Extracellular Vesicles as Markers of Liver Function: Optimized Workflow for Biomarker Identification in Liver Disease.

Liver diseases represent a significant global health burden, necessitating the development of reliable biomarkers for early detection, prognosis, and therapeutic monitoring. Extracellular vesicles (EVs) have emerged as promising candidates for liver disease biomarkers due to their unique cargo composition, stability, and accessibility in various biological fluids. In this study, we present an optimized workflow for the identification of EVs-based biomarkers in liver disease, encompassing EVs isolation, characterization, cargo analysis, and biomarker validation. Here we show that the levels of microRNAs miR-10a, miR-21, miR-142-3p, miR-150, and miR-223 were different among EVs isolated from patients with nonalcoholic fatty liver disease and autoimmune hepatitis. In addition, IL2, IL8, and interferon-gamma were found to be increased in EVs isolated from patients with cholangiocarcinoma compared with healthy controls. By implementing this optimized workflow, researchers and clinicians can improve the identification and utilization of EVs-based biomarkers, ultimately enhancing liver disease diagnosis, prognosis, and personalized treatment strategies.

Characterization of the scavenger cell proteome in mouse and rat liver.

The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly specialized hepatocyte subpopulations like glutamine synthetase (GS) expressing perivenous hepatocytes (scavenger cells). However, this cell population has not yet been characterized extensively regarding expression of other genes and potential subpopulations. This was investigated in the present study by proteome profiling of periportal GS-negative and perivenous GS-expressing hepatocytes from mouse and rat. Apart from established markers of GS+ hepatocytes such as glutamate/aspartate transporter II (GLT1) or ammonium transporter Rh type B (RhBG), we identified novel scavenger cell-specific proteins like basal transcription factor 3 (BTF3) and heat-shock protein 25 (HSP25). Interestingly, BTF3 and HSP25 were heterogeneously distributed among GS+ hepatocytes in mouse liver slices. Feeding experiments showed that RhBG expression was increased in livers from mice fed with high protein diet compared to standard chow. While spatial distributions of GS and carbamoylphosphate synthetase 1 (CPS1) were unaffected, periportal areas constituted by glutaminase 2 (GLS2)-positive hepatocytes were enlarged or reduced in response to high or low protein diet, respectively. The data suggest that the population of perivenous GS+ scavenger cells is heterogeneous and not uniform as previously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration.

O-GlcNAcylation-dependent upregulation of HO1 triggers ammonia-induced oxidative stress and senescence in hepatic encephalopathy.

BACKGROUND & AIMS: Cerebral oxidative stress plays an important role in the pathogenesis of hepatic encephalopathy (HE), but the underlying mechanisms are incompletely understood. Herein, we analyzed a role of heme oxygenase (HO)1, iron and NADPH oxidase 4 (Nox4) for the induction of oxidative stress and senescence in HE. METHODS: Gene and protein expression in human post-mortem brain samples was analyzed by gene array and western blot analysis. Mechanisms and functional consequences of HO1 upregulation were studied in NH4Cl-exposed astrocytes in vitro by western blot, qPCR and super-resolution microscopy. RESULTS: HO1 and the endoplasmic reticulum (ER) stress marker grp78 were upregulated, together with changes in the expression of multiple iron metabolism-related genes, in post-mortem brain samples from patients with liver cirrhosis and HE. NH4Cl elevated HO1 protein and mRNA in cultured astrocytes through glutamine synthetase (GS)-dependent upregulation of glutamine/fructose amidotransferases 1/2 (GFAT1/2), which blocked the transcription of the HO1-targeting miR326-3p in a O-GlcNAcylation dependent manner. Upregulation of HO1 by NH4Cl triggered ER stress and was associated with elevated levels of free ferrous iron and expression changes in iron metabolism-related genes, which were largely abolished after knockdown or inhibition of GS, GFAT1/2, HO1 or iron chelation. NH4Cl, glucosamine (GlcN) and inhibition of miR326-3p upregulated Nox4, while knockdown of Nox4, GS, GFAT1/2, HO1 or iron chelation prevented NH4Cl-induced RNA oxidation and astrocyte senescence. Elevated levels of grp78 and O-GlcNAcylated proteins were also found in brain samples from patients with liver cirrhosis and HE. CONCLUSION: The present study identified glucosamine synthesis-dependent protein O-GlcNAcylation as a novel mechanism in the pathogenesis of HE that triggers oxidative and ER stress, as well as senescence, through upregulation of HO1 and Nox4. LAY SUMMARY: Patients with liver cirrhosis frequently exhibit hyperammonemia and suffer from cognitive and motoric dysfunctions, which at least in part involve premature ageing of the astrocytes in the brain. This study identifies glucosamine and an O-GlcNAcylation-dependent disruption of iron homeostasis as novel triggers of oxidative stress, thereby mediating ammonia toxicity in the brain.

Tauroursodeoxycholate protects from glycochenodeoxycholate-induced gene expression changes in perfused rat liver.

Tauroursodeoxycholate (TUDC) is well known to protect against glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes. In the present study, we analyzed whether TUDC also exerts protective effects by modulating GCDC-induced gene expression changes. For this, gene array-based transcriptome analysis and quantitative polymerase chain reaction (qPCR) were performed on RNA isolated from rat livers perfused with GCDC, TUDC or a combination of both (each 20 µm for 2 h). GCDC led to a significant increase of lactate dehydrogenase (LDH) into the effluent perfusate, which was prevented by TUDC. GCDC, TUDC and co-perfusion induced distinct gene expression changes. While GCDC upregulated the expression of several pro-inflammatory genes, co-perfusion with TUDC increased the expression of pro-proliferative and anti-apoptotic p53 target genes. In line with this, levels of serine20-phosphorylated p53 and of its target gene p21 were elevated by GCDC in a TUDC-sensitive way. GCDC upregulated the oxidative stress surrogate marker 8OH(d)G and the pro-apoptotic microRNAs miR-15b/16 and these effects were prevented by TUDC. The upregulation of miR-15b and miR-16 in GCDC-perfused livers was accompanied by a downregulation of several potential miR-15b and miR-16 target genes. The present study identified changes in the transcriptome of the rat liver which suggest, that TUDC is hepatoprotective by counteracting GCDC-induced gene expression changes.

Real-world performance of the NeuMoDx™ HCV Quant Test for quantification of hepatitis C virus (HCV)-RNA.

Quantification of hepatitis C virus (HCV)-RNA in serum or plasma samples is an essential parameter in HCV diagnostics. Here, the NeuMoDx™Molecular System (Qiagen) was tested for the most common HCV genotypes and compared to the cobas c6800 system (Roche). HCV-RNA from 131 plasma/serum samples from chronically infected patients was determined in parallel on the NeuMoDx and c6800 systems. Linearity was analysed using the four most common HCV genotypes (1-4) in our cohort. The coefficient of variation (CV) within (intra-assay) and between (inter-assay) runs was calculated based on HCV-RNA concentration. Quantitative HCV-RNA results were highly correlated on both test systems (R2 = 0.7947; y = 0.94 x + 0.37). On average, the NeuMoDx and c6800 HCV RNA levels showed a mean difference of only 0.05 log10 IU/mL but with a broad distribution (±1.2 2 x SD). The NeuMoDx demonstrated very good linearity across all HCV genotypes tested at concentrations between 1.7 and 6.2 log10 IU/mL (R2 range: 0.9257-0.9991) with the highest mean coefficient of determination for genotype 1 (R2 = 0.9909). The mean intra- and inter-assay CV for both serum and plasma samples was <5 %. The NeuMoDx HCV-RNA Assay demonstrates high subtype-independent comparability, linearity, and reproducibility for the quantification of HCV-RNA in serum and plasma samples from chronically infected patients.

Uncovering Novel Roles of miR-122 in the Pathophysiology of the Liver: Potential Interaction with NRF1 and E2F4 Signaling.

MicroRNA miR-122 plays a pivotal role in liver function. Despite numerous studies investigating this miRNA, the global network of genes regulated by miR-122 and its contribution to the underlying pathophysiological mechanisms remain largely unknown. To gain a deeper understanding of miR-122 activity, we employed two complementary approaches. Firstly, through transcriptome analysis of polyribosome-bound RNAs, we discovered that miR-122 exhibits potential antagonistic effects on specific transcription factors known to be dysregulated in liver disease, including nuclear respiratory factor-1 (NRF1) and the E2F transcription factor 4 (E2F4). Secondly, through proteome analysis of hepatoma cells transfected with either miR-122 mimic or antagomir, we discovered changes in several proteins associated with increased malignancy. Interestingly, many of these proteins were reported to be transcriptionally regulated by NRF1 and E2F4, six of which we validated as miR-122 targets. Among these, a negative correlation was observed between miR-122 and glucose-6-phosphate dehydrogenase levels in the livers of patients with hepatitis B virus-associated hepatocellular carcinoma. This study provides novel insights into potential alterations of molecular pathway occurring at the early stages of liver disease, driven by the dysregulation of miR-122 and its associated genes.

Differential Modulation of miR-122 Transcription by TGFß1/BMP6: Implications for Nonresolving Inflammation and Hepatocarcinogenesis.

Chronic inflammation is widely recognized as a significant factor that promotes and worsens the development of malignancies, including hepatocellular carcinoma. This study aimed to explore the potential role of microRNAs in inflammation-associated nonresolving hepatocarcinogenesis. By conducting a comprehensive analysis of altered microRNAs in animal models with liver cancer of various etiologies, we identified miR-122 as the most significantly downregulated microRNA in the liver of animals with inflammation-associated liver cancer. Although previous research has indicated the importance of miR-122 in maintaining hepatocyte function, its specific role as either the trigger or the consequence of underlying diseases remains unclear. Through extensive analysis of animals and in vitro models, we have successfully demonstrated that miR-122 transcription is differentially regulated by the immunoregulatory cytokines, by the transforming growth factor-beta 1 (TGFß1), and the bone morphogenetic protein-6 (BMP6). Furthermore, we presented convincing evidence directly linking reduced miR-122 transcription to inflammation and in chronic liver diseases. The results of this study strongly suggest that prolonged activation of pro-inflammatory signaling pathways, leading to disruption of cytokine-mediated regulation of miR-122, may significantly contribute to the onset and exacerbation of chronic liver disease.

Sequence diversity of hepatitis D virus in Mongolia.

Introduction: The Hepatitis Delta Virus (HDV) is a defective, single-stranded RNA virusoid encoding for a single protein, the Hepatitis Delta Antigen (HDAg), which requires the hepatitis B virus (HBV) envelope protein (HBsAg) for its transmission. Currently, hepatitis D is the most aggressive form of viral hepatitis and treatment options are limited. Worldwide 12 million people are chronically infected with HDV being at high risk for progression to cirrhosis and development of liver cancer. Objectives: Although it is well established that Mongolia is the country with the highest prevalence of HDV infections, the information on the molecular epidemiology and factors contributing to HDV sequence diversity are largely unclear. The aim of the study was to characterize the sequence diversity of HDV in rural areas from Mongolia and to determine the extent of HLA class I-associated selection pressure. Patients and methods: From the HepMongolia cohort from rural areas in Mongolia, 451 HBsAg-positive individuals were selected and anti-HDV, HDV-RNA and the sequence of the large HDAg was determined. For all individuals the HLA class I locus was genotyped. Residues under selection pressure in the presence of individual HLA class I types were identified with the recently published analysis tool HAMdetector. Results: Of 431 HBsAg positive patients, 281 were anti-HDV positive (65%), and HDV-RNA could be detected in 207 of 281 (74%) of patients. The complete large HDAg was successfully sequenced from 131 samples. Phylogenetic analysis revealed that all Mongolian HDV isolates belong to genotype 1, however, they separate into several different clusters without clear regional association. In turn, from phylogeny there is strong evidence for recent local transmission events. Importantly, we found multiple residues with strong support for HLA class I-associated selection pressure consistent with a functional CD8+ T cell response directed against HDV. Conclusion: HDV isolates from Mongolia are highly diverse. The molecular epidemiology suggests circulation of multiple subtypes and provides evidence for ongoing recent transmissions.

Impaired humoral immunity to BQ.1.1 in convalescent and vaccinated patients.

Determining SARS-CoV-2 immunity is critical to assess COVID-19 risk and the need for prevention and mitigation strategies. We measured SARS-CoV-2 Spike/Nucleocapsid seroprevalence and serum neutralizing activity against Wu01, BA.4/5 and BQ.1.1 in a convenience sample of 1,411 patients receiving medical treatment in the emergency departments of five university hospitals in North Rhine-Westphalia, Germany, in August/September 2022. 62% reported underlying medical conditions and 67.7% were vaccinated according to German COVID-19 vaccination recommendations (13.9% fully vaccinated, 54.3% one booster, 23.4% two boosters). We detected Spike-IgG in 95.6%, Nucleocapsid-IgG in 24.0%, and neutralization against Wu01, BA.4/5 and BQ.1.1 in 94.4%, 85.0%, and 73.8% of participants, respectively. Neutralization against BA.4/5 and BQ.1.1 was 5.6- and 23.4-fold lower compared to Wu01. Accuracy of S-IgG detection for determination of neutralizing activity against BQ.1.1 was reduced substantially. We explored previous vaccinations and infections as correlates of BQ.1.1 neutralization using multivariable and Bayesian network analyses. Given a rather moderate adherence to COVID-19 vaccination recommendations, this analysis highlights the need to improve vaccine-uptake to reduce the COVID-19 risk of immune evasive variants. The study was registered as clinical trial (DRKS00029414).

Swelling-induced upregulation of miR-141-3p inhibits hepatocyte proliferation.

Background & Aims: MicroRNAs (miRNAs) act as a regulatory mechanism on a post-transcriptional level by repressing gene transcription/translation and play a central role in the cellular stress response. Osmotic changes occur in a variety of diseases including liver cirrhosis and hepatic encephalopathy. Changes in cell hydration and alterations of the cellular volume are major regulators of cell function and gene expression. In this study, the modulation of hepatic gene expression in response to hypoosmolarity was studied. Methods: mRNA analyses of normo- and hypoosmotic perfused rat livers by gene expression arrays were used to identify miRNA and their potential target genes associated with cell swelling preceding cell proliferation. Selected miR-141-3p was also investigated in isolated hepatocytes treated with miRNA mimic, cell stretching, and after partial hepatectomy. Inhibitor perfusion studies were performed to unravel signalling pathways responsible for miRNA upregulation. Results: Using genome-wide transcriptomic analysis, it was shown that hypoosmotic exposure led to differential gene expression in perfused rat liver. Moreover, miR-141-3p was upregulated by hypoosmolarity in perfused rat liver and in primary hepatocytes. In concert with this, miR-141-3p upregulation was prevented after Src-, Erk-, and p38-MAPK inhibition. Furthermore, luciferase reporter assays demonstrated that miR-141-3p targets cyclin dependent kinase 8 (Cdk8) mRNA. Partial hepatectomy transiently upregulated miR-141-3p levels just after the initiation of hepatocyte proliferation, whereas Cdk8 mRNA was downregulated. The mechanical stretching of rat hepatocytes resulted in miR-141-3p upregulation, whereas Cdk8 mRNA tended to decrease. Notably, the overexpression of miR-141-3p inhibited the proliferation of Huh7 cells. Conclusions: Src-mediated upregulation of miR-141-3p was found in hepatocytes in response to hypoosmotic swelling and mechanical stretching. Because of its antiproliferative function, miR-141-3p may counter-regulate the proliferative effects triggered by these stimuli. Lay summary: In this study, we identified microRNA 141-3p as an osmosensitive miRNA, which inhibits proliferation during liver cell swelling. Upregulation of microRNA 141-3p, controlled by Src-, Erk-, and p38-MAPK signalling, results in decreased mRNA levels of various genes involved in metabolic processes, macromolecular biosynthesis, and cell cycle progression.

Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver.

microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.