Role Of Hepatocytes Research Articles

Oval Cell Response Is Attenuated by Depletion of Liver Resident Macrophages in the 2-AAF/Partial Hepatectomy Rat

Background/AimsMacrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) in rats.Methodology/Principal FindingsWe depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy.ConclusionsThese data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1β, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.

Activation of human liver sinusoidal endothelial cell by human platelets induces hepatocyte proliferation

We previously reported that platelets promote hepatocyte proliferation. In this study, we focused on the role of platelets in liver sinusoidal endothelial cells (LSECs) in addition to their role in hepatocyte in liver regeneration. Immortalized human LSECs (TMNK-1) were used. The LSECs were co-cultured with human platelets, and the proliferation of LSECs and the excretion of growth factors and interleukin-6 (IL-6) were subsequently measured. The main factor from platelets which induced the excretion of IL-6 from LSECs was determined using inhibitors of each component contained in the platelets. The need for direct contact between platelets and LSECs was investigated using cell culture inserts. The proliferation of human primary hepatocytes was measured after the addition of the supernatant of LSECs cultured with or without platelets. The number of LSECs cocultured with platelets significantly increased. Excretion of IL-6 and vascular endothelial growth factor (VEGF) increased in LSECs with platelets. JTE-013, a specific antagonist for sphingosine 1-phosphate (S1P) 2 receptors, inhibited the excretion of IL-6 from LSECs after the addition of platelets. When the platelets and LSECs were separated by the cell culture insert, the excretion of IL-6 from LSECs was decreased. DNA synthesis was significantly increased in human primary hepatocytes cultured with the supernatant of LSECs with platelets. Platelets promote LSEC proliferation and induce IL-6 and VEGF production. Direct contact between the platelets and LSECs and S1P, that are contained in platelets, were involved in the excretion of IL-6 from LSECs. IL-6 from LSECs induced proliferation of parenchymal hepatocytes.

Hepatocyte Nuclear Factor 4 alpha (HNF4α) is involved in regulation of hepatocyte proliferation

HNF4α is the master regulator of hepatocyte differentiation and controls expression of a wide variety of hepatocyte‐specific genes. Here we report that HNF4α is involved in inhibiting hepatocyte proliferation. Decreased HNF4α expression was observed in hepatocyte specific integrin linked kinase knockout mice (ILK‐KO), which have sustained cell proliferation resulting in hepatomegaly. Following partial hepatectomy (PH), the ILK‐KO mice exhibited enhanced hepatocyte proliferation resulting in 60% higher liver mass as compared to their pre‐PH liver mass. The increased proliferation in ILK‐KO mice after PH was accompanied by further decrease in HNF4α expression. Further analysis revealed decrease in expression of HNF4α targets such as p21 and claudin1, which are involved in cell‐cycle regulation in ILK‐KO livers. Hepatocyte specific knockdown of HNF4α in HNF4α‐floxed mice using Cre recombinase carried by a adeno‐associated virus 8 (AAV‐8) vector resulted in induction of cell proliferation accompanied by decreased expression of p21 and claudin1. Over‐expression of rat HNF4α in JM1 cells resulted in decreased proliferation of these cells. Taken together, these data indicate that HNF4α plays an anti‐proliferative role in hepatocytes and may be involved in termination of liver regeneration.

S1602 The Role of Hepatocytes in Fibrosis Progression and Predisposition: Using BXD Lines and Systems Genetics to Unravel a Complex Network

S1602 The Role of Hepatocytes in Fibrosis Progression and Predisposition: Using BXD Lines and Systems Genetics to Unravel a Complex Network

C-Met Confers Protection Against Chronic Liver Tissue Damage and Fibrosis Progression After Bile Duct Ligation in Mice

The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been shown in acute liver regeneration, its cell-specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined. Hepatocyte-specific c-Met knockout mice (c-Met(Delta hepa)) using the Cre-loxP system were studied in a bile duct ligation (BDL) model. Microarray analyses were performed to define HGF/c-Met-dependent gene expression. Two strategies for c-Met deletion in hepatocytes to generate hepatocyte-specific c-Met knockout mice were tested. Early deletion during embryonic development was lethal, whereas post-natal Cre expression was successful, leading to the generation of viable c-Met(Delta hepa) mice. BDL in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-Met(Delta hepa) mice revealed a significant reduction of anti-apoptotic genes in c-Met-deleted hepatocytes. These findings could be tested functionally because c-Met(Delta hepa) mice showed a stronger apoptotic response after BDL and Jo-2 stimulation. The phenotype was associated with increased expression of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger profibrogenic response as evidenced by increased transforming growth factor-beta(1), alpha-smooth muscle actin, collagen-1alpha messenger RNA expression, and enhanced collagen-fiber staining in c-Met(Delta hepa) mice. Our results show that deletion of c-Met in hepatocytes leads to more liver cell damage and fibrosis in a chronic cholestatic liver injury model because c-Met triggers survival signals important for hepatocyte recovery.

Induction of Hepatitis by JNK-Mediated Expression of TNF-α

The c-Jun NH(2)-terminal kinase (JNK) signaling pathway has been implicated in the development of tumor necrosis factor (TNF)-dependent hepatitis. JNK may play a critical role in hepatocytes during TNF-stimulated cell death in vivo. To test this hypothesis, we examined the phenotype of mice with compound disruption of the Jnk1 and Jnk2 genes. Mice with loss of JNK1/2 expression in hepatocytes exhibited no defects in the development of hepatitis compared with control mice, whereas mice with loss of JNK1/2 in the hematopoietic compartment exhibited a profound defect in hepatitis that was associated with markedly reduced expression of TNF-alpha. These data indicate that JNK is required for TNF-alpha expression but not for TNF-alpha-stimulated death of hepatocytes. Indeed, TNF-alpha induced similar hepatic damage in both mice with hepatocyte-specific JNK1/2 deficiency and control mice. These observations confirm a role for JNK in the development of hepatitis but identify hematopoietic cells as the site of the essential function of JNK.

TGF-β induces IL-15 in hepatocytes. Is it then opposing TGFβ in a negative feedback manner? Does this mechanism play a role in liver disease?

Liver fibrogenesis is a process involving the participation of several cellular components. We described a new role for hepatocytes in the process of fibrogenesis, in which TGFbeta induces expression and secretion of CTGF, another potent profibrogenic cytokine. Recently Interleukin-15 has emerged as a new player in several models of liver damage, but its precise role has not yet been determined. Real Time PCR showed that TGFbeta induces IL-15 mRNA up to 80 fold after 24h in primary hepatocytes, and up to 10 fold in AML12 cells. Using chemical inhibitors we determined that although ALK5 activity is essential, ERK and JNK pathways cooperate in this induction. Two isoforms are expressed, differing in the length of the signal peptide. By PCR we observed induction of both splice variants of IL-15. Using siRNA we determined that Smad3 but not Smad2 is partially necessary for IL-15 mRNA induction. Software analysis of a -1,8kb region of mouse IL-15 promoter revealed binding sites for Smad3, Fast-1 and AP-1 highlighting the potential cooperation between Smad3 and AP-1 complexes. By ELISA we detected IL-15 in cell pellets but not in supernatants, indicating that IL-15 and IL-15/IL-15R complex are not secreted. Immunohistochemistry analysis of mouse and human livers revealed expression of IL-15 in hepatocytes found increased in TGFbeta transgenic mice, and in patients with liver fibrosis due to HBV, Schistosoma japonicum and gallstone bladder. Our results indicate that a new potent cytokine is induced in hepatocytes by TGFbeta. We are currently investigating the biological significance of this finding with regard to TGFbeta induced hepatocyte apoptosis, EMT and fibrotic processes.

Proapoptotic BH3-Only Protein Bid Is Essential For Death Receptor–Induced Apoptosis of Pancreatic β-Cells

Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Ceruloplasmin Is Essential for the Mobilization of Tissue Iron Stores.

In extracellular fluid, iron is in the ferric (oxidized form) but the intracellular form is ferrous iron (reduced). The outflow of iron from cells is dependent on oxidase activity that converts ferrous to ferric iron. Iron-absorbing enterocytes possess a unique iron oxidase, hephaestin. It is presumed that the circulating hephaestin paralog ceruloplasmin fulfils this role in hepatocytes and macrophages. The DiSnA mouse lacks ceruloplasmin. We hypothesized that iron homeostasis in this mouse would be unusually dependent on dietary iron because the mouse would not be able to mobilize iron from tissue stores in hepatocytes and macrophages. We fed 4-week-old DiSnA and wildtype (WT) mice a high iron (1%) diet for 4 weeks to load tissue stores. We then switched them to an iron-deficient diet and analyzed them weekly to measure iron and hemoglobin concentrations. Even on the high iron diet, DiSnA mice had lower serum iron concentrations than WT control (32.9±17.9 vs. 53.5±17.6 μM, p=0.05) but after two weeks on the iron deficient diet, the DiSnA mice had almost undetectable serum iron (4.2±1.8 μM) whereas the WT controls had only declined slightly (43.2±9.6 μM, p<0.001). Iron saturation followed a similar trend. Neither WT nor DiSnA mice were anemic at baseline (Hgb = 13.9±0.3 and 13.9±1.1 g/dL, respectively; p=0.994) but by the end of two weeks, the DiSnA mice had developed anemia whereas the WT mice had not (Hgb = 10.4±0.5 vs. 12.6±0.5 g/dL; p<0.001). The difference in hemoglobin concentrations persisted to the 6-week timepoint (Hgb = 8.4±0.5 vs. 12.6±1.4; p<0.001). After 6-weeks on a low iron diet, iron was still present in livers and spleens of both groups. Ceruloplasmin is essential for the mobilization of iron stores to protect against iron deficiency anemia in response to periods of dietary iron deficiency.

In Vitro and Ex Vivo Permissivity of Hepatocytes for Leishmania donovani

Using models of ex vivo infection of murine, rat, and human primary hepatocytes by Leishmania donovani, we showed that hepatocytes are permissive for Leishmania at a low level. We then modeled the in vitro infection of a human hepatoma-derived cell line to examine the parasite's capability to proliferate and to cause direct damage to hepatocytes. Results showed that L. donovani can infect hepatocytes, but do not massively proliferate. This slight infection under our experimental conditions resulted in limited damage to hepatocytes. These results bring into question a possible role for hepatocytes as a parasite reservoir during latent infection.

Hepatocyte transplantation

Hepatocyte transplantation

Role of hepatocytes in adaptation of muscle tissue architecture in hypertrophied muscle of rat.

Role of hepatocytes in adaptation of muscle tissue architecture in hypertrophied muscle of rat.

A DNA microarray study of nitric oxide-induced genes in mouse hepatocytes: implications for hepatic heme oxygenase-1 expression in ischemia/reperfusion

Nitric oxide (NO) can modulate numerous genes directly; however, some genes may be modulated only in the presence of the inflammatory stimuli that increase the expression of the inducible nitric oxide synthase (iNOS). One method by which to examine changes in NO-mediated gene expression is to carry out a gene array analysis on NO-naı̈ve cells. Herein, we report a gene array analysis on mRNA from iNOS-null (iNOS −/−) mouse hepatocytes harvested from mice exposed to NO by infection with an adenovirus expressing human iNOS (Ad-iNOS). Of the 6500 genes on this array, only approximately 200 were modulated either up or down by the increased iNOS activity according to our criteria for significance. Several clearly defined families of genes were modulated, including genes coding for proinflammatory transcription factors, cytokines, cytokine receptors, proteins associated with cell proliferation and cellular energetics, as well as proteins involved in apoptosis. Our results suggest that iNOS has a generally anti-inflammatory and anti-apoptotic role in hepatocytes but also acts to suppress proliferation and protein synthesis. The expression of iNOS results in increased expression of stress-related proteins, including heme oxygenase-1 (HO-1). We used HO-1 to confirm that a significant change identified by an analysis could be demonstrated as significant in cells and tissues. The elevation of HO-1 was confirmed at the protein level in hepatocytes in vitro. Furthermore, iNOS −/− mice experienced greatly increased liver injury subsequent to intestinal ischemia/reperfusion injury, associated with an inability to upregulate HO-1. This is the first study to address the global gene changes induced by iNOS in any cell type, and the findings presented herein may have clinical relevance for conditions such as septic or hemorrhagic shock in which hepatocytes, NO, and HO-1 play a crucial role.

Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver grafts.

In liver transplantation, it is currently hypothesized that nonparenchymal cell damage and/or activation is the major cause of preservation-related graft injury. Because parenchymal cells (hepatocytes) appear morphologically well preserved even after extended cold preservation, their injury after warm reperfusion is ascribed to the consequences of nonparenchymal cell damage and/or activation. However, accumulating evidence over the past decade indicated that the current hypothesis cannot fully explain preservation-related liver graft injury. We review data obtained in animal and human liver transplantation and isolated perfused animal livers, as well as isolated cell models to highlight growing evidence of the importance of hepatocyte disturbances in the pathogenesis of normal and fatty graft injury. Particular attention is given to preservation time-dependent decreases in high-energy adenine nucleotide levels in liver cells, a circumstance that (1) sensitizes hepatocytes to various stimuli and insults, (2) correlates well with graft function after liver transplantation, and (3) may also underlie the preservation time-dependent increase in endothelial cell damage. We also review damage to bile duct cells, which is increasingly being recognized as important in the long-lasting phase of reperfusion injury. The role of hydrophobic bile salts in that context is particularly assessed. Finally, a number of avenues aimed at preserving hepatocyte and bile duct cell integrity are discussed in the context of liver transplantation therapy as a complement to reducing nonparenchymal cell damage and/or activation.

The role of C/EBP(epsilon) in the terminal stages of granulocyte differentiation.

As a consequence of its characterization using both in vitro and knockout mouse models, the myeloid-specific transcription factor, CCAAT/enhancer binding protein (C/EBP)epsilon, has been identified as a critical regulator of terminal granulopoiesis and one of the causative mutations in the human disease, neutrophil-specific granule deficiency. C/EBPs are a family of transcription factors sharing numerous structural and functional features and to date include C/EBPalpha, -beta, -gamma, -delta, -epsilon, and -zeta. C/EBPalpha was the first family member isolated and characterized, its essential role in hepatocyte and adipocyte differentiation demonstrated in knockout mouse models. Subsequent analysis of the hematopoietic elements in fetal mouse liver revealed its critical role in myelopoiesis. Understanding the role of C/EBPepsilon in terminal granulopoiesis in the context of other known transcription factors is ongoing with analysis of deficient and conditionally expressing cell lines and knockout models. Mouse models with targeted gene disruptions have contributed greatly to our understanding of the transcriptional regulation of granulopoiesis. Further manipulation of these models and other conditional expression systems have bypassed some of the limitations of knockout models and helped delineate the interactions of different transcription factors in affecting granulocyte development. Phenotypic expression of the loss of C/EBPepsilon in mice is extreme, resembling absolute neutropenia with systemic infection with P. aeruginosa. Future work will need to explore the regulation of C/EBPepsilon expression, its functional interactions with other transcriptional regulators such as PU.1, and its role in monocyte differentiation and function in the mouse.

Role of hepatocyte nuclear factor-1A and B in the transcriptional regulation of human dipeptidyl peptidase ivduring differentiation of Caco-2 cells

Role of hepatocyte nuclear factor-1A and B in the transcriptional regulation of human dipeptidyl peptidase ivduring differentiation of Caco-2 cells

Liposome Elimination by Non-Phagocytic Cells of the Liver

In this contribution we summarize our observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. We demonstrate that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in accessibility is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to hypothesize at least four different mechanisms of interaction of liposomes with hepatocytes.

Intracerebroventricular Administration of Antisense Oligodeoxynucleotide against GLUT2 Glucose Transporter mRNA Reduces Food Intake, Body Weight Change and Glucoprivic Feeding Response in Rats ,

The GLUT2 glucose transporter, which may play a glucose-sensing role in hepatocyte and islet beta cells because of its low affinity and high Km for glucose, has been identified in some discrete brain areas that are related to feeding behavior and energy metabolism. We tested the hypothesis that brain GLUT2 may play a role in the control of food intake by antisense technology loss-of-function analysis. Antisense oligonucleotides directed against GLUT2 mRNA were administered intracerebroventricularly to eight rats daily for 13 days. Another eight rats were administered intracerebroventricularly with missense oligonucleotides as the control. Food intake was monitored by a computerized feeding system. Data were analyzed using a one-way general linear model or Mann-Whitney U test when appropriate. Cumulative food intake and body weight change in antisense-treated rats were significantly lower (18 and 160%, respectively) in the group treated with antisense oligonucleotides than in the group treated with missense control oligonucleotides. There was no increase in cumulative food intake in response to 2-deoxyglucose challenge in rats treated with antisense oligonucleotide, but in those treated with missense control oligonucleotide, cumulative food intake was fivefold greater in response to 2-deoxyglucose. These data suggest a possible role of brain GLUT2 in the regulation of food intake and body energy stores.

NO-mediated vasodilation in the rat liver: Role of hepatocytes and liver endothelial cells

Background/Aims: Nitric oxide (NO) is a potent vasodilator. We investigated the mechanisms responsible for this effect in the liver. Methods: Isolated perfused rat liver and cultures of endothelial sinusoidal cells and hepatocytes were used. Results: L-arginine (10 −3 M) and NO donor Sin-1 (10 −5 M) respectively increased the liver flow by 52% ( p<0.01) and 93% ( p<0.01) vs controls. The NO synthase inhibitor N w-nitro-L-arginine (10 −3 M) and the guanylate cyclase inhibitor methylene blue (10 −5 M) respectively decreased the basal liver flow by 26% and 16% ( p<0.05) and inhibited the vasodilating effects of L-arginine. L-arginine (10 −3 M) increased nitrite concentration in hepatocyte culture (77.25±7.40 μmol · 1 −1 vs 14.70±3.55 μmol · 1 −1 in controls; p<0.01) and in liver endothelial cell culture (0.36±0.09 μmol · 1 −1 vs 0.12±0.05 μmol · 1 −1 in controls; p<0.05). N w-nitro-L-arginine inhibited the basal production and abolished the L-arginine-induced production of nitrites both in hepatocyte and in liver endothelial cell cultures. The concentration of nitrites in the hepatocyte supernatant rose from 14.70±3.55 μmol −1 · 1 − to 150.50±45.55 μmol · 1 −1 in the presence of a combination of interleukin-1β, TNF a and interferon γ. Conclusions: Under basal conditions, NO regulates the vascular tone of liver circulation. Both liver endothelial cells and hepatocytes can be implicated. NO production by hepatocytes may increase during inflammation.