High risk of misinterpreting liver and spleen stiffness using 2D shear-wave and transient elastography after a moderate or high calorie meal.

Food intake increases liver stiffness, but it is believed that liver stiffness returns to baseline two hours after a meal. The aim of this study was to investigate the impact of different sized meals on liver stiffness. Liver and spleen stiffness was measured with transient elastography (TE) and real-time 2-dimensional shear wave elastography (2D-SWE). Patients ingested a 625 kcal and a 1250 kcal liquid meal on two consecutive days. We measured liver and spleen elasticity, Controlled attenuation parameter (CAP) and portal flow at baseline and after 20, 40, 60, 120 and 180 minutes. Sixty patients participated, 83% with alcoholic liver disease. Twenty-eight patients had METAVIR fibrosis score F0-3 and 32 patients had cirrhosis. Liver stiffness, spleen stiffness and CAP increased after both meals for all stages of fibrosis. False positive 2D-SWE liver stiffness measurements caused 36% and 52% of patients with F0-3 fibrosis to be misclassified with higher stages of fibrosis after the moderate and high caloric meal. Likewise, 10% and 13% of compensated cirrhosis patients were misclassified with clinically significant portal hypertension after the two meals. We observed similar misclassification rates with TE. After three hours, liver stiffness remained elevated more than 20% from baseline in up to 50% of patients. IN CONCLUSION: Liver stiffness, spleen stiffness and CAP increase after a meal across all stages of fibrosis and across elastography techniques. Up to half of patients may be misclassified with higher stages of fibrosis, if they are assessed after less than three hours fasting period.

Shear-wave elastography of the liver and spleen identifies clinically significant portal hypertension: A prospective multicentre study.

BACKGROUND & AIMS: Clinically significant portal hypertension (CSPH) is associated with severe complications and decompensation of cirrhosis. Liver stiffness measured either by transient elastography (TE) or Shear-wave elastography (SWE) and spleen stiffness by TE might be helpful in the diagnosis of CSPH. We recently showed the algorithm to rule-out CSPH using sequential liver- (L-SWE) and spleen-Shear-wave elastography (S-SWE). This study investigated the diagnostic value of S-SWE for diagnosis of CSPH. METHODS: One hundred and fifty-eight cirrhotic patients with pressure gradient measurements were included into this prospective multicentre study. L-SWE was measured in 155 patients, S-SWE in 112 patients, and both in 109 patients. RESULTS: Liver-shear-wave elastography and S-SWE correlated with clinical events and decompensation. SWE of liver and spleen revealed strong correlations with the pressure gradient and to differentiate between patients with and without CSPH. The best cut-off values were 24.6 kPa:L-SWE and 26.3 kPa:S-SWE. L-SWE ≤16.0 kPa and S-SWE ≤21.7 kPa were able to rule-out CSPH. Cut-off values of L-SWE >29.5 kPa and S-SWE >35.6 kPa were able to rule-in CSPH (specificity >92%). Patients with a L-SWE >38.0 kPa had likely CSPH. In patients with L-SWE ≤38.0 kPa, a S-SWE >27.9 kPa ruled in CSPH. This algorithm has a sensitivity of 89.2% and a specificity of 91.4% to rule-in CSPH. Patients not fulfilling these criteria may undergo HVPG measurement. CONCLUSIONS: Liver and spleen SWE correlate with portal pressure and can both be used as a non-invasive method to investigate CSPH. Even though external validation is still missing, these algorithms to rule-out and rule-in CSPH using sequential SWE of liver and spleen might change the clinical practice.

Antithrombotic treatment with direct-acting oral anticoagulants in patients with splanchnic vein thrombosis and cirrhosis.

BACKGROUND: Direct-acting oral anticoagulants (DOACs) are used in patients with splanchnic vein thrombosis (SVT) and cirrhosis, but evidence for safety and efficacy in this setting is limited. Our aim was to identify indications and reasons for starting or switching to DOACs and to report adverse effects, complications and short-term outcome. METHODS: Data collection including demographic information, laboratory values, treatment and complications through the Vascular Liver Disease Interest Group Consortium. RESULTS: Forty-five centres (90%) of the consortium completed the initial eCRF. We report here a series of 94 patients from 17 centres. Thirty-six patients (38%) had cirrhosis. Child-Pugh score was 6 (range 5-8), and MELD score 10.2 (range 6-19). Indications for anticoagulation were splanchnic vein thrombosis (75%), deep vein thrombosis (5%), atrial fibrillation (14%) and others (6%). DOACs used were rivaroxaban (83%), dabigatran (11%) and apixaban (6%). Patients were followed up for a median duration of 15 months (cirrhotic) and 26.5 months (non-cirrhotic). Adverse events occurred in 17% of patients and included one case of recurrent portal vein thrombosis and five cases of bleeding. Treatment with DOACs was stopped in three cases. The major reasons for choosing DOACs were no need for monitoring or inadequacy of INR to guide anticoagulation in cirrhotic patients. Renal and liver function did not change during treatment. CONCLUSIONS: A consistent number of patients with SVT and/or cirrhosis are currently treated with DOACs, which seem to be effective and safe. These data provide a basis for performing randomized clinical trials of DOACs vs. low molecular weight heparin or vitamin K antagonists.

γ-Glutamyl Transferase Is an Independent Biomarker of Splanchnic Thrombosis in Patients With Myeloproliferative Neoplasm.

Myeloproliferative neoplasms (MPNs) are associated with an increased risk of thrombotic events and constitute the major risk factor of splanchnic venous thrombosis (SVT) in Western countries. Although timely anticoagulation resolves SVT, unrecognized SVT frequently leads to portal hypertension and, potentially, variceal bleeding, which may render anticoagulation difficult. Thus, early identification of SVT development is clinically relevant in MPN patients.In this retrospective analysis, we included 126 patients with MPN and/or SVT referred to our hospital between 2009 and 2014. A total of 86 patients diagnosed with MPN formed the first cohort (PV n = 18, ET n = 16, and MF n = 40), whereas 40 patients who had SVT without adjunct MPN formed a control cohort. Median follow-up period was 960 days. Clinical and laboratory data were collected and analyzed for the identification of potential biomarkers applying descriptive statistics, nonparametric testing, Kaplan-Meier, and logistic regression analysis. The relevance of the identified biomarkers was evaluated in an independent 2nd cohort of 181 patients from the MPN registry of the Study Alliance of Leukemia (SAL-MPN).Thirty-three MPN patients (38%) in the 1st cohort had SVT. Elevated levels of aspartate aminotransferase, alanine aminotransferase, serum bilirubin, or γ-GT were significantly correlated to the presence of SVT. In multivariate testing, CRP and aspartate aminotransferase were predictors for survival and γ-GT remained the only significant variable associated with SVT in MPN patients (P < 0.05). These findings were confirmed in the 2nd cohort comprising 42% of patients with MPN suffering from SVT.Elevated γ-GT levels indicate SVT in MPN patients, whereas CRP levels are independent predictors of patient survival.

The Royal Free Hospital-Nutritional Prioritizing Tool Is an Independent Predictor of Deterioration of Liver Function and Survival in Cirrhosis.

BACKGROUND AND AIMS: Malnutrition might affect survival and severity of complications in cirrhotic patients. However, adequate evaluation of the nutritional status is a difficult task since the common assessment tools are either inappropriate or too complicated. A simpler method could evaluate the patient's risk for malnutrition instead of the nutritional status itself. This study evaluated the prediction of clinical deterioration and transplant-free survival in patients with chronic liver disease by two nutritional risk scores. METHODS: In 84 cirrhotic patients, Nutritional Risk Screening (NRS), Royal Free Hospital-Nutritional Prioritizing Tool (RFH-NPT), and the chronic liver disease questionnaire have been assessed. These patients were evaluated at a second time point after a median observation time of 500 days. Another cohort of 64 patients was collected to validate the findings. RESULTS: Of the included patients, 67.7 % were male with a median age of 57 years and a median Child score of 9. RFH-NPT classified 50.7 % of the patients as high-risk patients, and NRS assessed 44.6 % of the patients as moderate- to high-risk patients. RFH-NPT correlated with clinical deterioration, severity of disease (Child score, MELD score), and clinical complications such as ascites, hepatorenal syndrome, and episodes of hepatic encephalopathy. RFH-NPT was an independent predictor of clinical deterioration and transplant-free survival. Furthermore, improvement in RFH-NPT within 500 days was associated with improved survival. CONCLUSION: Assessing the patients' risk for malnutrition by RFH-NPT may be a useful predictor of disease progression and outcome for patients with chronic liver disease.

Chemokine (C-X-C motif) ligand 11 levels predict survival in cirrhotic patients with transjugular intrahepatic portosystemic shunt.

BACKGROUND & AIMS: Chemokines, such as CXCR3-ligands, have been identified to play an important role during hepatic injury, inflammation and fibrosis. While CXCL9 is associated with survival in patients receiving transjugular intrahepatic portosystemic shunt (TIPS), the role of CXCL11 in severe portal hypertension remains unknown. METHODS: CXCL11-levels were measured in 136 patients with liver diseases, and 63 healthy controls. In further 47 cirrhotic patients receiving TIPS, CXCL11 levels were measured in portal and hepatic veins at TIPS insertion by cytometric bead array. CXCL11-levels were measured in 23 patients in cubital vein and right atrium, whereas in 24 patients in portal and hepatic blood at an invasive reevaluation. RESULTS: CXCL11-levels were increased with the severity of liver fibrosis. CXCL11-levels from portal, hepatic and cubital veins and right atrium showed a highly significant correlation among each other in these patients. Furthermore, levels of CXCL11 from the right atrium were significantly higher than those from cubital vein. Interestingly, patients with alcoholic cirrhosis had significantly lower CXCL11-levels, than other aetiologies of cirrhosis. After TIPS, CXCL11 levels correlated with the degree of portal pressure and patients with higher CXCL11-levels in portal and hepatic veins showed higher mortality. Multivariate analysis revealed hepatic CXCL11-levels before TIPS, creatinine and age as independent predictors for survival in TIPS patients, whereas MELD score and low portal CXCL11-levels after TIPS predicted long-term survival. CONCLUSION: CXCL11 levels are mainly increased in patients with non-alcoholic cirrhosis and high portal pressure. Moreover, levels of CXCL11 might predict long-time survival of cirrhotic patients bearing TIPS.

Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis.

INTRODUCTION: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions. METHODS: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot. RESULTS: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated. CONCLUSIONS: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

CXCL9 is a prognostic marker in patients with liver cirrhosis receiving transjugular intrahepatic portosystemic shunt.

BACKGROUND & AIMS: Inflammation, collagen deposition and tissue remodelling are involved in the pathogenesis and complications of cirrhosis with portal hypertension. CXCL9 and other chemokines play an important role in these processes and have been associated with liver injury and complications of liver disease in humans. However, their predictive value in patients with cirrhosis and portal hypertension remains to be established. METHODS: 103 patients with liver cirrhosis who had received TIPS (transjugular intrahepatic portosystemic shunt) were included into this study. The TIPS indication was either refractory ascites or recurrent bleeding. Before and after the TIPS procedure portal and hepatic venous blood samples were obtained in 78 patients. In 25 patients blood samples were obtained from the portal vein, hepatic vein, right atrium and cubital vein at TIPS insertion. Serum levels of CXCL9 were measured by cytometric bead array and correlated with clinical parameters and overall outcome. RESULTS: Portal venous levels of CXCL9 decreased after TIPS. Child-Pugh score, refractory ascites, renal dysfunction and alcoholic aetiology of cirrhosis were associated with increased CXCL9 levels. Importantly, low levels of CXCL9 in portal and hepatic vein samples were prognostic factors for the survival of patients receiving TIPS during long-time follow-up. CONCLUSIONS: The CXCR3 ligand CXCL9 affects the liver and/or is released by the liver and thereby might contribute to hepatic and extrahepatic organ dysfunction. Elevated levels of CXCL9 are associated with shorter survival in cirrhotic patients with severe portal hypertension receiving TIPS. This chemokine should be further evaluated as a novel biomarker for the outcome in patients with cirrhosis and portal hypertension and its modulation as a new therapeutic strategy.

Angiotensin-II type 1 receptor-mediated Janus kinase 2 activation induces liver fibrosis.

UNLABELLED: Activation of the renin angiotensin system resulting in stimulation of angiotensin-II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real-time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human-derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild-type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects were prevented in AT1R(-/-) mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. CONCLUSION: Our study substantiates the important cell-intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis.