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The progression of pancreatic ductal adenocarcinoma (PDAC) is significantly affected by transforming growth factor (TGF)-β but targeting TGF-β can also compromize physiological effects in patients. Our study examined the functions of the ubiquitously expressed protein, PDCD10, as a modulator of TGF-β signaling in PDAC. Using in silico analyses we found that in patient samples, PDCD10 is significantly higher expressed in PDAC tumor tissue compared with normal pancreas and it is highly correlated with reduced survival. We created stable KO's of PDCD10 in two PDAC lines, PaTu 8902 (SMAD4 +/+) and PaTu 8988t (SMAD4 -/-), and found that KO lines are more sensitive to 5-FU and Gemcitabine treatment than their wild-type counterparts. Performing viability and wound closure assays we further found that PDCD10 promotes cell survival and proliferation by enhancing specifically the mitogenic functions of TGF-β. The molecular mechanism underlying this effect was further investigated using Western blots and with primary organoid lines derived from patient PDAC tissue samples. The data imply that PDCD10 mediates an increase in p-ERK through a non-SMAD4 pathway, leading to EMT promotion. Furthermore, PDCD10 facilitates deactivation of RB via a SMAD4-dependent pathway, thereby counter-acting the anti-proliferative actions of TGF-β. By performing proximity ligation assays (PLA) we found that PDCD10 associates with the kinase MST4, translocates it intracellularly and thereby facilitates phosphorylations of RB and ERK1/2. Our study indicates that PDCD10 promotes the proliferative function and EMT induction of TGF-β in pancreatic cancer cells. Therefore, targeting PDCD10 in PDAC patients could represent a promising new strategy to optimize TGF-β targeted therapies. [ABSTRACT FROM AUTHOR]

Ghafoory S, Breitkopf-Heinlein K, Li Q, Dzieran J, Scholl C, Dooley S & Wölfl S (2012) Histopathology 61, 306-313 A fast and efficient polymerase chain reaction-based method for the preparation of in situ hybridization probes Aims: In situ hybridization (ISH) is the method of choice for analysis of the local distribution of gene expression in tissue samples at the cellular level. In this study we present a rapid and efficient protocol for the generation of labelled cRNA probes. Methods and results: The protocol is based on the preparation of DNA in vitro transcription templates using polymerase chain reaction (PCR), using primers that include RNA polymerase promoter sequences and size-based purification of PCR fragments containing the target gene-specific cDNA and promoter elements for T7 and SP6 RNA polymerase. The optimized purification protocols ensure high transcription efficiency and target specificity of the labelled cRNA. The cRNA hybridization probes obtained are compatible with established in situ hybridization protocols. Conclusions: Purified PCR fragment-based in vitro transcription enables preparation of in situ hybridization probes which allow the rapid detection of gene expression distribution in tissue slices from any gene of interest. [ABSTRACT FROM AUTHOR]

Liver inflammation and macrophage infiltration are critical steps in the progression of non-alcoholic fatty liver to the development of non-alcoholic steatohepatitis. Bone morphogenetic protein-9 is a cytokine involved in the regulation of chemokines and lipogenesis. However, the function of bone morphogenetic protein-9 in non-alcoholic steatohepatitis is still unknown. The present study hypothesized that bone morphogenetic protein-9 may contribute to steatohepatitis in mice fed a methionine choline deficiency diet (MCD). C57BL/6 mice overexpressing bone morphogenetic protein-9 and control mice were fed the MCD diet for 4 weeks. Liver tissue and serum samples were obtained for subsequent measurements. Bone morphogenetic protein-9 overexpression exacerbated steatohepatitis in mice on the MCD diet, as indicated by liver histopathology, increased serum alanine aminotransferase activity, aspartate transaminase activity, hepatic inflammatory gene expression and M1 macrophage recruitment. Although bone morphogenetic protein-9 overexpression did not affect the expression of pro-fibrogenic genes, including Collagen I (α)1 or matrix metalloproteinase (MMP) 9, it did upregulate the expression of transforming growth factor-β and plasminogen activator inhibitor 1, and downregulated the expression of MMP2. The above results indicate that bone morphogenetic protein-9 exerts a pro-inflammatory role in MCD diet-induced non-alcoholic steatohepatitis. [ABSTRACT FROM AUTHOR]

Zonation of metabolic activities within specific structures and cell types is a phenomenon of liver organization and ensures complementarity of variant liver functions like protein production, glucose homeostasis and detoxification. To analyze damage and regeneration of liver tissue in response to a toxic agent, expression of liver specific enzymes was analyzed by in situ hybridization in mouse over a 6 days time course following carbon tetrachloride (CCl4) injection. CCl4 mixed with mineral oil was administered to BALB/c mice by intraperitoneal injection, and mice were sacrificed at different time points post injection. Changes in the expression of albumin (Alb), arginase (Arg1), glutaminase 2 (Gls2), Glutamine synthetase (Gs), glucose-6-phosphatase (G6pc), glycogen synthase 2 (Gys2), Glycerinaldehyd-3-phosphat-Dehydrogenase (Gapdh), Cytochrom p450 2E1 (Cyp2e1) and glucagon receptor (Gcgr) genes in the liver were studied by in situ hybridization and qPCR. We observed significant changes in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution following CCl4 injury. We also found that Cyp2e1, the primary metabolizing enzyme for CCl4, was strongly expressed in the pericentral zone during recovery. Furthermore, cells in the damaged area displayed distinct gene expression profiles during the analyzed time course and showed complete recovery with strong albumin production 6 days after CCl4 injection. Our results indicate that despite severe damage, liver cells in the damaged area do not simply die but instead display locally adjusted gene expression supporting damage response and recovery. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMP-2 to BMP-15) belong to the Transforming Growth Factor (TGF)-ß superfamily and, besides their well-documented roles during embryogenesis and bone formation, some of them have recently been described to be involved in the pathogenesis of different organs, including the liver. The role of BMPs in liver damage responses including hepatocellular carcinoma (HCC) development has only begun to be addressed and strong evidence supports the concept of a pro-tumorigenic role of BMP signaling in HCC cells. BMP-9 (also termed Growth and Differentiation Factor (GDF)-2) represents the most recently discovered member of the BMP family. We have previously demonstrated that in HCC patient samples BMP-9 expression was positively associated with the tumor seize ("T stage") and that it enhanced cell migration and induced epithelial to mesenchymal transition (EMT) in HCC cells in vitro. In another study we recently found that BMP-9 promotes growth in HCC cells, but not in non-transformed hepatocytes. Published as well as unpublished results obtained with primary hepatocytes support the concept of a dual function of BMP-9 in the liver: while in primary, non-malignant cells BMP-9 stabilizes the epithelial phenotype and inhibits proliferation, in HCC cells it induces cell growth and the acquisition of a migratory phenotype. In this review article we summarize current knowledge about BMPs in liver diseases, with special focus on the role of BMP-9 in HCC development and progression, that may provide new clues for a better understanding of the contribution of BMP-signaling to chronic liver diseases. [ABSTRACT FROM AUTHOR]

Background: Biliary tract cancers (BTCs) have a poor prognosis. BTCs are characterized by a prominent desmoplastic reaction which possibly contributes to the aggressive phenotype of this tumor. The desmoplastic reaction includes excessive production and deposition of extracellular matrix proteins such as periostin, secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1, as well as accumulation of α-smooth muscle actin-positive cancer-associated fibroblasts and immune cells, secreting growth factors and cytokines including transforming growth factor (TGF)-β. In the present study, we investigated the expression of SPARC in BTC as well as its possible regulation by TGF-β. Methods: Expression levels of Sparc,TGF-β1 and its receptor ALK5 were evaluated by quantitative real-time PCR in 6 biliary tract cell lines as well as 1 immortalized cholangiocyte cell line (MMNK-1). RNAs from tumor samples of 7 biliary tract cancer patients were analyzed for expression of Sparc, TGF-β type II receptor (TbRII) as well as Twist and ZO-1. MMNK-1 cells were stimulated with TGF-β for 24 h, and Sparc,ZO-1 and E-Cadherin expressions were determined. The presence of SPARC protein was analyzed by immunohistochemistry in tumor specimens from 10 patients. Results: When comparing basal Sparc transcript levels in diverse BTC cell lines to MMNK-1 cells, we found that it was strongly downregulated in all cancer cell lines. The remaining expression levels were higher in highly differentiated cell lines (CCSW1, MZChA1, MZChA2 and TFK-1) than in less differentiated and undifferentiated ones (BDC, SKChA1). Expression of Sparc in BTC patient samples showed a significant positive correlation with expression of the epithelial marker ZO-1. In contrast, the mesenchymal marker Twist and the TbRII showed a trend of negative correlation with expression of Sparc in these samples. TGF-β exposure significantly downregulated Sparc expression in MMNK-1 cholangiocytes in vitro in parallel to downregulation of epithelial markers (E-Cadherin and ZO-1). Finally, SPARC immunostaining was performed in 10 patient samples, and the correlation between absence of SPARC and survival times was analyzed. Conclusions: These data imply that a decrease in SPARC expression is correlated with dedifferentiation of BTC cells resulting in enhanced EMT being possibly mediated by TGF-β. Thereby SPARC levels might be a marker for individual prognosis of a patient, and strategies aiming at inhibition of SPARC downregulation might have potential for new future therapies. [ABSTRACT FROM AUTHOR]

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems. [ABSTRACT FROM AUTHOR]

Liver sinusoidal endothelial cells (LSEC) represent a unique, organ-specific type of discontinuous endothelial cells. LSEC instruct the hepatic vascular niche by paracrine-acting angiocrine factors. Recently, we have shown that LSEC-specific transcriptional regulator GATA4 induces expression of BMP2 in cultured endothelial cells (EC) in vitro. Furthermore, angiocrine Bmp2 signaling in the liver in vivo was demonstrated to control iron homeostasis. Here, we investigated GATA4-dependent autocrine BMP2 signaling in endothelial cells by gene expression profiling. GATA4 induced a large cluster of inflammatory endothelial response genes in cultured EC, which is similar to previously identified virus-induced and interferon-associated responses. Treating the cells with the BMP2 inhibitor Noggin counter-regulated the GATA4-dependent inflammatory phenotype of EC, indicating that BMP2 is indeed the major driver. In contrast to continuous EC, LSEC were less prone to activation by BMP2. Notably, GATA4-dependent induction of the inflammatory EC response gene cluster was attenuated by over-expression of the LSEC-specific transcriptional modifier LMO3 while hepatocyte activation was fully preserved, indicating conserved BMP2 synthesis. In summary, our data suggest that transcriptional counter-regulation by GATA4 and LMO3 in LSEC prevents autocrine induction of an inflammatory phenotype, while maintaining angiocrine BMP2-mediated cell-cell communication in the liver vascular niche. [ABSTRACT FROM AUTHOR]

The segmentation of cell nuclei is an important step towards the automated analysis of histological images. The presence of a large number of nuclei in whole-slide images necessitates methods that are computationally tractable in addition to being effective. In this work, a method is developed for the robust segmentation of cell nuclei in histological images based on the principles of persistent homology. More specifically, an abstract simplicial homology approach for image segmentation is established. Essentially, the approach deals with the persistence of disconnected sets in the image, thus identifying salient regions that express patterns of persistence. By introducing an image representation based on topological features, the task of segmentation is less dependent on variations of color or texture. This results in a novel approach that generalizes well and provides stable performance. The method conceptualizes regions of interest (cell nuclei) pertinent to their topological features in a successful manner. The time cost of the proposed approach is lower-bounded by an almost linear behavior and upper-bounded by O ( n 2 ) in a worst-case scenario. Time complexity matches a quasilinear behavior which is O ( n 1 + ɛ ) for ε < 1. Images acquired from histological sections of liver tissue are used as a case study to demonstrate the effectiveness of the approach. The histological landscape consists of hepatocytes and non-parenchymal cells. The accuracy of the proposed methodology is verified against an automated workflow created by the output of a conventional filter bank (validated by experts) and the supervised training of a random forest classifier. The results are obtained on a per-object basis. The proposed workflow successfully detected both hepatocyte and non-parenchymal cell nuclei with an accuracy of 84.6%, and hepatocyte cell nuclei only with an accuracy of 86.2%. A public histological dataset with supplied ground-truth data is also used for evaluating the performance of the proposed approach (accuracy: 94.5%). Further validations are carried out with a publicly available dataset and ground-truth data from the Gland Segmentation in Colon Histology Images Challenge (GlaS) contest. The proposed method is useful for obtaining unsupervised robust initial segmentations that can be further integrated in image/data processing and management pipelines. The development of a fully automated system supporting a human expert provides tangible benefits in the context of clinical decision-making. [ABSTRACT FROM AUTHOR]

Promotion of rhBMP2 and rhBMP7 for the routine use to support fracture healing has been hampered by high costs, safety concerns and reasonable failure rates, imposing restrictions in its clinical use. Since there is little debate regarding its treatment potential, there is rising need for a better understanding of the mode of action of these BMPs to overcome its drawbacks and promote more efficacious treatment strategies for bone regeneration. Recently, BMP9, owing to its improved osteogenic potential, is gaining attention as a promising therapeutic alternative. Our study aimed at identifying specific gene expression patterns which may predict and explain individual responses to rhBMP7 and rhBMP9 treatments. Therefore, we investigated the effect of rhBMP7 and rhBMP9 on primary human osteoblasts from 110 donors and corresponding THP-1-derived osteoclasts. This was further compared with each other and our reported data on rhBMP2 response. Based on the individual donor response, we found three donor groups profiting from rhBMP treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclasts. The response on rhBMP7 treatment correlated with expression levels of the genes BAMBI, SOST, Noggin, Smad4 and RANKL, while the response of rhBMP9 correlated to the expression levels of Alk6, Endoglin, Smurf1, Smurf2, SOST and RANKL in these donors. Noteworthy, rhBMP9 treatment showed significantly increased osteogenic activity (AP activity and Smad nuclear translocation) when compared to the two clinically used rhBMPs. Based on patient's respective expression profiles, clinical application of rhBMP9 either solely or in combination with rhBMP2 and/or rhBMP7 can become a promising new approach to fit the patient's needs to promote fracture healing. [ABSTRACT FROM AUTHOR]

Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis. [ABSTRACT FROM AUTHOR]

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrineBmp2 signalingin the liver,weconditionallydeletedBmp2 inLSECsusing EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serumironlevelsand iron deposition in several organs similar to classic hereditaryhemochromatosis. Ironoverloadwasmediatedbydecreasedhepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism. [ABSTRACT FROM AUTHOR]

The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/ 3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5- mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels. [ABSTRACT FROM AUTHOR]

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) are members of the TGFβ family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFβ receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and timedependent manner, which can provide an explanation of some properties of the BMP regulatory network. [ABSTRACT FROM AUTHOR]

Abstract: The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe−/− and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe. [Copyright &y& Elsevier]

Genetic factors contribute to progression and modulation of hepatic fibrosis. High throughput genomics/transcriptomics approaches aiming at identifying key regulators of fibrosis development are tainted with the difficulty of separating essential biological 'driver' from modifier genes. We applied a comparative transcriptomics approach and investigated fibrosis development in different organs to identify overlapping expression changes, since these genes may be part of core pathways in fibrosis development. Gene expression was analysed on publicly available microarray data from liver, lung and kidney fibrosis. RARRES1, AGER and S100A2 were differentially regulated in all fibrosis experiments. RARRES1 was extensively analysed by means of advanced bioinformatics analyses and functional studies. Microarray and Western Blot analysis of a standard liver fibrosis model (CCl) demonstrated an early induction of RARRES1 mRNA and protein expression. In addition, quantitative RT-PCR in tissue samples from patients with advanced liver fibrosis showed higher expression as compared to non-fibrotic biopsies. Microarray analysis of RARRES1 overexpressing cells identified an enrichment of a major signature associated with fibrosis. Furthermore, RARRES1 expression increased during in vitro activation of hepatic stellate cells. To further verify the pro-fibrogenic role across organs, we demonstrated an increase in RARRES1 expression in a rat lung fibrosis model induced by adenoviral TGF-β1 induction. We have performed a comparative transcriptomics analysis in order to identify core pathways of liver fibrogenesis, confirmed a candidate gene and enlightened the up- and downstream mechanisms of its action leading to fibrosis across organs and species. [ABSTRACT FROM AUTHOR]

The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H2O2 (0.3-6 μM), concentrations that are comparable with levels of H2O2 released by inflammatory cells. In contrast, bolus treatment of H2O2 has no effect at low concentrations and even suppresses hepcidin at concentrations of>50μM.H2O2 treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H2O2-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H2O2 responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H2O2 released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H2O2. Thus, similar to cytokines, H2O2 provides an important regulatory link between inflammation and iron metabolism. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease remains a frequent and serious problem for increasing numbers of patients. Research has expanded our molecular understanding of the cellular basis of disease progression; however, translation into therapy is still hampered by a lack of suitable animal models for alcoholic liver disease, as well as from consequences of related liver damage due to malnutrition, hepatitis C virus infection, or abuse of other substances. Many patients with liver disease do not simply consume too much alcohol; they also suffer from comorbidities such as obesity or viral hepatitis, and/or may be addicted to other drugs besides alcohol. This review will summarize the currently available animal models to study liver disease due to either single causes or combinations of liver toxic substances/infections and alcohol. [ABSTRACT FROM AUTHOR]

• Bone morphogenetic protein 9 (BMP9) promotes MCD diet-induced nonalcoholic steatohepatitis. • BMP9 accelerates inflammation via macrophage M1 polarization. • BMP9 plays a pro-inflammatory role by activating NF-κB pathway. Our previous study confirmed that bone morphogenetic protein 9 (BMP9) participated in the development of nonalcoholic steatohepatitis (NASH) by affecting macrophage polarization. The focus of this study was to further confirm the role of macrophages in BMP9-mediated NASH and to analyze the underlying mechanism. In vivo , mice that were administered adeno-associated viral (AAV) vectors containing a null transgene (AAV-null) or the BMP9 transgene (AAV-BMP9) were divided into methionine- and choline-deficient (MCD) and control diet (CD) groups, and they were administered either control liposomes or clodronate liposomes via tail vein injection, the latter to deplete macrophages. The mice were sacrificed after 4 weeks of MCD diet feeding. In vitro , RAW264.7 cells were pretreated with or without BAY11-7085 (an NF-κB inhibitor) and stimulated with recombinant human BMP9 (rh-BMP9). To explore the underlying mechanism of action of BMP9, primary human monocyte-derived macrophages were additionally investigated and immunohistochemistry, biochemical assays, qRT-PCR, and Western blotting were used. The characteristics of NASH-related inflammation were assessed by hepatic histological analysis. Serum AST and ALT and hepatic triglyceride were examined by biochemical assays. We found that the expression of M1 macrophage genes (including CD86, IL1β, IL6, MCP-1 and TNFα) and the number of M1 macrophages (iNOS+ macrophages) in the liver were significantly elevated after BMP9 overexpression and BMP9 directly upregulated TLR4 expression in MCD-induced NASH. These effects were eliminated by macrophage depletion. In vitro , we discovered that BMP9 enhanced the nuclear translocation of NF-κB to induce macrophage M1 polarization in RAW264.7 cells and it promoted LPS-mediated activation of the NF-κB pathway in primary human macrophages. Taken together, this study demonstrates that BMP9 promotes NASH development by directly acting on macrophages. [ABSTRACT FROM AUTHOR]

It was previously shown that Bone Morphogenetic Protein (BMP)-9 is constitutively produced and secreted by hepatic stellate cells (HSC). Upon acute liver damage, BMP-9 expression is transiently down-regulated and blocking BMP-9 under conditions of chronic damage ameliorated liver fibrogenesis in C57BL/6 mice. Thereby, BMP-9 acted as a pro-fibrogenic cytokine in the liver but without directly activating isolated HSC in vitro. Lipopolysaccharide (LPS), an endotoxin derived from the membrane of Gram-negative bacteria in the gut, is known to be essential in the pathogenesis of diverse kinds of liver diseases. The aim of the present project was therefore to investigate how high levels of BMP-9 in the context of LPS signalling might result in enhanced liver damage. For this purpose, we stimulated human liver sinusoidal endothelial cells (LSEC) with LPS and incubated primary human liver myofibroblasts (MF) with the conditioned medium of these cells. We found that LPS led to the secretion of factors from LSEC that upregulate BMP-9 expression in MF. At least one of these BMP-9 enhancing factors was defined to be IL-6. High BMP-9 in turn, especially in combination with LPS stimulation, induced the expression of certain capillarization markers in LSEC and enhanced the LPS-mediated induction of pro-inflammatory cytokines in primary human macrophages. In LSEC, pre-treatment with BMP-9 reduced the LPS-mediated activation of the NfkB pathway, whereas in macrophages, LPS partially inhibited the BMP-9/Smad-1 signaling cascade. In vivo, in mice, BMP-9 led to the enhanced presence of F4/80-positive cells in the liver and it modulated the LPS-mediated regulation of inflammatory mediators. In summary, our data point to BMP-9 being a complex and highly dynamic modulator of hepatic responses to LPS: Initial effects of LPS on LSEC led to the upregulation of BMP-9 in MF but sustained high levels of BMP-9 in turn promote pro-inflammatory reactions of macrophages. Thereby, the spatial and timely fine-tuned presence (or absence) of BMP-9 is needed for efficient wound-healing responses in the liver. [ABSTRACT FROM AUTHOR]