Consequences of Amyloid-ß Deficiency for the Liver.

The hepatic content of amyloid beta (Aß) decreases drastically in human and rodent cirrhosis highlighting the importance of understanding the consequences of Aß deficiency in the liver. This is especially relevant in view of recent advances in anti-Aß therapies for Alzheimer's disease (AD). Here, it is shown that partial hepatic loss of Aß in transgenic AD mice immunized with Aß antibody 3D6 and its absence in amyloid precursor protein (APP) knockout mice (APP-KO), as well as in human liver spheroids with APP knockdown upregulates classical hallmarks of fibrosis, smooth muscle alpha-actin, and collagen type I. Aß absence in APP-KO and deficiency in immunized mice lead to strong activation of transforming growth factor-ß (TGFß), alpha secretases, NOTCH pathway, inflammation, decreased permeability of liver sinusoids, and epithelial-mesenchymal transition. Inversely, increased systemic and intrahepatic levels of Aß42 in transgenic AD mice and neprilysin inhibitor LBQ657-treated wild-type mice protect the liver against carbon tetrachloride (CCl4)-induced injury. Transcriptomic analysis of CCl4-treated transgenic AD mouse livers uncovers the regulatory effects of Aß42 on mitochondrial function, lipid metabolism, and its onco-suppressive effects accompanied by reduced synthesis of extracellular matrix proteins. Combined, these data reveal Aß as an indispensable regulator of cell-cell interactions in healthy liver and a powerful protector against liver fibrosis.

Saturated Fat-Mediated Upregulation of IL-32 and CCL20 in Hepatocytes Contributes to Higher Expression of These Fibrosis-Driving Molecules in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases, ranging from liver steatosis to metabolic dysfunction-associated steatohepatitis (MASH), increasing the risk of developing cirrhosis and hepatocellular carcinoma (HCC). Fibrosis within MASLD is critical for disease development; therefore, the identification of fibrosis-driving factors is indispensable. We analyzed the expression of interleukin 32 (IL-32) and chemokine CC ligand 20 (CCL20), which are known to be linked with inflammation and fibrosis, and for their expression in MASLD and hepatoma cells. RT-PCR, ELISA and Western blotting analyses were performed in both human liver samples and an in vitro steatosis model. IL-32 and CCL20 mRNA expression was increased in tissues of patients with NASH compared to normal liver tissue. Stratification for patatin-like phospholipase domain-containing protein 3 (PNPLA3) status revealed significance for IL-32 only in patients with I148M (rs738409, CG/GG) carrier status. Furthermore, a positive correlation was observed between IL-32 expression and steatosis grade, and between IL-32 as well as CCL20 expression and fibrosis grade. Treatment with the saturated fatty acid palmitic acid (PA) induced mRNA and protein expression of IL-32 and CCL20 in hepatoma cells. This induction was mitigated by the substitution of PA with monounsaturated oleic acid (OA), suggesting the involvement of oxidative stress. Consequently, analysis of stress-induced signaling pathways showed the activation of Erk1/2 and p38 MAPK, which led to an enhanced expression of IL-32 and CCL20. In conclusion, cellular stress in liver epithelial cells induced by PA enhances the expression of IL-32 and CCL20, both known to trigger inflammation and fibrosis.

Gas6 in chronic liver disease-a novel blood-based biomarker for liver fibrosis.

The expression of the receptor tyrosine kinase Axl and its cleavage product soluble Axl (sAxl) is increased in liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In this multicenter study, we evaluated the diagnostic value of Gas6, the high-affinity ligand of Axl, in patients with chronic liver disease. Levels of sAxl and Gas6, and their albumin (alb) ratios were analyzed in serum samples of patients with biopsy-proven liver fibrosis, end-stage liver disease, HCC, and healthy controls, and were compared to Fibrosis-4 (FIB-4), enhanced liver fibrosis (ELF™) test, Child-Pugh score (CPS), model of end-stage liver disease (MELD) score, hepatic venous pressure gradient, and α-fetoprotein, respectively. A total of 1111 patients (median age 57.8 y, 67.3% male) was analyzed. Gas6/alb showed high diagnostic accuracy for the detection of significant (≥F2: AUC 0.805) to advanced fibrosis (≥F3: AUC 0.818), and was superior to Fib-4 for the detection of cirrhosis (F4: AUC 0.897 vs. 0.878). In addition, Gas6/alb was highly predictive of liver disease severity (Odds ratios for CPS B/C, MELD ≥ 15, and clinically significant portal hypertension (CSPH) were 16.534, 10.258, and 12.115), and was associated with transplant-free survival (Hazard ratio 1.031). Although Gas6 and Gas6/alb showed high diagnostic accuracy for the detection of HCC in comparison to chronic liver disease patients without cirrhosis (AUC 0.852, 0.868), they failed to discriminate between HCC in cirrhosis versus cirrhosis only. In conclusion, Gas6/alb shows a high accuracy to detect significant to advanced fibrosis and cirrhosis, and predicts severity of liver disease including CSPH.

Interleukin-1ß Attenuates Expression of Augmenter of Liver Regeneration (ALR) by Regulating HNF4α Independent of c-Jun.

Inflammasomes and innate immune cells have been shown to contribute to liver injury, thereby activating Kupffer cells, which release several cytokines, including IL-6, IL-1ß, and TNFα. Augmenter of liver regeneration (ALR) is a hepatotropic co-mitogen that was found to have anti-oxidative and anti-apoptotic properties and to attenuate experimental non-alcoholic fatty liver disease (NAFLD) and cholestasis. Additionally, hepatic ALR expression is diminished in patients with NAFLD or cholestasis, but less is known about the mechanisms of its regulation under these conditions. Therefore, we aimed to investigate the role of IL-1ß in ALR expression and to elucidate the molecular mechanism of this regulation in vitro. We found that ALR promoter activity and mRNA and protein expression were reduced upon treatment with IL-1ß. Early growth response protein-1 (Egr-1), an ALR inducer, was induced by IL-1ß but could not activate ALR expression, which may be attributed to reduced Egr-1 binding to the ALR promoter. The expression and nuclear localization of hepatocyte nuclear factor 4 α (HNF4α), another ALR-inducing transcription factor, was reduced by IL-1ß. Interestingly, c-Jun, a potential regulator of ALR and HNF4α, showed increased nuclear phosphorylation levels upon IL-1ß treatment but did not change the expression of ALR or HNF4α. In conclusion, this study offers evidence regarding the regulation of anti-apoptotic and anti-oxidative ALR by IL-1ß through reduced Egr-1 promoter binding and diminished HNF4α expression independent of c-Jun activation. Low ALR tissue levels in NAFLD and cholestatic liver injury may be caused by IL-1ß and contribute to disease progression.

Hepatocyte expressed chemerin-156 does not protect from experimental non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine-choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.

Preclinical Efficacy and Toxicity Analysis of the Pan-Histone Deacetylase Inhibitor Gossypol for the Therapy of Colorectal Cancer or Hepatocellular Carcinoma.

Gossypol, a sesquiterpenoid found in cotton seeds, exerts anticancer effects on several tumor entities due to inhibition of DNA synthesis and other mechanisms. In clinical oncology, histone deacetylase inhibitors (HDACi) are applied as anticancer compounds. In this study, we examined whether gossypol harbors HDAC inhibiting activity. In vitro analyses showed that gossypol inhibited class I, II, and IV HDAC, displaying the capability to laterally interact with the respective catalytic center and is, therefore, classified as a pan-HDAC inhibitor. Next, we studied the effects of gossypol on human-derived hepatoma (HepG2) and colon carcinoma (HCT-116) cell lines and found that gossypol induced hyperacetylation of histone protein H3 and/or tubulin within 6 h. Furthermore, incubation with different concentrations of gossypol (5-50 µM) over a time period of 96 h led to a prominent reduction in cellular viability and proliferation of hepatoma (HepG2, Hep3B) and colon carcinoma (HCT-116, HT-29) cells. In-depth analysis of underlying mechanisms showed that gossypol induced apoptosis via caspase activation. For pre-clinical evaluation, toxicity analyses showed toxic effects of gossypol in vitro toward non-malignant primary hepatocytes (PHH), the colon-derived fibroblast cell line CCD-18Co, and the intestinal epithelial cell line CCD 841 CoN at concentrations of ≥5 µM, and embryotoxicity in chicken embryos at ≥2.5 µM. In conclusion, the pronounced inhibitory capacity of gossypol on cancer cells was characterized, and pan-HDACi activity was detected in silico, in vitro, by inhibiting individual HDAC isoenzymes, and on protein level by determining histone acetylation. However, for clinical application, further chemical optimization is required to decrease cellular toxicity.

Chemerin Overexpression in the Liver Protects against Inflammation in Experimental Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is marked by macrophage infiltration and inflammation. Chemerin is a chemoattractant protein and is abundant in hepatocytes. The aim of this study was to gain insight into the role of hepatocyte-produced prochemerin in NASH. Therefore, mice were infected with adeno-associated virus 8 to direct hepatic overexpression of prochemerin in a methionine-choline deficient dietary model of NASH. At the end of the study, hepatic and serum chemerin were higher in the chemerin-expressing mice. These animals had less hepatic oxidative stress, F4/80 and CC-chemokine ligand 2 (CCL2) protein, and mRNA levels of inflammatory genes than the respective control animals. In order to identify the underlying mechanisms, prochemerin was expressed in hepatocytes and the hepatic stellate cells, LX-2. Here, chemerin had no effect on cell viability, production of inflammatory, or pro-fibrotic factors. Notably, cultivation of human peripheral blood mononuclear cells (PBMCs) in the supernatant of Huh7 cells overexpressing chemerin reduced CCL2, interleukin-6, and osteopontin levels in cell media. CCL2 was also low in RAW264.7 cells exposed to Hepa1-6 cell produced chemerin. In summary, the current study showed that prochemerin overexpression had little effect on hepatocytes and hepatic stellate cells. Of note, hepatocyte-produced chemerin deactivated PBMCs and protected against inflammation in experimental NASH.

Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

Serum Amyloid Beta42 Is Not Eliminated by the Cirrhotic Liver: A Pilot Study.

Amyloid-beta (Aß) deposition in the brain is the main pathological hallmark of Alzheimer disease. Peripheral clearance of Aß may possibly also lower brain levels. Recent evidence suggested that hepatic clearance of Aß42 is impaired in liver cirrhosis. To further test this hypothesis, serum Aß42 was measured by ELISA in portal venous serum (PVS), systemic venous serum (SVS), and hepatic venous serum (HVS) of 20 patients with liver cirrhosis. Mean Aß42 level was 24.7 ± 20.4 pg/mL in PVS, 21.2 ± 16.7 pg/mL in HVS, and 19.2 ± 11.7 pg/mL in SVS. Similar levels in the three blood compartments suggested that the cirrhotic liver does not clear Aß42. Aß42 was neither associated with the model of end-stage liver disease score nor the Child-Pugh score. Patients with abnormal creatinine or bilirubin levels or prolonged prothrombin time did not display higher Aß42 levels. Patients with massive ascites and patients with large varices had serum Aß42 levels similar to patients without these complications. Serum Aß42 was negatively associated with connective tissue growth factor levels (r = -0.580, p = 0.007) and a protective role of Aß42 in fibrogenesis was already described. Diabetic patients with liver cirrhosis had higher Aß42 levels (p = 0.069 for PVS, p = 0.047 for HVS and p = 0.181 for SVS), which is in accordance with previous reports. Present analysis showed that the cirrhotic liver does not eliminate Aß42. Further studies are needed to explore the association of liver cirrhosis, Aß42 levels, and cognitive dysfunction.

Liver Lipids of Patients with Hepatitis B and C and Associated Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) levels are not associated with severity of liver disease and are inversely related to cholesterol in a cohort of thirty eight patients with liver cirrhosis.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is of particular importance in cholesterol metabolism with high levels contributing to hypercholesterolemia. Cholesterol and sphingolipids are low in patients with liver cirrhosis. Purpose of this study was to find associations of plasma PCSK9 with circulating cholesterol and sphingolipid species and measures of liver disease severity in patients with liver cirrhosis. METHODS: PCSK9 protein levels were determined by ELISA in systemic vein (SVP), hepatic vein (HVP) and portal vein plasma of patients with mostly alcoholic liver cirrhosis. PCSK9 and LDL-receptor protein expression were analysed in cirrhotic and non-cirrhotic liver tissues. RESULTS: Serum PCSK9 was reduced in patients with liver cirrhosis in comparison to non-cirrhotic patients. In liver cirrhosis, plasma PCSK9 was not correlated with Child-Pugh score, Model for End-Stage Liver Disease score, bilirubin or aminotransferases. A negative association of SVP PCSK9 with albumin existed. PCSK9 protein in the liver did not change with fibrosis stage and was even positively correlated with LDL-receptor protein levels. Ascites volume and variceal size were not related to PCSK9 levels. Along the same line, transjugular intrahepatic shunt to lower portal pressure did not affect PCSK9 concentrations in the three blood compartments. Serum cholesterol, sphingomyelin and ceramide levels did not correlate with PCSK9. Stratifying patients by high versus low PCSK9 levels using the median as cut-off, several cholesteryl ester species were even low in the subgroup with high PCSK9 levels. A few sphingomyelin species were also reduced in the patients with PCSK9 levels above the median. PCSK9 is highly expressed in the liver but systemic, portal and hepatic vein levels were similar. PCSK9 was not correlated with the inflammatory proteins C-reactive protein, IL-6, galectin-3, resistin or pentraxin 3. Of note, HVP PCSK9 was positively associated with HVP chemerin and negatively with HVP adiponectin levels. CONCLUSIONS: In the cohort of patients with liver cirrhosis mostly secondary to alcohol consumption high PCSK9 was associated with low levels of certain cholesteryl ester and sphingomyelin species. Positive correlations of PCSK9 and LDL-receptor protein in the liver of patients with chronic liver injury are consistent with these findings.

Augmenter of Liver Regeneration (ALR) regulates bile acid synthesis and attenuates bile acid-induced apoptosis via glycogen synthase kinase-3ß (GSK-3ß) inhibition.

Bile acid synthesis is restricted to hepatocytes and is rate-limited by CYP7A1 (cholesterol 7α hydroxylase). CYP7A1 expression undergoes tight regulation and is repressed after partial hepatectomy to prevent the accumulation of toxic bile acids. Augmenter of Liver Regeneration (ALR) is a hepatotrophic factor shown to support liver regeneration by augmenting cell proliferation and reducing apoptosis. Nevertheless, less is known about ALR's role in protecting hepatocytes from bile acid accumulation and bile acid-induced apoptosis. Therefore, HepG2 and Huh-7 cells were incubated with recombinant human ALR (rALR) and the expression of CYP7A1, bile acid-induced apoptosis as well as potential molecular mechanisms were analyzed. We found that rALR reduces CYP7A1 expression by increasing nuclear NFκB levels. Moreover, rALR reduced glycochenodeoxycholate (GCDC)-induced-apoptosis by decreased expression of pro-apoptotic Bax and enhanced expression of anti-apoptotic Mcl-1, which is regulated by phosphatidylinositol-3-kinase (PI3K)/Akt activation and glycogen synthase kinase-3ß (GSK3ß) phosphorylation. Inhibitors for PI3K/Akt (GSK690693) and GSK3ß (SB415286) confirmed the specificity of rALR treatment for this pathway. In addition, rALR reduces pro-death signaling by decreasing GCDC-induced JNK phosphorylation. Taken all together, rALR might contribute to protecting hepatocytes from toxic concentrations of bile acids by down-regulating their denovo synthesis, attenuating apoptosis by activation of PI3K/Akt - GSK3ß pathway and inhibition of JNK signaling. Thereby this suggests a new role of ALR in augmenting the process of liver regeneration.

Chemerin Is Induced in Non-Alcoholic Fatty Liver Disease and Hepatitis B-Related Hepatocellular Carcinoma.

Chemerin is protective in experimental models of hepatocellular carcinoma (HCC). Noteworthy, chemerin mRNA and protein were reduced in HCC tissues of Asian patients with mostly hepatitis B disease etiology. The current study nevertheless showed that chemerin protein was induced in tumor tissues of European HCC patients with non-alcoholic fatty liver disease (NAFLD) and patients with unclear disease etiology. A similar regulation was observed in hepatitis B virus (HBV), but not in hepatitis C virus (HCV), related HCC. The apparent discrepancy between the regulation of chemerin in HBV-HCC obtained from our study and recent reports led us to use the chemerin antibodies applied in the previous assays. These antibodies could not equally detect different chemerin isoforms, which were overexpressed in HepG2 cells. Higher chemerin protein in HCC was nevertheless confirmed by the use of all antibodies. Chemerin protein was low in Huh7 and PLC/PRF/5 cells whereas HepG2 and Hep3B cells had chemerin protein similar as primary human hepatocytes. Besides, the anti-tumor effects of retinoids in hepatocyte cell lines did not enclose upregulation of chemerin, which was initially discovered as a tazarotene induced protein in the skin. Finally, protein levels of the chemerin receptor, chemokine-like receptor 1 (CMKLR1), declined in non-viral, and tended to be lower in HBV-HCC tissues suggesting reduced chemerin activity in the tumors. To sum up, our work showed an opposite regulation of chemerin and CMKLR1 in NAFLD and HBV associated HCC. In HCV-HCC neither chemerin nor its receptor were changed in the tumor tissues. Current findings do not support a critical role of total chemerin protein levels in HCC of non-viral and viral etiology. Accordingly, tumor-localized chemerin protein was not associated with tumor-node-metastasis classification.

Antifibrotic Effects of Amyloid-Beta and Its Loss in Cirrhotic Liver.

The function and regulation of amyloid-beta (Aß) in healthy and diseased liver remains unexplored. Because Aß reduces the integrity of the blood-brain barrier we have examined its potential role in regulating the sinusoidal permeability of normal and cirrhotic liver. Aß and key proteins that generate (beta-secretase 1 and presenilin-1) and degrade it (neprilysin and myelin basic protein) were decreased in human cirrhotic liver. In culture, activated hepatic stellate cells (HSC) internalized Aß more efficiently than astrocytes and HSC degraded Aß leading to suppressed expression of α-smooth muscle actin (α-SMA), collagen 1 and transforming growth factor ß (TGFß). Aß also upregulated sinusoidal permeability marker endothelial NO synthase (eNOS) and decreased TGFß in cultured human liver sinusoidal endothelial cells (hLSEC). Liver Aß levels also correlate with the expression of eNOS in transgenic Alzheimer's disease mice and in human and rodent cirrhosis/fibrosis. These findings suggest a previously unexplored role of Aß in the maintenance of liver sinusoidal permeability and in protection against cirrhosis/fibrosis via attenuation of HSC activation.

Pentraxin-3 is not related to disease severity in cirrhosis and hepatocellular carcinoma patients.

The acute-phase protein pentraxin-3 (PTX3) is a component of the innate immune system. Inflammation and tissue injury increased PTX3 in the injured liver, and accordingly, circulating PTX3 was induced in patients with chronic liver diseases. In the present study, PTX3 protein was determined in systemic, hepatic, and portal vein plasma of patients with liver cirrhosis to assess a possible association between hepatic PTX3 release and extent of liver injury. However, PTX3 levels were not related to disease severity. Of note, portal PTX3 levels were higher than concentrations in the hepatic vein. PTX3 in the hepatic and portal veins was negatively correlated with factor V, antithrombin 3, and prothrombin time. PTX3 did neither correlate with C-reactive protein nor galectin-3 or resistin, whereby the latter two proteins are associated with hepatic injury. PTX3 levels were not changed in cirrhosis patients with ascites or varices and did not correlate with the hepatic venous pressure gradient. Likewise, serum PTX3 was not correlated with histological steatosis, inflammation, or fibrosis stage in patients with hepatocellular carcinoma (HCC). Moreover, PTX3 was not associated with tumor node metastasis classification in HCC. Above all, PTX3 increased in hepatic, portal, and systemic blood immediately after transjugular intrahepatic portosystemic shunt (TIPS). Higher PTX3 in portal than hepatic vein plasma and further increase after TIPS suggests that the liver eliminates PTX3 from the circulation. In summary, PTX3 is not of diagnostic value in cirrhosis and HCC patients.

TGF-ß2 silencing to target biliary-derived liver diseases.

OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.

Inflammation-associated suppression of metabolic gene networks in acute and chronic liver disease.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.

Serum Adiponectin Levels Do Not Distinguish Primary from Metastatic Liver Tumors.

BACKGROUND/AIM: Adiponectin protects from metabolic disease and cancer. Accordingly, serum adiponectin was reduced in patients with colorectal cancer (CRC). This hepatoprotective factor was definitely increased in hepatocellular carcinoma (HCC). CRC metastases to the liver are common and the aim of the present study was to evaluate whether serum adiponectin discriminates primary from secondary liver cancers. MATERIALS AND METHODS: Adiponectin was measured by ELISA in the serum of 36 patients with colorectal liver metastases, 32 patients with HCC and 49 patients without cancer. RESULTS: Serum adiponectin levels were higher in cancer than non-tumor patients. Adiponectin was not related to TNM stage in HCC nor to the levels of serum tumor markers. Moreover, hepatic inflammation and liver fibrosis were not correlated with serum adiponectin levels. Metabolic diseases are associated with low adiponectin and a higher risk of cancer. In HCC, but not in CRC serum, adiponectin was increased in patients with hypertension and hyperuricemia. In this cohort, adiponectin positively correlated with chemerin, an adipokine supposed to contribute to metabolic disturbances. CONCLUSION: Serum adiponectin cannot discriminate primary from secondary liver tumors.