Albumin mRNA Levels Research Articles

Comparison of two hepatocyte differentiation protocols in human umbilical cord mesenchymal stem cells: In vitro study

Human umbilical cord mesenchymal stromal cells (HUCMSCs) are an emerging source of cell therapy due to their self-renew and differentiation ability. They can differentiate into three germ layers, including the potential to generate hepatocytes. This study determined the transplantation efficiency and suitability of HUCMSCs-derived hepatocyte-like cells (HLCs) for their therapeutic application for liver diseases. This study aims to formulate ideal conditions to induce HUCMSCs into the hepatic lineage and investigate the efficiency of the differentiated HLCs based on their expression characteristics and capacity to integrate into the damaged liver of CCl4-challenged mice. Hepatocyte growth factor (HGF) and Activin A, Wnt3a were found to optimally promote the endodermal expansion of HUCMSCs, which showed phenomenal expression of hepatic markers upon differentiation in the presence of oncostatin M and dexamethasone. HUCMSCs expressed MSC-related surface markers and could undergo tri-lineage differentiations. Two hepatogenic differentiation protocols (differentiated hepatocyte protocol 1 [DHC1]: 32 days and DHC2: 15 days) were experimented with. The proliferation rate was faster in DHC2 than in DHC1 on day 7 of differentiation. The migration capability was the same in both DHC1 and DHC2. Hepatic markers like CK18, CK19, ALB, and AFP were upregulated. The mRNA levels of albumin, α1AT, αFP, CK18, TDO2, CYP3A4, CYP7A1, HNF4A, CEBPA, PPARA, and PAH were even higher in the HUCMSCs-derived HCLs than in the primary hepatocytes. Western blot confirmed HNF3B and CK18 protein expression in a step-wise manner differentiated from HUCMSCs. The metabolic function of differentiated hepatocytes was evident by increasing PAS staining and urea production. Pre-treating HUCMSCs with a hepatic differentiation medium containing HGF can drive their differentiation towards endodermal and hepatic lineages, enabling efficient integration into the damaged liver. This approach represents a potential alternative protocol for cell-based therapy that could enhance the integration potential of HUCMSC-derived HLCs.

Hypothyroidism in hibernating brown bears

Brown bears hibernate throughout half of the year as a survival strategy to reduce energy consumption during prolonged periods with scarcity of food and water. Thyroid hormones are the major endocrine regulators of basal metabolic rate in humans. Therefore, we aimed to determine regulations in serum thyroid hormone levels in hibernation compared to the active state to investigate if these are involved in the adaptions for hibernation.We used electrochemiluminescence immunoassay to quantify total triiodothyronine (T3) and thyroxine (T4) levels in hibernation and active state in paired serum samples from six subadult Scandinavian brown bears. Additionally, we determined regulations in the liver mRNA levels of three major thyroid hormone-binding proteins; thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin, by analysis of previously published grizzly bear RNA sequencing data.We found that bears were hypothyroid when hibernating with T4 levels reduced to less than 44% (P = 0.008) and T3 levels reduced to less than 36% (P = 0.016) of those measured in the active state. In hibernation, mRNA levels of TBG and albumin increased to 449% (P = 0.031) and 121% (P = 0.031), respectively, of those measured in the active state. TTR mRNA levels did not change.Hibernating bears are hypothyroid and share physiologic features with hypothyroid humans, including decreased basal metabolic rate, bradycardia, hypothermia, and fatigue. We speculate that decreased thyroid hormone signaling is a key mediator of hibernation physiology in bears. Our findings shed light on the translational potential of bear hibernation physiology to humans for whom a similar hypometabolic state could be of interest in specific conditions.

Prenatal glucocorticoid administration accelerates the maturation of fetal rat hepatocytes.

Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because their livers are immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, hepatocyte nuclear factor-4 alpha (HNF4α), hepatocyte growth factor (HGF), thymus cell antigen 1 (Thy-1), cyclin B, and Cyclin-dependent kinase 1 (CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in preterm fetuses.

Chemerin reverses the malignant phenotype and induces differentiation of human hepatoma SMMC7721 cells.

Chemerin exhibits an inhibitory effect on hepatocellular carcinoma; however, the underlying mechanism is unclear. Here, low chemerin expression was confirmed in samples of liver cancer patients and hepatoma cells. Chemerin altered hepatoma cell morphology but had no effect on normal hepatocytes. Chemerin inhibited proliferation of several human hepatoma cell lines. Real-time PCR detection of hepatocellular carcinoma markers showed that mRNA levels of albumin and A-type gamma-glutamyl transferase increased whereas those of alpha-fetoprotein, alkaline phosphatase, B-type gamma-glutamyl transferase, insulin-like growth factor II, and human telomerase reverse transcriptase decreased in chemerin-treated SMMC7721 cells. Western blotting revealed that chemerin up-regulated albumin and vimentin expressions, and downregulated alpha-fetoprotein expression. Phosphorylated STAT3 was significantly up-regulated, whereas phosphorylated ERK and AKT were significantly downregulated by chemerin. Chemerin decreased phosphorylated ERK and AKT expression and the cell proliferation induced by PI3K activator 740 Y-P but could not significantly alter phosphorylated STAT3 expression and the cell growth induced by STAT3 inhibitor NSC74859. In conclusion, chemerin reversed the malignant phenotype and induced SMMC7721 cell differentiation by inhibiting MAPK/ERK and PI3K/AKT signaling; growth inhibition by chemerin is not directly related to the JAK/STAT signaling pathway. Our study provides novel evidence that chemerin could be utilized for liver cancer treatment.

Expression of genes related to lipid transport in meat-type ducks divergent for low or high residual feed intake.

ObjectiveThis study examined the effects of divergence in residual feed intake (RFI) on expression profiles of key genes related to lipid transport in the liver and duodenal epithelium and their associations with feed efficiency traits in meat-type ducks.MethodsA total of 1,000 male ducks with similar body weight (1,042.1±87.2 g) were used in this study, and their individual RFI was calculated from 21 to 42 d of age. Finally, the 10 highest RFI (HRFI) and 10 lowest RFI (LRFI) ducks were chosen for examining the expression of key genes related to lipid transport in the liver and duodenal epithelium using quantitative polymerase chain reaction.ResultsIn the liver, expression levels of albumin (ALB), CD36 molecule (CD36), fatty acid hydroxylase domain containing 2 (FAXDC2), and choline kinase alpha (CHKA) were significantly higher in LRFI ducks than in HRFI ducks (p<0.01); negative correlations (p<0.05) between expression levels of ALB, CD36, FAXDC2, and CHKA and RFI were detected in the liver. Additionally, ALB expression was strongly positively correlated (p<0.05) with CD36, FAXDC2, CHKA, and apolipoprotein H (APOH) expression in the liver. In duodenal epithelium, we found that mRNA levels of ALB, CD36, FAXDC2, and APOH were significantly higher in LRFI ducks than in HRFI ducks (p<0.01); RFI was strongly negatively correlated (p<0.05) with ALB, FAXDC2, and APOH expression, while ALB expression was strongly positively correlated with APOH expression (p<0.01) in duodenal epithelium. Furthermore, expression levels of both ALB and FAXDC2 genes were significantly associated with feed conversion ratio and RFI in both liver and duodenal epithelium (p<0.05).ConclusionOur findings therefore suggest that ALB and FAXDC2 genes might be used as potential gene markers designed to improve feed efficiency in future meat-type duck breeding programs.

Downregulation of miR-152 contributes to the progression of liver fibrosis via targeting Gli3 in vivo and in vitro.

The Gli family is known to be required for the activation of hedgehog signalling, which participates in the pathogenesis of liver fibrosis. The aim of the present study was to identify the association between microRNA (miR)-152 and GLI family zinc finger 3 (Gli3) and their roles in liver fibrosis. In a carbon tetrachloride (CCl4)-treated rat model, fibrogenesis-associated indexes, including hydroxyproline content, collagen deposition, and α-smooth muscle actin (α-SMA) and albumin expression, were examined in in vivo and in vitro models. The expression of miR-152 and Gli3 in cells and tissues was determined by reverse transcription quantitative polymerase chain reaction and western blot analysis. The interaction of Gli3 and miR-152 was evaluated by bioinformatical analysis and a dual-luciferase reporter assay. The results demonstrated that miR-152 was significantly downregulated in serum samples from clinical patients, liver tissues from CCl4-treated rats and activated LX2 cells. Furthermore, at the cellular level, the mRNA and protein expression levels of α-SMA and albumin were increased and decreased, respectively, in LX2 cells. Nevertheless, following transfection with an miR-152 mimic, the expression levels of α-SMA and albumin were reversed, and Gli3 expression was notably decreased in LX2 cells. Additionally, the target interaction between miR-152 and Gli3 was demonstrated. Finally, an miR-152 mimic was introduced into the rat model and additionally demonstrated that the changes in α-SMA, albumin and Gli3 expression levels were similar to the expression pattern in LX2 cells following miR-152 mimic transfection. These data provided insight into the potential function of miR-152 as an anti-fibrotic therapy through the modulation of Gli3.

Therapeutic effect of hepatocyte growth factor-overexpressing bone marrow-derived mesenchymal stem cells on CCl4-induced hepatocirrhosis.

Hepatocirrhosis is one of the most severe complications of chronic hepatic disease in terms of medical intervention, and the available therapies are limited and not very successful. In this study, bone marrow-derived mesenchymal stem cells (BM-MSCs) from host rats were transduced with an adenoviral vector labelled with green fluorescent protein (EGFP) to overexpress hepatocyte growth factor (HGF). The therapeutic effect of these modified stem cells (HGF-BM-MSC group) transplanted intravenously into hepatocirrhosis model rats treated with CCl4 was evaluated using serological, biochemical and histological approaches. We compared the rats in the HGF-BM-MSC group with those in the other groups (rats treated with BM-MSCs, rats treated with HGF and untreated rats (Controls)) in detail. The localisation of EGFP-tagged BM-MSCs in the injured liver was evaluated using a microscope, and the cells co-expressed hepatocyte nuclear factor 4α, albumin and cytokeratin 18. After treatment for 4 weeks, the HGF-BM-MSC, BM-MSC and HGF groups exhibited increased protein and mRNA levels of hepatocyte nuclear factor 4α, albumin and cytokeratin 18, but decreased levels of aspartate aminotransferase, alanine aminotransferase and total bilirubin. These findings indicate that BM-MSC transplantation and HGF application have great potential for the treatment of hepatocirrhosis.

The impact of early-life stress on corticosteroid carrier protein levels and 11β-hydroxysteroid dehydrogenase 1 expression in adolescent rats.

Corticosteroid-binding globulin (CBG), albumin and 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes play crucial roles in the bioavailability of glucocorticoids. Downstream of the adrenal glands, these proteins affect glucocorticoid levels in target tissues. Early-life stress (ELS) is known to program glucocorticoid action at many levels. The effects of ELS on the concentrations and synthesis of CBG and albumin and on the expression of 11β-HSD remain unclear. The maternal separation (MS) procedure in rats on postnatal days 1-14 was used as a model of ELS. On postnatal day 35 (adolescence), the serum corticosterone, CBG and albumin concentrations of male rats were measured by ELISA, while the mRNA and protein levels of CBG, albumin and 11β-HSD1 in the liver and brain were examined by RT-qPCR and Western blot, respectively. Under basal conditions, MS rats displayed lower levels of serum CBG and albumin. However, MS did not affect CBG or albumin synthesis in the liver, suggesting that the half-life and/or secretion of these proteins were influenced by MS. Additionally, MS rats showed increased protein expression of 11β-HSD1, specifically in the medial prefrontal cortex. These results indicate that ELS may potentially program glucocorticoid action through its effects on glucocorticoid bioavailability in tissues.

Enhanced hepatogenic differentiation of bone marrow derived mesenchymal stem cells on liver ECM hydrogel.

Bone marrow derived mesenchymal stem cells (BM-MSC) is a promising alternative cell source to primary hepatocytes because of their ability to differentiate into hepatocyte-like cells. However, their inability to differentiate efficiently and potential to turn into myofibroblasts restrict their applications. This study developed a plate coating from the liver extracellular matrix (ECM) and investigated its ability in facilitating the BM-MSCs proliferation, hepatic differentiation, and hepatocyte-specific functions during in vitro culture. After 28-day culture, BM-MSCs on the ECM coating showed hepatocyte-like morphology, and certain cells took up low-density lipoprotein. Synthesis of albumin, urea, and anti-alpha-fetoprotein, as well as expression of certain hepatic markers, in cells cultured on ECM were higher than cells cultured on non-coated and Matrigel-coated plates. mRNA levels of CYP3A4, albumin, CK18, and CYP7A1 in cells on ECM coating were significantly higher than cells cultured on the non-coating environment. In conclusion, viability and hepatogenic differentiation of BM-MSCs cultured on both Matrigel and ECM coating were significantly enhanced compared with those cultured on non-coated plates. Moreover, the liver ECM coating induced additional metabolic functions relative to the Matrigel coating. The liver ECM hydrogel preserves the natural composition, promotes simple gelling, induces efficient stem cell hepatogenic differentiation, and may have uses as an injectable intermedium for hepatocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 829-838, 2018.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Quantitative Assessment of Population Variability in Hepatic Drug Metabolism Using a Perfused Three-Dimensional Human Liver Microphysiological System.

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro–in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process.

A study of changes in ET-1 gene expression in peripheral blood mononuclear cells in patients with diabetic retinopathy

Objectives The objective of this study was to assess the usefulness of relative quantification of endothelin-1 (ET-1) in peripheral blood mononuclear cells in diabetic retinopathy (DR). Background DR is one of the most serious complications of diabetes mellitus (DM) that can lead to blindness. Alterations in activity of the ET system are believed to underlie the development of chronic complications of type 2 diabetes mellitus (T2DM). Patients and methods This is a clinical study carried out at Medical Biochemistry and Ophthalmology Departments. It included 50 patients divided into three groups: group I (included 14 diabetics without DR), group II (included 26 diabetics with DR), and group III (included 10 normal healthy controls). All studied patients were subjected to full history taking, clinical and ophthalmological examination, and laboratory investigations including fasting blood glucose, glycated hemoglobin (HbA1c), lipid profile, serum creatinine, urinary albumin, and creatinine and ET-1 mRNA level in peripheral blood mononuclear cells using real-time quantitative reverse transcription PCR. Results The mRNA level of ET-1 was significantly higher in diabetics with DR than those without retinopathy and was higher in diabetics than in controls. In diabetics with DR, the blood mRNA level of ET-1 was significantly higher in patients with proliferative diabetic retinopathy than in those with nonproliferative diabetic retinopathy. Increased expression of ET-1 mRNA was associated with severity of DR, duration of DM, insulin therapy, and serum creatinine level. Decreased expression of ET-1 mRNA was associated with sulfonylureas treatment. Expression of ET-1 mRNA was not affected by age, sex, BMI, smoking, and control of blood sugar. Conclusion Expression of ET-1 is associated with severity of DR in T2DM. Expression of ET-1 is positively correlated with duration of DM, serum creatinine, and urinary albumin. A larger study is needed to confirm the role of ET-1 mRNA determination in the progression of retinal changes in patients with T2DM.

Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis.

Introduction: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.Methods: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.Results: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.Conclusions: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

Influence of Rictor and Raptor Expression of mTOR Signaling on Long-Term Outcomes of Patients with Hepatocellular Carcinoma.

Aberrant signaling mediated by the mammalian target of rapamycin (mTOR) occurs at high frequency in hepatocellular carcinoma (HCC), indicating that mTOR is a candidate for targeted therapy. mTOR forms two complexes called mTORC1 (mTOR complexed with raptor) and mTORC2 (mTOR complexed with rictor). There are minor studies of the expression kinetics of mTORC1 and mTORC2 in HCC. We studied 62 patients with HCC who underwent curative resection. We used univariate and multivariate analyses to identify factors that potentially influence disease and overall survival after hepatectomy. The mRNA and protein levels of mTOR, rictor and raptor in cancer and non-cancer tissues were analyzed using quantitative RT-PCR, immunohistochemistry and Western blotting. High ratio of the levels of rictor and raptor mRNAs in tumors was identified as independent prognostic indicators for disease-free survival. Low and high levels of preoperative serum albumin and mTOR mRNA in the tumor, respectively, were identified as independent indicators of overall survival. HCC is likely to recur early after hepatic resection in patients with high levels of mTOR and rictor mRNAs and high rictor/raptor ratios in cancer tissues. We conclude that analysis of mTOR expression in cancer tissues represents an essential strategy to predict HCC recurrence after curative treatment.

Cubilin Maintains Blood Levels of HDL and Albumin

Cubilin is an endocytic receptor highly expressed in renal proximal tubules, where it mediates uptake of albumin and filtered forms of apoA-I/HDL. Cubilin deficiency leads to urinary loss of albumin and apoA-I; however, the consequences of cubilin loss on the homeostasis of blood albumin and apoA-I/HDL have not been studied. Using mice heterozygous for cubilin gene deletion (cubilin HT mice), we show that cubilin haploinsufficiency leads to reduced renal proximal tubular uptake of albumin and apoA-I and significantly increased urinary loss of albumin and apoA-I. Moreover, cubilin HT mice displayed significantly decreased blood levels of albumin, apoA-I, and HDL. The levels of albumin and apoA-I protein or mRNA expressed in the liver, kidney, or intestine of cubilin HT mice did not change significantly. The clearance rate of small HDL3 particles (density>1.13 g/ml) from the blood increased significantly in cubilin HT mice. In contrast, the rate of clearance of larger HDL2 particles from the blood did not change significantly, indicating a decreased half-life for HDL particles capable of filtering through the glomerulus. On the basis of these findings, we conclude that cubilin deficiency reduces renal salvage and delivery back to the blood of albumin and apoA-I, which decreases blood levels of albumin and apoA-I/HDL. These findings raise the possibility that therapeutic increase of renal cubilin expression might reduce proteinuria and increase blood levels of albumin and HDL.

Constitutive Notch2 signaling induces hepatic tumors in mice

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2 regulates cellular differentiation in the developing and adult liver. Although aberrant Notch signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to up-regulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation, and expression of pro-proliferative genes when compared with HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 messenger RNA (mRNA) levels and reduced Albumin and Alpha-fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium, and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of hepatocyte nuclear factor 4α(HNF4α), possibly indicating dedifferentiation of BECs. Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.

Endoplasmic reticulum stress in HepG2 cells inhibits apolipoprotein A-I secretion

AimsEndoplasmic reticulum (ER) stress modulates gene expression and has been implicated in causing dyslipidemias. To determine if ER stress may contribute to hypoalphalipoproteinemia through suppression of apo A-I gene expression, human hepatoma cell line Hep G2 was treated with ER stress inducers and the changes in apo A-I gene expression were compared to albumin gene expression. Main methodsHepG2 cells were treated with tunicamycin (TM) and thapsigargin (TG), two potent inducers of ER stress, and apo A-I and albumin protein levels, mRNA levels, and promoter activity were measured. ER stress was measured using the ER stress-responsive alkaline phosphatase assay and by Western blot quantitation of ER stress markers such as glucose-regulated protein-78 (GRP-78), phosphorylated Jun N-terminal kinase (phospho-JNK), total JNK, phosphorylated eukaryotic initiation factor 2 alpha (phospho eIF2α), and total eIF2α. Key findingsTM and TG induced ER stress in HepG2 cells and reduced apo A-I and albumin secretion in a dose-dependent manner. Intracellular albumin levels increased in cells treated with TM and TG while intracellular apo A-I levels decreased. Albumin mRNA and albumin gene promoter activity were reduced in proportion to the decrease in albumin secreted while changes in the apo A-I mRNA levels and promoter activity were modest and did not account for the reduction in apo A-I secretion. SignificanceThese results indicate that apo A-I secretion is inhibited by ER stress possibly by affecting cellular degradation pathways. However, ER stress does not affect apo A-I secretion by regulating gene expression.

Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model

When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-β1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ∼100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19+ area), but significant in AAF/PH livers (8.5±7.2% of CK19+), and inversely correlated to LMR (r2=0.63, P<0.0001). A quarter of AAF/PH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67+ LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (P<0.0001). Amongst those, rare LPC with an intermediate hepatocyte-like phenotype were seen. Also, less than 2% of hepatocytes were engaged into the cell cycle (Ki67+), while more numerous (∼25% of hepatocytes) in the livers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes.

Intrasplenic Transplantation of Isolated Adult Rat Hepatocytes

Adult male and female rat hepatocytes were individually transplanted into the spleens of adult male and female rats. The recipients were euthanized at either eight, sixteen, thirty, or forty-five weeks following transplantation, at which time hepatic and splenic levels of liver-specific rat albumin mRNA as well as sex-dependent transcript levels of CYP2C11, -2C12, -2C7, -2A1, and -3A2-which accounts for > 60% of the total concentration of hepatic constituent cytochrome P450-were determined. Whereas the pre-infused hepatocytes expressed their expected cytochrome P450 sexual dimorphisms (female-specific CYP2C12, male-specific CYP3A2, and female-predominant CYP2A1), their post-transplantational competence now reflected the sexual dimorphisms of the recipient (as observed in the host's liver), which supports the concept that the sex-dependent growth hormone circulating profiles are the determinants regulating the expression levels of hepatic cytochrome P450. Also expressed at normal concentrations in the pre-infused hepatocytes, male-specific CYP2C11 and female-predominant CYP2C7 were inexplicably undetectable in the spleens of both recipient males and females, regardless of the sex of the donor hepatocytes, almost one year after transplantation.

Enhanced Differentiation of Adult Bone Marrow-Derived Stem Cells to Liver Lineage in Aggregate Culture

Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology.