Liver-specific Fas silencing prevents galactosamine/lipopolysaccharide-induced liver injury.

Acute liver failure (ALF) is a life threatening disease for which only few treatment options exist. The molecular pathways of disease progression are not well defined, but the death receptor Fas (CD95/Apo-1) appears to play a pivotal role in hepatocyte cell death and the development of ALF. Here, we explored posttranscriptional gene silencing of Fas by RNAi to inhibit pathophysiological gene expression. For targeting Fas expression in mice, Fas siRNA was formulated with the liver-specific siRNA delivery system DBTC. Treatment of mice with DBTC/siRNA(Fas) reduced Fas expression in the liver, but not in the spleen, lung, kidney or heart. Furthermore, silencing of Fas receptor was effective in blocking or reducing several aspects of ALF when it was tested in mice exposed to galactosamine/lipopolysaccharide (G/L), a well-known model of ALF. The application of DBTC/siRNA(Fas) 48 h prior G/L exposure resulted in amelioration of hepatic perfusion, reduction of hepatocellular death and increase of survival rate. The administration of DBTC/siRNA(Fas) formulation further diminished the inflammatory response upon G/L challenge, as indicated by a marked decrease of TNFα mRNA expression. However, IL-6 plasma concentration remained unaffectedly by DBTC/siRNA(Fas) formulation. Since the specific silencing of hepatic Fas expression only partially protected from inflammation, but completely attenuated apoptotic and necrotic cell death as well as microcirculatory dysfunction, the development of therapeutic strategies with DBTC lipoplex formulations to treat ALF should be combined with anti-inflammatory strategies to reach maximal therapeutic efficacy.

Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.

Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1-dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluronan-mediated motility receptor RHAMM. E2F1 directly up-regulates RHAMM, which in turn acts as a co-activator of E2F1 to stimulate expression of the extracellular matrix protein fibronectin. Enhanced fibronectin secretion links E2F1/RHAMM transcriptional activity to integrin-ß1-FAK signalling associated with cytoskeletal remodelling and enhanced tumour cell motility. RHAMM depletion abolishes fibronectin expression and cell transmigration across the endothelial layer in E2F1-activated cells. In a xenograft model, knock-down of E2F1 or RHAMM in metastatic cells protects the liver parenchyma of mice against extravasation of CTCs, whereas the number of transmigrated cells increases in response to E2F1 induction. Expression data from clinical tissue samples reveals high E2F1 and RHAMM levels that closely correlate with malignant progression. These findings suggest a requirement for RHAMM in late-stage metastasis by a mechanism involving cooperative stimulation of fibronectin, with a resultant tumourigenic microenvironment important for enhanced extravasation and distant organ colonization. Therefore, stimulation of the E2F1-RHAMM axis in aggressive cancer cells is of high clinical significance. Targeting RHAMM may represent a promising approach to avoid E2F1-mediated metastatic dissemination.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC.

Differential Effects of Axin2 Deficiency on the Fibrogenic and Regenerative Response in Livers of Bile Duct-Ligated Mice.

BACKGROUND: Wnt signaling is involved in the pathogenesis of liver fibrosis. Axin2 is a negative regulator of the canonical Wnt pathway by promoting ß-catenin degradation. ß-Catenin-activating and loss-of-function mutations of Axin2 are thought to be functionally relevant for liver diseases and cancer. Thus, we hypothesized that Axin2 deficiency promotes fibrogenesis. METHODS: As the functions and mechanisms of how Axin2/ß-catenin signaling participates in the progression of liver fibrosis are unclear, we investigated the progression of liver fibrosis in Axin2-deficient mice using Axin2-LacZ reporter mice (Axin2+/-, Axin2-/-, and Axin2+/+) which underwent bile duct ligation (BDL). RESULTS: Here, we show that the expression of Axin2 is downregulated during fibrogenesis in wild-type mice, which is consistent with a decreased expression of the reporter gene LacZ in Axin2+/- and Axin2-/- mice. Surprisingly, no alteration in active ß-catenin/Wnt signaling occurs in Axin2-deficient mice upon BDL. Despite a less pronounced liver injury, Axin2 deficiency had only minor and no significant effects on the fibrogenic response upon BDL, i.e. slightly reduced hepatic stellate cell activity and collagen mRNA expression. However, livers of Axin2-/- mice shared a stronger cell proliferation both already at baseline as well as immediately after BDL. CONCLUSION: Our results strongly suggest, contrary to expectation, that a deficiency in Axin2 is not equivalent to an increase in active ß-catenin and target genes, indicating no functional relevance of Axin2-dependent regulation of the canonical Wnt/ß-catenin pathway in the progression of cholestatic liver injury. This also suggests that the negligible effects of Axin2 deficiency during fibrogenesis may be related to an alternative pathway.

Anti-apoptotic therapeutic approaches in liver diseases: do they really make sense?

A variety of data suggesting apoptotic cell death as a key feature of liver injury stimulated researchers to investigate the therapeutic potential of anti-apoptotic strategies in experimental models. However, the overestimated role of apoptotic cell death in liver injury has tempered the clinical translation of the protection afforded by anti-apoptotic regimes in experimental models. Thus, the hope for apoptosis modulation as potential treatment strategy for injured liver in humans could not be confirmed. Herein, we evaluated the degree of apoptosis in different hepatic stress models which are relevant for the human pathophysiology. Using morphological criteria of apoptosis, caspase-3 activation as well as TUNEL assay in combination with a positive control of apoptosis in liver injury, we quantified apoptotic cell death discriminating between parenchymal and non-parenchymal cells and confirmed these results by cleaved caspase-3 and PARP-1 protein expression. Discussing our findings and relating them to the existing literature on the potential role of apoptotic cell death, we strongly recommend reconsidering anti-apoptotic strategies to ameliorate liver injury efficiently.

7 Tesla MRI Liver Fat Quantification in Mice: Data Quality Assessment.

PURPOSE: The objective of this study was to evaluate the robustness of proton density fat fraction (PDFF) data determined by magnetic resonance imaging (MRI) and spectroscopy (MRS) via spatially resolved error estimation. MATERIALS AND METHODS: Using standard T2* relaxation time measurement protocols, in-vivo and ex-vivo MRI data with water and fat nominally in phase or out of phase relative to each other were acquired on a 7 T small animal scanner. Based on a total of 24 different echo times, PDFF maps were calculated in a magnitude-based approach. After identification of the decisive error-prone variables, pixel-wise error estimation was performed by simple propagation of uncertainty. The method was then used to evaluate PDFF data acquired for an explanted mouse liver and an in vivo mouse liver measurement. RESULTS: The determined error maps helped excluding measurement errors as cause of unexpected local PDFF variations in the explanted liver. For in vivo measurements, severe error maps gave rise to doubts in the acquired PDFF maps and triggered an in-depth analysis of possible causes, yielding abdominal movement or bladder filling as in vivo occurring reasons for the increased errors. CONCLUSION: The combination of pixel-wise acquisition of PDFF data and the corresponding error maps allows for a more specific, spatially resolved evaluation of the PDFF value reliability.

Erythropoietin as additive of HTK preservation solution in cold ischemia/reperfusion injury of steatotic livers.

BACKGROUND: Organ shortage in liver transplantation has justified usage of marginal donor livers to expand the donor organ pool. The particular susceptibility of steatotic livers to I/R injury necessitates optimal preservation conditions in order to minimize preservation-reperfusion injury for successful transplantation. METHODS: The effect of erythropoietin (EPO) as additive to HTK preservation solution was studied in a mouse model. Lean and steatotic livers were harvested, stored for 24 h in 4°C HTK solution containing either EPO or saline and reperfused for 2 h with 37°C Krebs-Henseleit buffer. Livers without cold storage served as sham controls. RESULTS: Flushing of livers upon cold storage revealed a transaminase release, which was 2- to 10-fold higher in steatotic versus lean livers. EPO was effective in reducing the enzyme release to 50% in steatotic but not in lean livers. EPO prevented cold storage-induced denudation of the endothelial lining in steatotic livers, but aggravated it in lean livers. During reperfusion, steatotic livers presented with lower oxygen consumption and higher enzyme release than lean livers. In all livers, parameters of reperfusion injury remained unaffected by EPO. Expression of UCP2 was found markedly higher in steatotic livers. After I/R, steatotic livers revealed a significant drop of UCP2, whereas expression in lean livers was only slightly affected. EPO diminished Erk phosphorylation to almost the same extent in both mouse strains. CONCLUSION: Fortification of the preservation solution by EPO ameliorates cold ischemic injury of steatotic livers and may thus be considered for use as an adjunctive agent to increase the success of transplanting steatotic livers.

A critical appraisal of the hemodynamic signal driving liver regeneration.

BACKGROUND: Many aspects of the signaling mechanisms involved in the initiation of hepatic regeneration are under current investigation. Nevertheless, the actual mechanisms switching liver regeneration on and off are still unknown. Hemodynamic changes in the liver following partial hepatectomy have been suggested to be a primary stimulus in triggering liver regeneration. Most of the new knowledge about the impact of hemodynamic changes on liver regeneration is both conceptually important and directly relevant to clinical problems. PURPOSE: The purpose of this review is therefore to exclusively address the hemodynamic signal driving the liver regeneration process.

Darbepoetin-α inhibits the perpetuation of necro-inflammation and delays the progression of cholestatic fibrosis in mice.

Biliary obstruction and cholestasis result in hepatocellular necro-inflammation and lead to the development of liver fibrosis. The objective of this study was to analyze whether the multiple tissue-protective properties of erythropoietin are salutary in an experimental model of liver fibrosis. For this purpose, C57BL/6J mice underwent common bile duct ligation (BDL) and were treated with either darbepoetin-α (10 µg/kg i.p.) or physiological saline every third day, beginning 24 h after BDL. Mice were killed at 2, 5, 14, and 28 days after BDL. Beside hematological parameters, markers of inflammation and fibrosis were assessed histomorphometrically and immunohistochemically as well as by quantitative real-time PCR. In addition, a 7-week survival study was performed. BDL provoked cholestatic hepatitis characterized by biliary infarcts with accumulation of macrophages followed by marked collagen deposition and increased expression of profibrotic gene transcripts. Darbepoetin-α treatment significantly diminished the area of focal necrosis, reduced the infiltration of macrophages, decreased levels of profibrotic genes, and lowered collagen deposition. Moreover, darbepoetin-α significantly reduced systemic anemia caused by BDL. Finally, darbepoetin-α treatment significantly prolonged the survival time after BDL. This study suggests that darbepoetin-α, which is a clinically well-established substance, might be used as an efficient therapeutic option for patients with chronic cholestatic liver disease.

Splenectomy improves survival by increasing arterial blood supply in a rat model of reduced-size liver.

Prevention of acute portal hyperperfusion in small-for-size livers by inflow modulation results in beneficial postoperative outcome. The objective of this study was to unravel the underlying mechanism, emphasizing the intimate relationship between portal venous (PV) and hepatic arterial (HA) blood flow (BF). Rats underwent partial hepatectomy (pHx), splenectomy before pHx or splenectomy and ligation of the A. hepatica before pHx. Portal venous blood flow (PVBF), hepatic arterial blood flow (HABF), and tissue pO2 were assessed during stepwise resection from 30% to 90%. Hepatic regeneration and hypoxia-responsive gene expression were analyzed in each group after nonlethal 85% pHx. 90% pHx caused a fourfold rise in PVBF, a slight decrease in HABF with a 50% reduction in pO2, and high mortality. Splenectomy before pHx reduced the PVBF and caused a rise in HABF with doubling in tissue pO2. An attenuation of hypoxia-responsive gene expression turned into enhanced hepatocellular regeneration and improved survival. A. hepatica ligation abolished the beneficial effect of splenectomy on tissue oxygenation, proliferation, and outcome. In conclusion, the beneficial effect of splenectomy in small-for-size livers can be attributed to a rise in HABF with sufficient oxygen supply rather than to a reduced portal venous hyperperfusion to the remnant liver.

Limited potential of resolvin D1 in treatment of cholestatic liver fibrosis.

BACKGROUND: Several studies suggest a role for EPA- and DHA-derived pro-resolving mediators like resolvins in reversing metabolic and inflammatory disturbances seen in various chronic diseases. Here, we investigated the effects of resolvin D1 (RvD1) on bile duct ligation (BDL)-induced cholestatic liver injury. METHODS: Mice were treated daily with RvD1 or 0.1% ethanol (control) from the day of BDL until the final observation time points. Blood and liver tissue were collected 2, 5 and 14 days after BDL for different analyses. RESULTS: RvD1 treatment of mice had no impact on the extent of cholestatic liver injury upon BDL, neither in the acute phase nor in the progressive state of liver fibrosis. Although RvD1 treatment resulted in a significantly reduced activity of hepatic stellate cells as well as reduced deposition of extracellular matrix 2 days after BDL, mice were not protected from inflammation and further fibrosis progression. CONCLUSIONS: These data indicate that RvD1 has a limited therapeutic potential to treat cholestatic liver diseases, as it has no significant impact on regression of hepatic necroinflammation and fibrotic changes in bile duct-ligated mice.

Comparing distress of mouse models for liver damage.

In order to foster animal welfare as well as high quality of research, many countries regulate by law that the severity of animal experiments must be evaluated and considered when performing biomedical research. It is well accepted that multiple parameters rather than a single readout parameter should be applied to describe animal distress or suffering. However, since the performance of readout parameters for animal distress is rarely defined and methods for multivariate analysis have only in rare cases been used, it is not known which methodology is most appropriate to define animal distress. This study used receiver operating characteristic curve analysis to quantify the performance of burrowing activity, body weight change and a distress score of mice after induction of liver damage by bile duct ligation or carbon tetrachloride. In addition, Support Vector Machine classification was used to compare the distress of these mouse models. This approach demonstrated that bile duct ligation causes much more distress than carbon tetrachloride-induced liver damage. This study, therefore, provides a prototype how to compare two animal models by considering several readout parameters. In the future these or similar methods for multivariate analysis will be necessary, when assessing and comparing the severity of animal models.

A Human REPIN1 Gene Variant: Genetic Risk Factor for the Development of Nonalcoholic Fatty Liver Disease.

OBJECTIVES: We tested the hypothesis that a genetic deletion (Del) variant in the REPIN1 gene is associated with the severity of nonalcoholic fatty liver disease (NAFLD) in humans. METHODS: Sixty-three donors of liver biopsies from individuals with obesity and different degrees of NAFLD and fibrosis were screened for a Del REPIN1 gene variant and liver REPIN1 mRNA expression. RESULTS: In 8 homozygous Del carriers, we found significantly lower NAFLD activity and fibrosis scores compared with 55 wild-type allele carriers. DISCUSSION: A Del variant of REPIN1 may be associated with a lower risk of the development of NAFLD.

A rational approach of early humane endpoint determination in a murine model for cholestasis.

Reduction of animal suffering during in vivo experiments is usually ensured by continuously monitoring the health status using a score sheet and by applying humane endpoints. However, most studies do not evaluate the plausibility of score sheets and do not attempt to reduce the suffering of animals by determining earlier and, therefore, more humane endpoints. The present study uses data from BALB/cANCrl mice after bile duct ligation to retrospectively analyze which score sheet criteria are informative to determine humane endpoints. The performance of each single as well as com­binations of multiple animal welfare parameters was analyzed by a Cox proportional-hazards model followed by Harrell's concordance index. The addition of behavioral parameters, such as burrowing activity, helped to define a more humane early endpoint for euthanizing these animals. Using this approach, we determined that a body weight loss of 10-20% combined with a reduction of burrowing activity by more than 79.4% was able to predict that these animals would die within two days. Thus, this approach successfully determined an earlier humane endpoint and will reduce the suffering of animals in future experiments. Application of such an approach or similar methods can contribute to the refinement of various animal experiments.

Uncoupling protein-2 deficiency provides protection in a murine model of endotoxemic acute liver failure.

OBJECTIVE: Liver injury and cell death are prominent features in the pathogenesis of acute liver failure. Mitochondrial uncoupling protein 2 plays a controversial role in liver cell death through its involvement in the production of reactive oxygen species and adenosine triphosphate. DESIGN: This randomized controlled animal study was designed to investigate the exact role of uncoupling protein 2 in the pathogenesis of endotoxemic acute liver failure. SETTING: Research laboratory of an academic institution. SUBJECTS, INTERVENTIONS, AND MEASUREMENTS: Uncoupling protein 2+/+ and uncoupling protein 2-/- mice were challenged with D-galactosamine (Gal, 720 mg/kg intraperitoneally) and Escherichia coli lipopolysaccharide (10 microg/kg intraperitoneally) and studied 6 hrs thereafter (n = 5 per group). Control mice received physiologic saline (n = 5 per group). Analysis included in vivo fluorescence microscopy of hepatic microcirculation and hepatocellular apoptosis as well as plasma malondialdehyde concentrations as reactive oxygen species-dependent lipid peroxidation product and hepatic adenosine triphosphate levels. MAIN RESULTS: Administration of Gal-lipopolysaccharide in uncoupling protein 2+/+ mice caused systemic cytokine release and malondialdehyde production. Further, it provoked marked hepatic damage, characterized by intrahepatic leukocyte recruitment (10.5 +/- 1.3 n/mm2 vs. 3.3 +/- 0.5 n/mm2), microvascular perfusion failure (33.1% +/- 1.6% vs. 2.3% +/- 0.4%), and adenosine triphosphate depletion (3.4 +/- 0.9 micromol/g vs. 6.4 +/- 0.9 micromol/g). Furthermore, uncoupling protein +/+ mice revealed a huge rise in cell apoptosis, given by high numbers of hepatocytes exhibiting nuclear chromatin fragmentation (44.9 +/- 11.5 n/mm2 vs. 0.0 +/- 0.0 n/mm2) and cleaved caspase-3 expression (1.24 +/- 0.24 vs. 0.06 +/- 0.04). Liver injury was coexistent with enzyme release (alanine aminotransferase 442 +/- 126 U/L vs. 57 +/- 12 U/L) and necrotic cell death. Of interest, Gal-lipopolysaccharide-exposed uncoupling protein 2-/- mice exhibited higher rates of hepatocellular apoptosis (135.6 +/- 46.0 n/mm2) as well as cleaved caspase-3 expression (1.75 +/- 0.25), however, preserved hepatic adenosine triphosphate (6.4 +/- 1.7), milder perfusion failure (24.5 +/- 2.4) and decreased leukocyte recruitment (2.7 +/- 0.2), less necrotic injury, lower transaminase levels (340 +/- 91), and finally better survival rates. CONCLUSION: The higher adenosine triphosphate availability in uncoupling protein 2-deficient mice might allow hepatocytes to undergo apoptosis as an energy-consuming mode of cell death, while at the same time cellular adenosine triphosphate levels seem to increase hepatic resistance against harmful effects upon Gal-lipopolysaccharide exposure. As net result, uncoupling protein 2 deficiency provided protection under endotoxemic stress conditions, underlining the significant role of the bioenergetic status in critical illness.

Endogenously increased n-3 PUFA levels in fat-1 transgenic mice do not protect from non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis. Possible reasons for the NAFLD epidemic in industrialized countries are the high intake of pro-inflammatory n-6 polyunsaturated fatty acids (n-6 PUFAs) and low consumption of healthy n-3 PUFAs. Due to their anti-inflammatory properties, n-3 PUFAs may have the potential to alleviate chronic liver disease. Herein, we examined the therapeutic effect of increased n-3 PUFA tissue levels in fat-1 transgenic mice on progressive NASH. METHODS: Disease was induced in mice by streptozotocin and high fat diet (STZ/HFD) resulting in NASH. NAFLD in 6 and 8 weeks old wild type and fat-1 transgenic STZ/HFD treated mice was analyzed. Unlike all other mammals, fat-1 transgenic mice ubiquitously express an n-3 fatty acid desaturase, which converts n-6 to n-3 PUFAs, leading to increased n-3 and decreased n-6 PUFA tissue contents. RESULTS: Liver damage, NAFLD activity score (NAS), hepatic lipid accumulation and inflammation were significantly reduced in fat-1 transgenic STZ/HFD treated mice in the early (6 weeks) but not late (8 weeks) phase of NASH. Simultaneously, mRNA expression of genes involved in fatty acid uptake and storage (Cd36 and Plin3, respectively) was significantly down-regulated in 6 week old but not 8 week old fat-1 transgenic STZ/HFD treated mice. CONCLUSIONS: Endogenously elevated n-3 PUFA levels in fat-1 transgenic mice transiently delay the onset of STZ/HFD induced NASH but failed to efficiently protect from NASH development.

n-3 PUFAs reduce tumor load and improve survival in a NASH-tumor mouse model.

BACKGROUND: With 9.1% of all cancer deaths, hepatocellular carcinoma is the second leading cause of cancer deaths worldwide. Due to the increasing prevalence of metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) has evolved into a major risk factor for hepatocellular carcinoma development. Herein, we investigated whether a dietary n-3 polyunsaturated fatty acid (PUFA) supplementation improves the outcome of progressive NAFLD. METHODS: Feeding three high-fat diets, differing in n-3 and n-6 PUFA contents and ratios (n-3/n-6: 1:8, 1:1, 5:1), the impact of n-3 PUFAs and n-3/n-6 PUFA ratios on NAFLD-related liver fibrosis and tumorigenesis was analyzed in 12- and 20-week-old streptozotocin/high-fat diet (STZ/HFD)-treated mice. RESULTS: Feeding of n-3 PUFA-rich diets (1:1 and 5:1) resulted in increased hepatic n-3 PUFA content and n-3/n-6 PUFA ratio with decreased hepatic lipid accumulation. In 20-week-old mice, n-3 PUFA-rich diets alleviated tumor load significantly, with reduced liver/body weight index, tumor size, and tumor number. Finally, these effects were accompanied by a significant improvement of survival of these mice. CONCLUSIONS: Herein, we showed that increased n-3 PUFA content and n-3/n-6 PUFA ratios lead to improved survival and attenuated tumor progression in STZ/HFD-treated mice. Thus, n-3 PUFAs could be the basis for new therapeutic options against NAFLD-related tumorigenesis.

Repin1 deficiency in liver tissue alleviates NAFLD progression in mice.

There is an increasing prevalence of obesity and metabolic syndrome, which promote the development of non-alcoholic fatty liver disease (NAFLD), a disease that can evolve into cirrhosis and hepatocellular carcinoma. Repin1 loss was previously shown to have beneficial effects on lipid and glucose metabolism and obesity regulation. Herein, we characterized NAFLD in mice with hepatic deletion of Repin1 (LRep1-/-). For this purpose, liver disease was analysed in male LRep1-/- and wild-type mice treated with streptozotocin/high fat diet or a control diet over a period of 20 wks. Streptozotocin/high fat diet treated LRep1-/- mice showed a significant decrease in systemic and hepatic lipid accumulation, accompanied by diminished chronic inflammation and a subsequent reduction in liver injury. Remarkably, Repin1-deficient mice exhibited a lower tumour prevalence and tumour frequency, as well as a reduced liver weight/body weight index. A therapeutic approach using Repin1 siRNA in the early phase of NAFLD verified the observed beneficial effects of Repin1 deficiency. This study provides evidence that loss of Repin1 in the liver attenuates NAFLD progression, most likely by reducing fat accumulation and alleviating chronic tissue inflammation. Thus, modulating Repin1 expression may become a novel strategy and potential tool to inhibit NAFLD progression.

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

Hepatocellular apoptosis is mediated by TNFalpha-dependent Fas/FasLigand cytotoxicity in a murine model of acute liver failure.

There is increasing evidence that the active contribution of hepatocytes to liver disease is strongly dependent on local cytokine environment. It has been shown in vitro that TNFalpha can enhance hepatocyte FasLigand (FasL)-mediated cytotoxicity. Here, we demonstrate that TNFalpha-induced apoptosis was associated with Fas and FasL upregulation and that a FasL-neutralizing antibody prevented TNFalpha-induced apoptosis. We further examined in vivo the relevance of the Fas/FasL pathway to hepatocellular apoptosis in a TNFalpha-driven model of acute liver failure. Livers of galactosamine/lipopolysaccharide (Gal/LPS)-exposed Fas wild-type mice highly expressed both Fas and FasL and revealed marked hepatocellular apoptosis that was almost completely blocked by soluble TNFalpha-receptor; this was also almost absent in Gal/LPS-exposed Fas lymphoproliferation mutant mice. Our data provide evidence for a direct link between TNFalpha and Fas/FasL in mediating hepatocyte apoptosis. Fratricidal death by Fas-FasL interactions of neighbouring hepatocytes may actively contribute to acute liver failure.