R Hall

Genetic Determinants in Hepatic Fibrosis: From Experimental Models to Fibrogenic Gene Signatures in Humans

Hepatic fibrosis, or scarring of the liver, is a nonspecific reaction to chronic liver injury. Hepatic fibrosis is commonly caused by exogenous factors such as viral hepatitis or alcohol abuse, but recent studies also indicate a genetic predisposition. Although some patients who have chronic liver diseases show only minor morphologic and functional alterations of the liver and are characterized by slow progression of disease with mild clinical symptoms, others develop pronounced hepatic fibrosis rapidly, culminating in cirrhosis, liver failure, or hepatocellular carcinoma, respectively. These well known differences in progression of hepatic fibrosis persist when controlling for age (at infection), gender, and exogenous factors in multivariate analysis, indicating that genetic factors might play important roles in the modulation of hepatic fibrosis and contribute to the variability in fibrosis progression. This review summarizes genetic determinants in hepatic fibrosis.

Inhibition of endothelial nitric oxide synthase induces and enhances myocardial fibrosis

AIMS The endothelial nitric oxide synthase (eNOS) contributes to cardiac remodelling. We studied the role of eNOS in the development of myocardial fibrosis during cardiac overload. METHODS AND RESULTS Ten-week-old male C57/Bl6 wildtype (WT) and eNOS mice (eNOS(-/-)) were subjected to transverse aortic constriction (TAC, 360 μm) and WT were treated with L-N(G)-nitroarginine methyl ester (L-NAME, 100 mg/kg/day) for 35 days. Inhibition of eNOS by L-NAME induced interstitial fibrosis, augmented replacement fibrosis, and induced apoptosis of cardiac fibroblasts and cardiomyocytes. L-NAME and eNOS(-/-) markedly increased the fibrosis induced by TAC and enhanced the myocardial prevalence of CXCR4(pos) fibroblasts. Myocardial stromal-derived factor-1 (SDF-1) expression was up-regulated by l-NAME and down-regulated after TAC. Blood pressure lowering by co-treatment with hydralazine (250 mg/L/day) did not reverse the L-NAME effects. In mice transplanted with green fluorescent protein (GFP)(pos) bone marrow, L-NAME increased the percentage of GFP(pos) fibroblasts in the myocardium to 45-70%. Strain-mismatched BMT of eNOS(-/-)-BM increased and of WT-BM decreased the percentage of CXCR4(pos) fibroblasts in all groups. The number of fibrocytes (CD45(pos) collagen I(pos) cells) in the peripheral blood and in the bone marrow was increased both by TAC and L-NAME. L-NAME but not the inhibitor of inducible NOS 1400 W and of neuronal NOS 7-nitroindazole increased hydroxyproline and collagen Iα1. L-NAME up-regulated SDF-1 mRNA in cultured neonatal rat cardiac fibroblasts as well as their migratory capacity. CONCLUSION eNOS inhibition induces and enhances cardiac fibrosis independently of blood pressure by activating SDF-1/CXCR4, extracellular matrix production in cardiac fibroblasts and by increasing recruitment of fibrocytes from the bone marrow.

C-kit(+) resident cardiac stem cells improve left ventricular fibrosis in pressure overload.

To investigate the effect of resident cardiac stem cells (RCSC) on myocardial remodeling, c-kit(+) RCSC were isolated from hearts of C57Bl/6-Tg (ACTb-EGFP)1Osb/J mice expressing green fluorescent protein and expanded in vitro. C57/Bl6N wildtype mice were subjected to transverse aortic constriction (TAC, 360 μm) or sham-operation. 5 × 10(5) c-kit(+) RCSC or c-kit(-) cardiac cells or cell buffer were infused intravenously 24 h post-surgery (n = 11-24 per group). Hypoxia-inducible factor-1α-mRNA in left ventricles of TAC mice was enhanced 24 h after transplantation. 35 days post-TAC, the density of c-kit(+) RCSC in the myocardium was increased by two-fold. Infusion of c-kit(+) resident cardiac stem cells post-TAC markedly reduced myocardial fibrosis and the expression of collagen Iα2 and connective tissue growth factor. Infusion of c-kit(-) cardiac cells did not ameliorate cardiac fibrosis. In parallel, expression of pro-angiogenic mediators (FGFb, IL-4, IL-6, TGFß, leptin) and the density of CD31(+) and CD31(+) GFP(+) endothelial cells were increased. Transplantation reduced brain- and atrial natriuretic peptides and the cardiomyocyte cross-sectional area. Infusion of c-kit(+) resident cardiac stem reduced the rate of apoptosis and oxidative stress in cardiomyocytes and in non-cardiomyocyte cells.

Systems genetics of liver fibrosis: identification of fibrogenic and expression quantitative trait loci in the BXD murine reference population.

The progression of liver fibrosis in response to chronic injury varies considerably among individual patients. The underlying genetics is highly complex due to large numbers of potential genes, environmental factors and cell types involved. Here, we provide the first toxicogenomic analysis of liver fibrosis induced by carbon tetrachloride in the murine ‘genetic reference panel’ of recombinant inbred BXD lines. Our aim was to define the core of risk genes and gene interaction networks that control fibrosis progression. Liver fibrosis phenotypes and gene expression profiles were determined in 35 BXD lines. Quantitative trait locus (QTL) analysis identified seven genomic loci influencing fibrosis phenotypes (pQTLs) with genome-wide significance on chromosomes 4, 5, 7, 12, and 17. Stepwise refinement was based on expression QTL mapping with stringent selection criteria, reducing the number of 1,351 candidate genes located in the pQTLs to a final list of 11 cis-regulated genes. Our findings demonstrate that the BXD reference population represents a powerful experimental resource for shortlisting the genes within a regulatory network that determine the livers vulnerability to chronic injury.

Expression of the megalin C-terminal fragment by macrophages during liver fibrogenesis in mice

The low-density-lipoprotein receptor megalin (LRP2, gp330) is strongly expressed in the kidney, where it is responsible for the resorption of metabolites from primary urine. One of the main ligands is the complex of retinol and retinol binding protein. Megalin has been hypothesized to be part of the retinol storage system in liver. Considering the role of hepatic stellate cells in retinol storage and fibrogenesis we investigated mouse strains that developed different degrees of fibrosis after challenge with CCl(4). Immunoblotting revealed the invariable expression of the megalin C-terminal fragment independent of liver damage in all strains. However, only a specific cell population in centrilobular areas of fibrotic livers from DBA/2J mice, which were most susceptible for CCl(4)-induced fibrogenesis in our study, was stained using megalin-specific antibodies. Double immunostaining indicated that a subset of hepatic macrophages might represent the megalin-expressing cells in fibrotic liver. Fluorescence activated cell sorting based isolation of hepatic macrophages and megalin specific expression analysis demonstrated the transcription of the whole megalin gene in liver macrophages. We argue that megalin might exhibit a proinflammatory effect by the uptake of retinoids in recruited monocytes, which thereby differentiate to liver macrophages and potentiate fibrogenesis by the release of proinflammatory mediators. Otherwise, megalin might be activated in macrophages during advanced fibrogenesis and act as a negative regulator of proinflammatory genes.

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum.

AbstractWe describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease. Graphical Abstractᅟ

Systems genetics of hepatocellular damage in vivo and in vitro: identification of a critical network on chromosome 11 in mouse

Quantitative trait locus (QTL) mapping is a powerful method to find modifier loci that influence disease risk and progression without prior knowledge of underlying genetic mechanisms. The aim of this study is to identify gene loci that contribute to individual differences in liver fibrosis following chronic liver damage. For this purpose, we carried out a mapping study across a panel of 21 BXD recombinant inbred strains using primary hepatocytes challenged with transforming growth factor (TGF)-β for 48 h. We identified a 6 Mb interval on chromosome 11 that is a major modifier of TGF-β-induced hepatocyte injury. Corresponding in vivo genetic analysis of fibrosis after chronic hepatotoxic injury by carbon tetrachloride (CCl4 ip for 6 wk) highlighted the same locus. Expression QTL (eQTL) analysis in liver tissues in the BXD family identified six polymorphisms in this region that are associated with strong cis eQTLs and that correlate well with gene expression in liver after both 6 wk CCl4 treatment and acute ethanol damage of the liver. Within this interval we rank two genes containing coding sequence variants as strong candidates that may modulate the severity of liver fibrosis: 1) the extracellular proteinase inhibitor gene Expi (also known as Wdnm1 or Wfdc18) and 2) musashi RNA-binding protein 2 (Msi2). The powerful combination of experimental, genetics, and bioinformatics methods, as well as combined in vitro and in vivo approaches can be used to define QTLs, genes, and even candidate sequence variants linked to hepatotoxicity and fibrosis.

Systems Genetics of Liver Fibrosis

This systems genetics analysis comprises quantitative measurements of hepatic fibrogenesis in mouse models and mapping of quantitative traits in mouse genetic reference populations. It is part of a large mapping project of fibrogenic genes including the analyses of experimental crosses from different inbred mouse strains. Extensive quantitative trait loci (QTL) mapping of fibrosis phenotypes and liver expression profiling in combination with in silico mapping facilitated the identification of QTL regions and underlying candidate genes that confer fibrosis susceptibility also in humans. Moreover, the approach led to the identification of interacting QTLs and gene networks in liver fibrosis, providing a key experimental platform for the development of novel, more precise therapeutic interventions. Here, we provide a use case for the application of different analysis tools and the integration of multiple datasets determined in F2 intercrosses and BXD recombinant inbred lines to identify, finemap and affirm fibrosis susceptibility loci.

Exploring multiple quantitative trait loci models of hepatic fibrosis in a mouse intercross

Most common diseases are attributed to multiple genetic variants, and the feasibility of identifying inherited risk factors is often restricted to the identification of alleles with high or intermediate effect sizes. In our previous studies, we identified single loci associated with hepatic fibrosis (Hfib1–Hfib4). Recent advances in analysis tools allowed us to model loci interactions for liver fibrosis. We analysed 322 F2 progeny from an intercross of the fibrosis-susceptible strain BALB/cJ and the resistant strain FVB/NJ. The mice were challenged with carbon tetrachloride (CCl4) for 6 weeks to induce chronic hepatic injury and fibrosis. Fibrosis progression was quantified by determining histological fibrosis stages and hepatic collagen contents. Phenotypic data were correlated to genome-wide markers to identify quantitative trait loci (QTL). Thirteen susceptibility loci were identified by single and composite interval mapping, and were included in the subsequent multiple QTL model (MQM) testing. Models provided evidence for susceptibility loci with strongest association to collagen contents (chromosomes 1, 2, 8 and 13) or fibrosis stages (chromosomes 1, 2, 12 and 14). These loci contained the known fibrosis risk genes Hc, Fasl and Foxa2 and were incorporated in a fibrosis network. Interestingly the hepatic fibrosis locus on chromosome 1 (Hfib5) connects both phenotype networks, strengthening its role as a potential modifier locus. Including multiple QTL mapping to association studies adds valuable information on gene–gene interactions in experimental crosses and human cohorts. This study presents an initial step towards a refined understanding of profibrogenic gene networks.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Dedifferentiation of primary hepatocytes in vitro makes their application in long‐term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long‐term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up‐regulated to deal with this, whereas in the ML culture a down‐regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down‐regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down‐regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447–454, 2018.