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

Implication of free fatty acids in thrombin generation and fibrinolysis in vascular inflammation in Zucker rats and evolution with aging

Background: The metabolic syndrome (MetS) and aging are associated with modifications in blood coagulation factors, vascular inflammation, and increased risk of thrombosis. Objectives: Our aim was to determine concomitant changes in thrombin generation in the blood compartment and at the surface of vascular smooth muscle cells (VSMCs) and its interplay with adipokines, free fatty acids (FFA), and metalloproteinases (MMPs) in obese Zucker rats that share features of the human MetS. Methods: Obese and age-matched lean Zucker rats were compared at 25 and 80 weeks of age. Thrombin generation was assessed by calibrated automated thrombography (CAT). Results: Endogenous thrombin potential (ETP) was increased in obese rats independent of platelets and age. Clot half-lysis time was delayed with obesity and age. Interleukin (IL)-1β and IL-13 were increased with obesity and age respectively. Addition of exogenous fibrinogen, leptin, linoleic, or palmitic acid increased thrombin generation in plasma whereas adiponectin had an opposite effect. ETP was increased at the surface of VSMCs from obese rats and addition of exogenous palmitic acid further enhanced ETP values. Gelatinase activity was increased in aorta at both ages in obese rats and MMP-2 activity was increased in VSMCs from obese rats. Conclusions: Our study demonstrated in MetS an early prothrombotic phenotype of the blood compartment reinforced by procoagulant properties of dedifferentiated and inflammatory VSMCs. Mechanisms involved (1) increased fibrinogen and impaired fibrinolysis and (2) increased saturated fatty acids responsible for additive procoagulant effects. Whether specifically targeting this hypercoagulability using direct thrombin inhibitors would improve outcome in MetS is worth investigating

Highly fluorescent guanosine mimics for folding and energy transfer studies

Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photophysical properties of a small family of 8-substituted-2′-deoxyguanosines that have been incorporated into the human telomeric repeat sequence using phosphoramidite chemistry. These include 8-(2-pyridyl)-2′-deoxyguanosine (2PyG), 8-(2-phenylethenyl)-2′-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2′-deoxyguanosine (4PVG). On DNA folding and stability, 8-substituted guanosines can exhibit context-dependent effects but were better tolerated by G-quadruplex and duplex structures than pyrimidine mismatches. In contrast to previously reported fluorescent guanine analogs, 8-substituted guanosines exhibit similar or even higher quantum yields upon their incorporation into nucleic acids (Φ = 0.02–0.45). We have used these highly emissive probes to quantify energy transfer efficiencies from unmodified DNA nucleobases to 8-substituted guanosines. The resulting DNA-to-probe energy transfer efficiencies (ηt) are highly structure selective, with ηt(duplex) < ηt(single-strand) < ηt(G-quadruplex). These trends were independent of the exact structural features and thermal stabilities of the G-quadruplexes or duplexes containing them. The combination of efficient energy transfer, high probe quantum yield, and high molar extinction coefficient of the DNA provides a highly sensitive and reliable readout of G-quadruplex formation even in highly diluted sample solutions of 0.25 nM

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays

MMP-9 activity is increased by adiponectin in primary human hepatocytes but even negatively correlates with serum adiponectin in a rodent model of non-alcoholic steatohepatitis

Adiponectin protects from inflammation and fibrosis in metabolic liver disease. In the present study we analyzed whether this adipokine may directly affect the activity of matrix metalloproteinases (MMPs), central regulators of fibrinolysis, in hepatocytes. Global gene expression analysis indicated upregulation of MMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in primary human hepatocytes (PHH) in response to stimulation with adiponectin, and these results were confirmed by real-time RT-PCR. Furthermore, gelatin zymography revealed that MMP-9 activity was significantly induced in supernatants of adiponectin stimulated PHHs. In a murine model of hepatic steatosis and in human steatotic liver samples hepatic MMP-9 activity was not significantly altered. However, in two different murine models of non-alcoholic steatohepatitis (NASH) MMP-9 activity was significantly elevated compared to chow fed control mice. Of note, MMP-9 activity did not or even negatively, respectively, correlate with adiponectin serum levels in these models. The current data indicate that in NASH hepatic inflammation and fibrosis but not hepatic steatosis induce liver MMP-9 activity, and this induction seems to be related to the anti-inflammatory activity of adiponectin rather than its effect on hepatocellular MMP-9 expression

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

Simple Summary Hepatocellular carcinoma (HCC) is a frequent liver cancer and high expression of bioactive chemerin in hepatocytes was protective in experimental HCC models. The main risk factors for HCC are non-alcoholic fatty liver disease (NAFLD), hepatitis B and C infections. The current analysis showed that chemerin protein was induced in HCC tissues of NAFLD and hepatitis B infected patients. This upregulation was modest in patients with unknown disease etiology and not detected in hepatitis C infected patients. Protein levels of the chemerin receptor CMKLR1 strongly declined in the tumors of NAFLD patients and patients with unclear disease etiology but not in patients with viral infections. Our results demonstrate that the expression of chemerin in HCC is related to disease etiology and this could also apply to the role of chemerin in human HCC. In contrast to the present findings, chemerin was shown to be low in the HCC tissues of Asian patients with mostly viral disease etiology and this indicates ancestry-specific regulation of chemerin in HCC. 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

Adiponectin isoforms differentially affect gene expression and lipidome of primary human hepatocytes

Repetitive transcranial magnetic stimulation (rTMS) of the temporal cortex has been used to treat patients with subjective tinnitus. While rTMS is known to induce morphological changes in healthy subjects, no study has investigated yet whether rTMS treatment induces grey matter (GM) changes in tinnitus patients as well, whether these changes are correlated with treatment success, and whether GM at baseline is a useful predictor for treatment outcome. Therefore, we examined magnetic resonance images of 77 tinnitus patients who were treated with rTMS of the left temporal cortex (10 days, 2000 stimuli/day, 1 Hz). At baseline and after the last treatment session high-resolution structural images of the brain were acquired and tinnitus severity was assessed. For a subgroup of 41 patients, additional brain scans were done after a follow-up period of 90 days. GM changes were analysed by means of voxel based morphometry. Transient GM decreases were detectable in several brain regions, especially in the insula and the inferior frontal cortex. These changes were not related to treatment outcome though. Baseline images correlated with change in tinnitus severity in the frontal cortex and the lingual gyrus, suggesting that GM at baseline might hold potential as a possible predictor for treatment outcome