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Background & aims: Vitamin D, best known to regulate bone mineralization, has numerous additional roles including regulation inflammatory pathways. Recently, an increased incidence of 25-hydroxyvitamin D3 (25(OH)D3) deficiency has been found in subjects suffering from liver diseases. We here investigated if low vitamin D levels might be associated with prognosis, inflammation and infectious complications in patients with cirrhosis.
Methods: We performed a prospective cohort study investigating the relation between 25(OH)D3 levels and stages of cirrhosis, mortality and complications of cirrhosis, including infections.
Results: 251 patients with cirrhosis were enrolled into the present prospective cohort study. 25(OH)D3 levels were quantified by radioimmunoassay from serum samples obtained at study inclusion. The mean follow-up time was 411 ± 397 days with a range of 1-1382 days. 30 (12.0%) patients underwent liver transplantation and 85 (33.8%) individuals died within the study. The mean serum 25(OH)D3 concentration was 8.93 ± 7.1 ng/ml with a range of 1.0 to 46.0 ng/ml. 25(OH)D3 levels differed significantly between Child Pugh scores and showed a negative correlation with the model of end stage liver disease (MELD) score. Patients with decompensated cirrhosis and infectious complications, had significantly lower 25(OH)D3 levels compared to subjects without complications. Low 25(OH)D3 was associated with mortality in uni- as well as multivariate Cox regression models.
Conclusions: 25(OH)D3 deficiency is associated with advanced liver disease and low 25(OH)D3 levels are an indicator for a poor outcome and are associated with infectious complications.
Background: Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRs) circulating in the blood are an emerging new class of biomarkers. In particular, the serum level of the liver-specific miR-122 might be a clinically useful new parameter in patients with acute or chronic liver disease.
Aim: Here we investigated if the serum level of miR-122 might be a prognostic parameter in patients with liver cirrhosis.
Methods: 107 patients with liver cirrhosis in the test cohort and 143 patients in the validation cohort were prospectively enrolled into the present study. RNA was extracted from the sera obtained at the time of study enrollment and the level of miR-122 was assessed. Serum miR-122 levels were assessed by quantitative reverse-transcription PCR (RT-PCR) and were compared to overall survival time and to different complications of liver cirrhosis.
Results: Serum miR-122 levels were reduced in patients with hepatic decompensation in comparison to patients with compensated liver disease. Patients with ascites, spontaneous bacterial peritonitis and hepatorenal syndrome had significantly lower miR-122 levels than patients without these complications. Multivariate Cox regression analysis revealed that the miR-122 serum levels were associated with survival independently from the MELD score, sex and age.
Conclusions: Serum miR-122 is a new independent marker for prediction of survival of patients with liver cirrhosis.
Cyclic AMP analogs containing hydrophobic modification of C(8) at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2'-O-methyl-adenosine (8-pCPT-2'-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2'-O-methyl-ado, and, to a smaller extent, 8-pCPT-2'-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A(2A) adenosine receptor (A(2A)R)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A(2A)R-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A(2A)R-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.
Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.
Background: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study.
Methods: The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied.
Results: The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles.
Conclusions: Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.
The nuclear factor kappa beta (NFκB) signaling pathway plays an important role in liver homeostasis and cancer development. Tax1-binding protein 1 (Tax1BP1) is a regulator of the NFκB signaling pathway, but its role in the liver and hepatocellular carcinoma (HCC) is presently unknown. Here we investigated the role of Tax1BP1 in liver cells and murine models of HCC and liver fibrosis. We applied the diethylnitrosamine (DEN) model of experimental hepatocarcinogenesis in Tax1BP1+/+ and Tax1BP1−/− mice. The amount and subsets of non-parenchymal liver cells in in Tax1BP1+/+ and Tax1BP1−/− mice were determined and activation of NFκB and stress induced signaling pathways were assessed. Differential expression of mRNA and miRNA was determined. Tax1BP1−/− mice showed increased numbers of inflammatory cells in the liver. Furthermore, a sustained activation of the NFκB signaling pathway was found in hepatocytes as well as increased transcription of proinflammatory cytokines in isolated Kupffer cells from Tax1BP1−/− mice. Several differentially expressed mRNAs and miRNAs in livers of Tax1BP1−/− mice were found, which are regulators of inflammation or are involved in cancer development or progression. Furthermore, Tax1BP1−/− mice developed more HCCs than their Tax1BP1+/+ littermates. We conclude that Tax1BP1 protects from liver cancer development by limiting proinflammatory signaling.
Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
Background: Autotaxin (ATX) and its product lysophosphatidic acid (LPA) are considered to be involved in the development of liver fibrosis and elevated levels of serum ATX have been found in patients with hepatitis C virus associated liver fibrosis. However, the clinical role of systemic ATX in the stages of liver cirrhosis was unknown. Here we investigated the relation of ATX serum levels and severity of cirrhosis as well as prognosis of cirrhotic patients.
Methods: Patients with liver cirrhosis were prospectively enrolled and followed until death, liver transplantation or last contact. Blood samples drawn at the day of inclusion in the study were assessed for ATX content by an enzyme-linked immunosorbent assay. ATX levels were correlated with the stage as well as complications of cirrhosis. The prognostic value of ATX was investigated by uni- and multivariate Cox regression analyses. LPA concentration was determined by liquid chromatography-tandem mass spectrometry.
Results: 270 patients were enrolled. Subjects with liver cirrhosis showed elevated serum levels of ATX as compared to healthy subjects (0.814±0.42 mg/l vs. 0.258±0.40 mg/l, P<0.001). Serum ATX levels correlated with the Child-Pugh stage and the MELD (model of end stage liver disease) score and LPA levels (r = 0.493, P = 0.027). Patients with hepatic encephalopathy (P = 0.006), esophageal varices (P = 0.002) and portal hypertensive gastropathy (P = 0.008) had higher ATX levels than patients without these complications. Low ATX levels were a parameter independently associated with longer overall survival (hazard ratio 0.575, 95% confidence interval 0.365–0.905, P = 0.017).
Conclusion: Serum ATX is an indicator for the severity of liver disease and the prognosis of cirrhotic patients.
Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC.
Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%.
Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC.
Background and aims: Expression of carbonic anhydrase IX (CA9), an enzyme expressed in response to hypoxia, acidosis and oncogenic alterations, is reported to be a prognostic factor in HCC patients. Here we evaluated serum CA9 levels in HCC and cirrhosis patients.
Methods: HCC and cirrhosis patients were prospectively recruited and CA9 levels were determined. CA9 levels were compared to stages of cirrhosis and HCC stages. The association of the CA9 levels and overall survival (OS) was assessed. Furthermore, immunohistochemical CA9 expression in HCC and cirrhosis was evaluated.
Results: 215 patients with HCC were included. The median serum CA9 concentration in patients with HCC was 370 pg/ml and significantly higher than in a healthy cohort. Patients with advanced cancer stages (BCLC and ALBI score) had hid significant higher levels of CA9 in the serum. HCC patients with high serum CA9 concentrations (>400 pg/ml) had an increased mortality risk (hazard ratio (HR) 1.690, 95% confidence interval (CI) 1.017–2.809, P = 0.043). Serum CA9 concentration in cirrhotic patients did not differ significantly from HCC patients. Higher CA9 levels in cirrhotic patients correlated with portal hypertension and esophageal varices. Patients with ethanol induced cirrhosis had the highest CA9 levels in both cohorts. Levels of CA9 did not correlate with immunohistochemical expression.
Conclusions: We conclude that a high CA9 level is a possible prognostic indicator for a poor outcome in HCC patients. The high CA9 levels are probably mainly associated with portal hypertension. Ductular reactions might be a possible source of serum CA9.