Ameet Dhar

Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver

Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein–protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.

The role of hypercoagulability in liver fibrogenesis.

The development of hepatic fibrosis on a background of chronic liver injury represents a complex disease trait modulated through the interaction of host genetic factors and environmental influences. Early observations that hepatic inflammation and cirrhosis are associated with the presence of microthrombi within the hepatic vasculature and fibrin/fibrinogen deposition were followed by epidemiological studies showing that carriage of the Factor V Leiden (FvL) mutation, protein C deficiency and increased expression of factor VIII are associated with accelerated progression to cirrhosis in a chronic hepatitis C infection. Additional data suggest that these factors may influence fibrogenesis in many forms of chronic liver disease and extra-hepatic fibrotic processes. Drawing evidence both from liver research and studies of fibrogenesis in other organ systems, two hypotheses may explain how activity of the coagulation cascade influences the rate of hepatic fibrogenesis: tissue ischaemia and parenchymal extinction and direct thrombin mediated stellate cell activation via PAR-1 cleavage. Drawing on preclinical and clinical studies we discuss the evidence for a role for coagulation cascade activity in hepatic fibrogenesis and explore the proposed pathogenic mechanisms that lead to stellate cell activation. The corollary of an association between hypercoagulation and increased fibrosis is that interference with the coagulation cascade may reduce hepatic fibrosis. We conclude this article by examining the implications for future therapeutic intervention.

Review article: depression and the use of antidepressants in patients with chronic liver disease or liver transplantation

The scale of depression in patients with chronic liver disease (CLD) and those who have received orthotopic liver transplantation (OLT) is poorly characterised. Clinicians are uncertain of how best to manage depression within these patients.

Anticoagulation in chronic liver disease

In this Grand Round presentation, the case of a man with decompensated liver disease is described. He subsequently developed a fatal pulmonary embolism, which may not have occurred if he had been prescribed prophylactic anticoagulation to prevent venous thromboembolic disease. The burden of thrombotic disease in those with chronic liver disease is discussed, before a more detailed analysis of the current evidence, safety data, and clinical dilemmas regarding the use of anticoagulation in patients with chronic liver disease. Finally, the future directions within this field are explored.

Severe cholestatic jaundice after a single administration of ajmaline; a case report and review of the literature

BackgroundAjmaline is a pharmaceutical agent now administered globally for a variety of indications, particularly investigation of suspected Brugada syndrome. There have been previous reports suggesting that repetitive use of this agent may cause severe liver injury, but little evidence exists demonstrating the same effect after only a single administration.Case presentationA 33-year-old man of Libyan origin with no significant past medical history underwent an ajmaline provocation test for investigation of suspected Brugada syndrome. Three weeks later, he presented with painless cholestatic jaundice which peaked in severity at eleven weeks after the test. Blood tests confirmed no evidence of autoimmune or viral liver disease, whilst imaging confirmed the absence of biliary tract obstruction. A liver biopsy demonstrated centrilobular cholestasis and focal rosetting of hepatocytes, consistent with a cholestatic drug reaction. Over the course of the next few months, he began to improve clinically and biochemically, with complete resolution by one year post-exposure.ConclusionWhilst ajmaline-related hepatotoxicity was well-recognised in the era in which the drug was administered as a regular medication, clinicians should be aware that ajmaline may induce severe cholestatic jaundice even after a single dose administration.

P104 Coagulation proteins in liver fibrosis: a role for tissue factor and fibrin/fibrinogen

Introduction Recent evidence suggests a role for the coagulation cascade in promoting liver fibrosis, but with the exception of thrombin the expression and role of individual coagulation proteins in the pathogenesis of liver fibrosis is poorly understood. Furthering our understanding of the role of specific coagulation proteins is essential when considering viable targets for anti-fibrotic therapies. Aim To quantify and qualify the expression of tissue factor (TF) and fibrin/fibrinogen in both murine liver fibrosis and human hepatitis C (HCV) related liver fibrosis. Method C57BL/6J mice (n=7), aged 8 weeks old, were treated with carbon tetrachloride by intraperitoneal injection for a period of 4 weeks to induce liver fibrosis. Animals were then culled and livers extracted and fixed in formalin. Mice injected with normal saline acted as normal controls. For human tissue, archived liver biopsy specimens (n=11) performed for the clinical staging of chronic HCV infection were used. An indirect immunohistochemical detection technique was employed with digital image analysis to qualify and semi-quantify expression of TF and fibrin/fibrinogen in tissue sections. Results In murine liver tissue, TF and fibrin/fibrinogen were expressed in hepatic sinusoids, peri-fibrotic areas and fibrotic septa. Digital image analysis demonstrated significant upregulation of TF (p=0.002) and fibrin/fibrinogen (p=0.009) in fibrotic vs normal control liver tissue. In HCV human liver tissue, TF and fibrin/fibrinogen were expressed in hepatic sinusoids and fibrotic areas. Digital image analysis demonstrated a significant correlation between TF expression and both fibrosis grade (r=0.71; p=0.015) and inflammatory score (r=0.79; p=0.004). Fibrin/fibrinogen expression was significantly correlated with inflammatory score (r=0.82; p=0.007), with a borderline correlation with grade of fibrosis (r=0.66; p=0.056). A significant correlation between TF and fibrin/fibrinogen expression was demonstrated (r=0.82; p=0.024). Conclusion The hepatic expression of TF and fibrin/fibrinogen is upregulated with fibrosis and inflammation. These findings suggest that activation of the coagulation cascade occurs in and may contribute to the generation of hepatic fibrosis. The therapeutic potential of targeted inhibition of specific coagulation proteins need to be evaluated in fibrotic liver disease.

PTH-106 Plasma S100A8/A9: a novel mechanistic biomarker in innate immune activation in acute-on-chronic liver failure

Introduction Acute-on-chronic liver failure (ACLF) is driven by systemic inflammation but lacks reliable diagnostic or prognostic biomarkers. Circulating S100A8/A9 heterodimer (calprotectin) is secreted by activated myeloid cells to activate and propagate innate immune responses and organ dysfunction. This study aims to evaluate circulating levels of S100A8/A9 in ACLF and determine its effect on myeloid cell function. Methods Plasma S100A8/A9 concentration of 92 patients at admission was analysed using enzyme-linked immunosorbent assay (ELISA) in ACLF (n=62), cirrhosis without organ failure (n=28) and healthy control (n=30) groups. Baseline plasma cytokines were measured by multiplex immunoassay. Indices of disease severity and survival was evaluated with Kaplan Meier analysis. Phenotype (CD11b, HLA-DR, Mer tyrosine kinase [MerTK], CD163 and CD206) of healthy CD14 +monocytes cultured with S100A8/A9 in vitro for 24 hours at 0, 1000 and 2500 ng/ml was assessed using flow cytometry (n=6). Results Admission plasma S100A8/A9 was higher in ACLF (median 2000 ng/ml) compared with cirrhotics without organ failure (934.8 ng/ml p=0.007) and healthy control (963 ng/ml p=0.003) (figure 1). S100A8/A9 was higher in patients with systemic inflammatory response syndrome (SIRS) (p=0.045) and non-survivors (p=0.01). Baseline interleukin-1β (IL-1β) was elevated in ACLF compared to healthy (0.07 vs. 0.36 pg/ml p=0.009), correlating with S100A8/A9 concentration (r=0.508 p=0.01). Area under the receiver operating characteristic curve (AUROC) for S100A8/A9 to detect the presence of ACLF was 0.681 (p=0.009). For 90 day mortality in ACLF, AUROC was 0.694 (p=0.014) but highest for the CLIF-ACLF score (0.767, p=0.001). S100A8/A9>1406 ng/ml (sensitivity 0.73 specificity 0.61) was associated with decreased transplant-free survival (log rank p=0.02) (figure 2). S100A8/A9 predicted 90 day mortality (p=0.018) on univariate analysis, remaining significant in a multivariate logistic regression model (OR 1.0 p=0.04). In flow cytometric analysis, activated CD11b+HLA-DRhighMerTKlow myeloid cells (%) significantly increased (p=0.01, Friedman’s ANOVA) as S100A8/A9 concentration increased from 1000 to 2500 ng/ml with a trend to reduction in CD206 (p=0.13) (figure 3).Abstract PTH-106 Figure 1Abstract PTH-106 Figure 2Abstract PTH-106 Figure 3 Conclusions Plasma S100A8/A9 is significantly elevated in ACLF, correlating strongly with activation of pro-inflammatory mediators and indices of disease severity, extra-hepatic organ failure and outcome. Our in vitro data indicate that this mediator promotes inflammation and represents a novel therapeutic target in ACLF.

Letter: depression and the use of anti-depressants in patients with chronic liver disease or liver transplantation - authors' reply.

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390 TARGETED INHIBITION OF TISSUE FACTOR AND THROMBIN ON CD31 EXPRESSING CELLS SUPPRESSES HEPATIC FIBROSIS IN CCL4 TREATED MICE

Introduction Recent evidence suggests a role for the coagulation cascade in promoting liver fibrosis. However, the cellular basis for this relationship is unclear. In order to explore this relationship we employed two unique transgenic mice strains expressing membrane–tethered tissue factor pathway inhibitor (TFPI) or hirudin (anti-thrombin) fusion proteins driven by a CD31 promoter. These strains allow for the selective inhibition of tissue factor (TF) or thrombin on endothelial cells, monocytes and platelets expressing CD31. Aims To evaluate the impact of the targeted inhibition of tissue factor and thrombin on effector cells of liver fibrosis in murine hepatic fibrosis induced by CCl 4 . Methods Liver fibrosis was induced in CD31-TFPI, CD31-Hirudin and wild type control mice with 4 weeks carbon tetrachloride (CCl 4 ) administered by intraperitoneal injection. Animals were culled and livers extracted for histological and biochemical analysis. Fibrosis was scored using a four point semi-quantitative system and quantified by digital image analysis to determine percentage area of fibrosis. Immunohistochemistry to determine α-SMA expression, a marker of hepatic stellate cell activation was performed and the mean number of activated stellate cells was quantified per high power field. Results The percentage area of fibrosis was significantly less in CD31-TFPI (1.89%±0.26, p=0.001) and CD31-Hirudin mice (1.04%±0.16, p=0.00003) in comparison to control mice (3.66%±0.39). Semiquantitative fibrosis scoring showed a significant difference between the CD31-TFPI (median 2/4) and CD31-Hirudin (median 3/4) mice in comparison with control mice (median 3/4, p=0.009) but the difference between the two transgenic strains was not significant. Both transgenic strains demonstrated a significantly reduced mean number of α-SMA stellate cells per high power field in comparison to control mice (CD31-TFPI vs control, 7.4 vs 29.4, p=0.0002; CD31-hirudin vs control, 5.25 vs 29.4, p=0.0002). Conclusion CD31 targeted inhibition of TF and thrombin significantly reduces liver fibrosis and stellate cell activation in a murine CCl 4 model. The data supports the use of this novel murine model as a tool for investigating the cellular biology of the role of coagulation in liver fibrogenesis. It will also provide information for developing new treatments of human liver fibrosis. Competing interests None declared.

825 COAGULATION PROTEINS IN ACUTE LIVER INJURY: A ROLE IN INITIATION AND PROPAGATION

Background: Recent evidence suggests liver injury is in part activated by the coagulation cascade. With the exception of thrombin the function and expression of individual coagulation proteins in the pathogenesis of liver injury is poorly understood. Paracetamol toxicity is a leading cause of liver failure in humans, but the mechanisms of initiation and progression are not clear. Aims: To determine if tissue factor (TF) and fibrin are upregulated in an animal model of acute liver injury and to quantify and qualify the expression of these proteins. Methods: Acute liver injury was induced in mice (C57BL/6 background) by a single intraperitoneal injection of 300mg/kg paracetamol. Control mice received intraperitoneal saline injection. Mice were culled by 24 hours and whole livers extracted and fixed in formalin. Each liver injury was graded based on extent and distribution of necrosis (Grade 0: No injury; Grade 1: perivenular necrosis, no bridging; Grade 2: focal bridging necrosis; Grade 3: extensive bridging necrosis; Grade 4: panlobular necrosis). An indirect immunohistochemical detection technique was employed to qualify and quantify expression of TF and fibrin. Results: Fibrin was expressed in all grades of acute liver injury. Grade 3 and 4 injuries demonstrated confluent fibrin expression with a centrilobular distribution. Less widespread expression was noted in milder injuries. A very strong correlation was demonstrated between fibrin expression and grade of liver injury on visual analogue scale (r = 0.956, p 0.05). Centrilobular TF expression strongly correlated with both grade of injury (r = 0.77, p < 0.001) and fibrin expression (r = 0.731, p < 0.001). Conclusions: TF is expressed normally in a sinusoidal pattern and upregulated with fibrin in a centrilobular pattern of expression in paracetamol induced acute liver injury. These findings suggest a possible role for these proteins in the initiation and propagation of necrosis and the therapeutic potential of their targeted inhibition should be considered in this type of injury.