction; GSI; Liver repair; Notch signaling
IL28B polymorphisms have been associated with both treatment induced and spontaneous clearance of hepatitis C virus (HCV). We previously found that LDL cholesterol levels were higher in chronic hepatitis C (CHC) patients with the CC genotype at the rs12979860 polymorphism, located proximal to the IL28 gene. Here we analyzed the association of steatosis with IL28B genotype in treatment naïve patients with CHC.
Two independent cohorts of 145 genotype 1 infected patients from an antifibrotic study and 180 genotype 1 patients from Duke were analyzed for presence and severity of steatosis in relation to the rs12979860 polymorphism at the IL28B locus. TaqMan assay based genotyping classified three groups CC, CT and TT.
CC genotype was associated with a lower prevalence of steatosis. In the antifibrotic study steatosis was found in 47.6% (50/105) of IL28B non-CC versus 22.5% (9/40; p=0.008) in CC patients. Similarly, steatosis was found in 67.4% (89/132) of non-CC patients compared to only 39.6% (19/48; p=0.001) of CC patients in the Duke cohort.
IL28B CC genotype is associated with less pronounced disturbances of lipid metabolism, as reflected both in serum lipoprotein levels and hepatic steatosis, in HCV infection.
chronic hepatitis C; genetic; lipids; IL28B; steatosis
Background & Aims
Aberrant activation of the AKT oncogenic pathway and downregulation of the Sprouty 2 (Spry2) tumor suppressor gene are frequently observed molecular events in human hepatocarcinogenesis. The goal of the present study was to investigate the eventual biochemical and genetic crosstalk between activated AKT and inactivation of Spry2 during liver cancer development by using in vivo and in vitro approaches.
Activated AKT and/or Spry2Y55F, a dominant negative form of Spry2, were overexpressed in the mouse liver via hydrodynamic gene delivery. Histological and biochemical assays were applied to characterize the molecular features of AKT and AKT/Spry2Y55F liver tumors. The human HLE hepatocellular carcinoma (HCC) cell line, stably overexpressing AKT, was transfected with Spry2Y55F to study the molecular mechanisms underlying hepatocarcinogenesis driven by Spry2 loss.
Spry2Y55F overexpression significantly accelerated AKT induced hepatocarcinogenesis in the mouse. AKT/Spry2Y55F liver lesions had increased proliferation and glycolysis and decreased lipogenesis when compared with AKT corresponding lesions. At the molecular level, AKT/Spry2Y55F HCCs exhibited a significantly stronger induction of activated mitogen-activated protein kinase (MAPK) and Pyruvate Kinase M2 (PKM2) pathways than in AKT corresponding lesions. This phenotype was reproduced in HLE cells overexpressing AKT following transfection with Spry2Y55F. Furthermore, we found that concomitant suppression of the MAPK cascade and PKM2 strongly inhibited the growth induced by Spry2Y55F in AKT-overexpressing cells.
Inactivation of Spry2 accelerates AKT induced hepatocarcinogenesis via activation of MAPK and PKM2 pathways.
HCC; AKT; Spry2; MAPK; PKM2
Background & Aims
Liver biopsy, the current clinical gold standard for assessment of fibrosis, is invasive and has sampling errors, and is not optimal for screening, monitoring, or clinical decision-making. Fibrosis is characterized by excessive accumulation of extracellular matrix proteins including type I collagen. We hypothesize that molecular magnetic resonance imaging (MRI) with a probe targeted to type I collagen could provide a direct and non-invasive method for assessment of fibrosis.
Liver fibrosis was generated in rats with diethylnitrosamine and in mice with carbon tetrachloride. Animals were imaged prior to and immediately following i.v. administration of either collagen-targeted probe EP-3533 or non-targeted control Gd-DTPA. Magnetic resonance (MR) signal wash-out characteristics were evaluated from T1 maps and T1-weighted images. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline.
EP-3533 enhanced MR showed greater signal intensity on delayed imaging (normalized signal enhancement mice: control = 0.39±0.04, fibrotic = 0.55±0.03, p <0.01) and slower signal washout in fibrotic liver compared to controls (liver t1/2 = 51.3±3.6 vs 42.0±2.5 min, p <0.05 and 54.5±1.9 vs 44.1±2.9 min, p <0.01 for fibrotic vs controls in rat and mouse models, respectively). Gd-DTPA enhanced MR could not distinguish fibrotic from control animals. EP-3533 gadolinium concentration in liver showed strong positive correlations with hydroxyproline levels (r = 0.74 (rats), r = 0.77 (mice)) and with Ishak scoring (r = 0.84 (rats), r = 0.79 (mice)).
Molecular MRI of liver fibrosis with a collagen-specific probe identifies fibrotic tissue in two rodent models of disease.
Molecular Imaging; Gadolinium; Type I Collagen; Carbon Tetrachloride; Diethylnitrosamine
Background & Aims
Lysosomal Acid Lipase (LAL) deficiency is a rare metabolic storage disease, caused by a marked reduction in activity of LAL, which leads to accumulation of cholesteryl esters (CE) and triglycerides (TG) in lysosomes in many tissues. We used 1H magnetic resonance (MR) spectroscopy to characterize the abnormalities in hepatic lipid content and composition in patients with LAL deficiency, and in ex vivo liver tissue from a LAL deficiency rat model. Secondly, we used MR spectroscopy to monitor the effects of an enzyme replacement therapy (ERT), sebelipase alfa (a recombinant human lysosomal acid lipase), on hepatic TG and CE content in the preclinical model.
Human studies employed cohorts of LAL-deficient patients and NAFLD subjects. Rat experimental groups comprised ex vivo liver samples of wild type, NAFLD, LAL-deficient, and LAL-deficient rats receiving 4 weeks of sebelipase alfa treatment. Hepatic 1H MR spectroscopy was performed using 3T (human) and 7T (preclinical) MRI scanners to quantify hepatic cholesterol and triglyceride content.
CE accumulation was identified in LAL deficiency in both human and preclinical studies. A significant decrease in hepatic CE was observed in LAL-deficient rats following treatment with sebelipase alfa.
We demonstrate an entirely non-invasive method to identify and quantify the hepatic lipid signature associated with a rare genetic cause of fatty liver. The approach provides a more favorable alternative to repeated biopsy sampling for diagnosis and disease progression / treatment monitoring of patients with LAL deficiency and other disorders characterised by increased free cholesterol and/or cholesteryl esters.
CESD, cholesteryl ester storage disease; LAL, lysosomal acid lipase; CE, cholesteryl ester; TG, triglyceride; ERT, enzyme replacement therapy; NAFLD, non-alcoholic fatty liver disease; Wolman disease; Cholesteryl ester storage disease; 1H MR spectroscopy; 13C MR spectroscopy; Liver fat; Lysosomal acid lipase; LIPA; LAL deficiency; Enzyme replacement therapy; Sebelipase alfa
The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR(−/−)) by CCl(4) intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl(4)-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.
Medical etymology sometimes provides unexpected information about health concepts and medical practice in different times and cultures. We conducted an etymological analysis of the terms used to indicate “liver” in Germanic and Romance languages. The Greek word “hèpar” was originally connected to the concept of “pleasure”, showing that in antiquity the liver was considered to be the seat of soul and human feelings. In Romance languages, the Latin term “ficatum” was linked to the ancient practice of fattening geese with figs (ficus in Latin) to make their livers more delicious. This relationship between the liver, fat, and carbohydrates seems to indicate that ancient gourmets had clear knowledge of the nutritional mechanisms underlying “fatty liver” in animals. On the other hand, the Germanic term “lifere” was initially connected to “life”, underscoring the relation of the liver to health and existence. In the Early Modern Age, the liver became a recurring image in political reflection, especially within the Elizabethan tradition of the body politic, where the king was frequently described as the “liver” of his country. Finally, the liver was used to indicate courage, or the lack of it: some modern French and English idiomatic expressions derive from the ancient belief that people who had no blood in their liver (“lily-livered”) would thus be cowards or betrayers.
Medical etymology; Literature; Liver; Figs; History
Background & Aims
Nonalcoholic fatty liver disease (NAFLD) is a cardiovascular risk factor. Although modest alcohol consumption may reduce the risk for cardiovascular mortality, whether patients with NAFLD should be allowed modest alcohol consumption remains an important unaddressed issue. We aimed to evaluate the association between modest alcohol drinking and nonalcoholic steatohepatitis(NASH), among subjects with NAFLD.
In a Cross-sectional analysis of adult participants in the NIH NASH Clinical Research Network, only modest or non-drinkers were included: participants identified as 1) drinking > 20gm/day, 2) binge drinkers, or 3) non-drinkers with previous alcohol consumption were excluded. The odds of having a histological diagnosis of NASH and other histological features of NAFLD were analyzed using multiple ordinal logistic regression.
The analysis included 251 lifetime non-drinkers and 331 modest drinkers. Modest drinkers compared to nondrinkers had lower odds of having a diagnosis of NASH (Summary odds ratio 0.56, 95%CI 0.39–0.84, p=0.002). The odds of NASH decreased as the frequency of alcohol consumption increased within the range of modest consumption. Modest drinkers also had significantly lower odds for fibrosis (OR 0.56 95%CI 0.41–0.77) and ballooning hepatocellular injury (OR 0.66 95%CI 0.48–0.92) than lifetime non-drinkers.
In a large, well-characterized population with biopsy-proven NAFLD, modest alcohol consumption was associated with lesser degree of severity as determined by lower odds of the key features that comprise a diagnosis of steatohepatitis, as well as fibrosis. These findings demonstrate the need for prospective studies and a coordinated consensus on alcohol consumption recommendations in NAFLD.
nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; alcohol; liver biopsy
Since its discovery in the early 1990s, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway has been found to play key roles in regulating many key cellular processes such as survival, proliferation, and differentiation. There are seven known mammalian STAT family members: STAT1, 2, 3, 4, 5a, 5b, and 6. In the liver, activation of these STAT proteins is critical for anti-viral defense against hepatitis viral infection and for controlling injury, repair, inflammation, and tumorigenesis. The identification of functions for these STAT proteins has increased our understanding of liver disease pathophysiology and treatments, while also suggesting new therapeutic modalities for managing liver disease.
HCV; interferon; liver injury; liver regeneration; liver tumor
Background & Aims
Blocking intestinal bile acid (BA) absorption by inhibiting or inactivating the apical sodium-dependent BA transporter (Asbt) classically induces hepatic BA synthesis. In contrast, blocking intestinal BA absorption by inactivating the basolateral BA transporter, organic solute transporter alpha–beta (Ostα–Ostβ) is associated with an altered homeostatic response and decreased hepatic BA synthesis. The aim of this study was to determine the mechanisms underlying this phenotype, including the role of the farnesoid X receptor (FXR) and fibroblast growth factor 15 (FGF15).
BA and cholesterol metabolism, intestinal phenotype, expression of genes important for BA metabolism, and intestinal FGF15 expression were examined in wild type, Ostα−/−, Fxr−/−, and Ostα−/−Fxr−/− mice.
Inactivation of Ostα was associated with decreases in hepatic cholesterol 7α-hydroxylase (Cyp7a1) expression, BA pool size, and intestinal cholesterol absorption. Ostα−/− mice exhibited significant small intestinal changes, including altered ileal villus morphology, and increases in intestinal length and mass. Total ileal FGF15 expression was elevated almost 20-fold in Ostα−/− mice as a result of increased villus epithelial cell number and ileocyte FGF15 protein expression. Ostα−/−Fxr−/− mice exhibited decreased ileal FGF15 expression, restoration of intestinal cholesterol absorption, and increases in hepatic Cyp7a1 expression, fecal BA excretion, and BA pool size. FXR deficiency did not reverse the intestinal morphological changes or compensatory decrease for ileal Asbt expression in Ostα−/− mice.
These results indicate that signaling via FXR is required for the paradoxical repression of hepatic BA synthesis but not the complex intestinal adaptive changes in Ostα−/− mice.
Bile acids; Enterohepatic circulation; Transporters; Cholesterol; Cyp7a1; FXR
Background & Aims
Sinusoidal obstruction syndrome (SOS) following oxaliplatin based chemotherapy can have a significant impact on post-operative outcome following resection of colorectal liver metastases. To date no relevant experimental models of oxaliplatin induced SOS have been described. The aim of this project was to establish a rodent model which could be utilised to investigate mechanisms underlying SOS to aid the development of therapeutic strategies.
C57Bl/6 mice, maintained on a purified diet, were treated with intra-peritoneal FOLFOX (n = 10), or vehicle (n = 10), weekly for five weeks and culled one week following final treatment. Sections of the liver and spleen were fixed in formalin and paraffin embedded for histological analysis. The role of oxidative stress on experimental-induced SOS was determined by dietary supplementation with butylated hydroxyanisole and N-acetylcysteine.
FOLFOX treatment was associated with the development of sinusoidal dilatation and hepatocyte atrophy on H&E stained sections of the liver in keeping with SOS. Immunohistochemistry for p21 demonstrated the presence of replicative senescence within the sinusoidal endothelium.
FOLFOX induced endothelial damage leads to a pro-thrombotic state within the liver associated with upregulation of PAI-1 (p <0.001), vWF (p <0.01) and Factor X (p <0.001), which may contribute to the propagation of liver injury.
Dietary supplementation with the antioxidant BHA prevented the development of significant SOS.
We have developed the first reproducible model of chemotherapy induced SOS that reflects the pathogenesis of this disease in patients. It appears that the use of antioxidants alongside oxaliplatin based chemotherapy may be of value in preventing the development of SOS in patients with colorectal liver metastases.
AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BHA, butylated hydroxyanisole; CRLM, colorectal liver metastases; CXCL1/2, chemokine (C-X-C motif) ligand 1/2; GAPDH, gyceraldehyde 3-phosphate dehydrogenase; H&E, haematoxylin and eosin; HPF, high powered field; γH2AX, phosphorylated form of the H2A histone family, member X; i.p., intraperitoneal; IL-6, interleukin 6; MCP1, monocyte chemotactic protein-1; NAC, N-acetylcysteine; NRF2, nuclear factor (erythroid-derived 2)-like 2; NQO1, NAD(P)H dehydrogenase 1; PCNA, proliferating cell nuclear antigen; PAI-1, plasminogen activatior inhibitor 1; PAR 1/2, protease activated receptor 1/2; STAT3, signal transducer and activator of transcription 3; SOS, sinusoidal obstruction syndrome; TXN1, thioredoxin 1; VEGF-A/B/C, vascular endothelial growth factor A/B/C; VEGFR-1/2, vascular endothelial growth factor receptor ½; vWF, von Willebrand factor; Sinusoidal obstruction syndrome; Oxaliplatin; Colorectal liver metastases; Chemotherapy induced liver injury
Background & Aims
Administration of terlipressin plus albumin is effective in reversing type 1 HRS as compared to albumin alone. However, only about 1/3 of patients respond to treatment, therefore, predictors of response and survival would help identify the patients most likely to benefit from treatment.
We analyzed our controlled trial of terlipressin vs. placebo (Gastroenterology 2008;134:1360) to define factors predictive of a response and to correlate hemodynamic changes to changes in renal function.
Single variant analysis showed treatment with terlipressin, MELD score, and baseline serum creatinine to be predictive of HRS reversal. Alcoholic hepatitis, baseline serum creatinine, and MELD score were predictive of survival. When treatment was not considered as a variable, only baseline serum creatinine predicted HRS reversal. Baseline serum creatinine, presence of alcoholic hepatitis, and Child-Pugh score were also predictive of survival on multivariate analysis. The rise in mean arterial pressure (MAP) following terlipressin administration was not predictive of HRS reversal. However, in those who achieved HRS reversal from terlipressin, there was a significant rise in MAP from beginning to end of treatment.
The most consistent predictor of response to terlipressin and of survival is the baseline serum creatinine. Patients most likely to benefit from terlipressin have earlier onset renal failure (i.e. serum creatinine <5.0 mg/dl). A sustained rise in MAP is required for HRS reversal. As MAP is a surrogate marker for the hyperdynamic circulation, it is only with improvement in the hyperdynamic circulation that HRS reversal is observed.
Background & Aims
While the majority of HCV-infected patients progress to chronic hepatitis, a small fraction of individuals are able to clear the virus. Resolution of infection occurs within the first few weeks to months of infection, suggesting that innate immune functions may be critical for early control. Epidemiologic data support a role for particular NK cell receptor bearing populations in this control, yet the mechanism by which NK cells respond to HCV early in infection is unknown.
Changes in the phenotype and function of NK cells were investigated in a cohort of 43 individuals identified during various stages of HCV infection with different clinical outcomes.
Acute, chronic, and resolved HCV infections were characterized by an expansion of CD56neg NK cells. Furthermore, increased levels of HLA-C-binding KIR+ NK cells were observed in HCV resolvers, while all stages of HCV infection were associated with reduced percentages of NKG2D+, NKp30+, and NKp46+ NK cells, and a slight increase in the ability of NK cells to respond to target cells bearing the ligands for these receptors. In contrast, NKG2A+ and CD94+ NK cells were elevated in acute and chronic HCV infection, but not in resolved infection. Most importantly, in acute infection, lower frequencies of NKp30+, NKp46+, CD161+, and NKG2D+ NK cells were observed in patients who were subsequently able to clear HCV infection than in those becoming chronically infected.
These data implicate particular populations of NK cells in the early control and clearance of HCV infection.
Hepatitis C virus; Natural killer cells; Innate immunity; CD161; NK-p30; NKp46; Resolution of HCV infection; Acute infection
A total of 213 patients with compensated cirrhosis, portal hypertension and no varices were included in a trial evaluating beta-blockers in preventing varices. Predictors of the development of hepatocellular carcinoma (HCC), including hepatic venous pressure gradient (HVPG) were analyzed.
Baseline laboratory tests, ultrasound and HVPG measurements were performed. Patients were followed prospectively every three months until development of varices or variceal bleeding or end of the study in 09/02. The endpoint was HCC development according to standard diagnostic criteria. Univariate and multivariate Cox regression models were developed to identify predictors of HCC.
In a median follow-up of 58 months 26/213 (12.2%) patients developed HCC. Eight patients were transplanted and 28 patients died without HCC. Twenty-one (84%) HCC developed in patients with HCV. On multivariate analysis HVPG (HR 1.18; 95%CI 1.08–1.29), albumin (HR 0.34; 95%CI 0.14–0.83) and viral etiology (HR 4.59; 95%CI 1.51–13.92) were independent predictors of HCC development. ROC curves identified 10 mmHg of HVPG as the best cutoff; those who had an HVPG above this value had a 6-fold increase in the HCC incidence.
Portal hypertension is an independent predictor of HCC development. An HVPG >10 mmHg is associated with a 6-fold increase of HCC risk.
Portal hypertension; End-stage liver disease; Liver cancer; Albumin; Predictive factors; Multivariate analysis
Background & Aims
Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice.
PCI-24781 [50 mg/kg/day] and control solution were delivered to BK5.erbB2 mice for four weeks. The therapeutic effect of PCI-24781 was evaluated via ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms for the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild-type mice was analyzed.
Treatment of BK5.erbB2 mice with PCI-24781 for one month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with down-regulation of erbB2 mRNA and erbB2 protein/activity and up-regulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for erbB2, in BTC cells. PCI-24781 had more effect on inhibiting growth of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. MiRNA profiling revealed that the expression of several miRNAs were significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including up-regulated miR21, which was down-regulated by PCI-24781.
These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing erbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have potential value as a novel chemotherapeutic agent against human BTC in which erbB2 is overexpressed.
Background & Aims
Hepatocellular carcinoma (HCC) is a heterogeneous cancer in which sorafenib is the only approved systemic therapy. Histone deacetylases (HDAC) are commonly dysregulated in cancer and therefore represent promising targets for therapies, however their role in HCC pathogenesis is still unknown. We analyzed the expression of 11 HDACs in human HCCs and assessed the efficacy of the pan-HDAC inhibitor panobinostat alone and in combination with sorafenib in preclinical models of liver cancer.
Gene expression and copy number changes were analyzed in a cohort of 334 human HCCs, while the effects of panobinostat and sorafenib were evaluated in 3 liver cancer cell lines and a murine xenograft model.
Aberrant HDAC expression was identified and validated in 91 and 243 HCCs, respectively. Upregulation of HDAC3 and 5 mRNAs were significantly correlated with DNA copy number gains. Inhibiting HDACs with panobinostat led to strong anti-tumoral effects in vitro and vivo, enhanced by the addition of sorafenib. Cell viability and proliferation declined, while apoptosis and autophagy increased. Panobinostat increased Histone H3 and HSP90 acetylation, downregulated BIRC5 (survivin) and upregulated CDH1. Combination therapy with panobinostat and sorafenib significantly decreased vessel density, and most significantly decreased tumor volume and increased survival in HCC xenografts.
Aberrant expression of several HDACs and copy number gains of HDAC3 and HDAC5 occur in HCC. Treatment with panobinostat combined with sorafenib demonstrated the highest preclinical efficacy in HCC models, providing the rationale for clinical studies with this novel combination.
histone modification; signaling pathways; molecular therapies
Background & Aims
Intestinal dysbiosis and bacterial translocation is common in patients with advanced liver disease, and there is strong evidence that the translocation of bacteria and their products across the epithelial barrier drives experimental liver disease progression. The aims of our study were to investigate dynamics of bacterial translocation and changes in the enteric microbiome in early stages of liver disease.
Cholestatic liver injury was induced by ligation of the common bile duct (BDL) and toxic liver injury by injection of carbon tetrachloride (CCl4) in mice.
Increased intestinal permeability and bacterial translocation occurred one day following liver injury in both disease models. This was accompanied by decreased intestinal expression of the tight junction protein occludin. Although BDL resulted in a rapid onset of intestinal bacterial overgrowth, bacterial overgrowth was observed in mice injected with CCl4 only in advanced stages of liver fibrosis. To further assess the qualitative changes in the intestinal microbiome, massively parallel pyrosequencing of 16S rRNA genes revealed minor microbial changes following BDL, while CCl4 administration resulted in a relative abundance of Firmicutes and Actinobacteria compared with oil injected mice. Four different liver disease models (cholestasis, toxic, alcohol, obesity) show few similarities in their intestinal microbiome.
Acute liver injury is associated with an early onset of increased intestinal permeability and bacterial translocation that precede changes in the microbiome. The enteric microbiome differs with respect to the etiology of liver disease.
bacterial overgrowth; microbiome; dysbiosis; bacterial translocation; 16S rRNA sequencing