Oval Cell Proliferation Research Articles

Immunohistochemical Characterization of the Hepatic Progenitor Cell Compartment in Medaka (Oryzias latipes) following Hepatic Injury

Laboratory fish species are used increasingly in biomedical research and are considered robust models for the study of regenerative processes. Studies investigating the response of the fish liver to injury have demonstrated the presence of a ductular reaction and oval-like cells in injured and regenerating liver. To date, however, it is unclear if this cell population is the piscine equivalent of oval cells (OCs) or intermediate hepatobiliary cells (IHBCs) identified in rodents and man, respectively. The present study defines the process of OC differentiation in fish liver using histopathology, immunohistochemistry and transmission electron microscopy. To generate OC proliferation in Japanese medaka (Oryzias latipes), hepatic injury was induced by exposure of adult fish to either microcystin LR or dimethylnitrosamine. A transgenic strain of medaka expressing a red fluorescent protein (RFP) exclusively in hepatocytes was used. The morphological response to injury was characterized by a ductular reaction comprised of cytokeratin (CK) AE1/AE3(+) OCs progressing to IHBCs variably positive for CK and RFP and finally mature RFP(+) hepatocytes and CK(+) cholangiocytes. These observations support a bipotential differentiation pathway of fish OCs towards hepatocytes and cholangiocytes. Ultrastructural morphology confirmed the presence of OCs and differentiation towards hepatocytes. These results demonstrated clear similarities between patterns of reaction to injury in fish and mammalian livers. They also confirm the presence of, and support the putative bipotential lineage capabilities of, the fish OC.

Congenital melanocytic nevus with features of hybrid schwannoma/perineurioma

Neural differentiation by melanocytic nevi represents a well-recognized phenomenon, and melanocytic nevi with perineurial differentiation have been reported recently. We reported a case of a congenital melanocytic nevus with histopathologic features of hybrid schwannoma/perineurioma. The patient was a 36-year-old male who presented with a black tumor on his arm since birth. Histopathology showed a congenital melanocytic nevus in the superficial dermis, but more strikingly, in continuity with the melanocytic nevus, there was a well-circumscribed but unencapsulated nodule in the deep dermis. The nodule was composed of cellular and myxoid areas with storiform, laminated or whorled growth patterns. The cellular area was mainly composed of proliferation of plump spindle, oval or epithelioid cells. The myxoid area was mainly composed of proliferation of slender spindle cells with mucin deposition. Immunohistochemical stains showed that the cellular area was positive for S100 and CD34, weakly positive for EMA, negative for Glut-1 and collagen IV, the myxoid area was positive for S100, negative for CD34, strongly positive for EMA and focally positive for Glut-1 and collagen IV. Our results show that congenital melanocytic nevi may show neural differentiation with histopathologic features of hybrid schwannoma/perineurioma.

In vitro and in vivo characteristics of hepatic oval cells modified with human hepatocyte growth factor

Hepatocyte growth factor (HGF) is a multifunctional growth factor that controls cell scattering. It has been suggested that it regulates the proliferation of hepatic oval cells (HOCs). Using a HOC line that stably expresses the human HGF gene (hHGF), we investigated the in vitro proliferation and differentiation characteristics of hHGF-modified HOCs and explored their potential capacity for intrahepatic transplantation. A modified 2-acetylaminofluorene and partial hepatectomy (2-AAF/PH) model was established to activate the proliferation of oval cells in the rat liver. HOCs were transfected with the pBLAST2-hHGF plasmid and hHGF-carrying HOCs were selected based on blasticidin resistance. The level of hHGF secretion was determined via ELISA. Cell proliferation was determined using the MTT assay. Differentiation was induced by growth factor withdrawal. A two-cuff technique was used for orthotopic liver transplantation, and HOCs or hHGF-modified HOCs were transplanted into the recipients. The levels of biochemical indicators of liver function were measured after transplantation. An HOC line stably expressing hHGF was established. The transfected line showed greater hHGF secretion than normal HOCs. The hHGF gene promoted the proliferation capability of HOCs by reducing the peak time in vitro. The hHGF-modified HOCs differentiated into hepatocytes and bile duct epithelial cells upon growth factor withdrawal in vitro. In addition, hHGF-modified HOC transplantation significantly prolonged the median survival time (MST) and improved the liver function of recipients compared to HOC transplant recipients and nontransplanted controls. Our results indicate that hHGF-modified HOCs may have valuable properties for therapeutic liver regeneration after orthotopic liver transplantation.

Effects of glycogen synthase kinase 3β overexpression in rat and glycogen synthase kinase 3β inhibitor SB-216763 on proliferation of hepatic oval cells

To research the effects of glycogen synthase kinase (GSK3β) overexpression and GSK3β inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway. The hepatic oval cells WBF-344 were divided into the blank control group, GSK3β over-expression group, DMSO control group and GSK3β inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3β over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3β, β-catenin and cyclin D1 were detected by Western blot. The cells of GSK3β over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3β over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3β over-expression group. Western blot showed that the expression of GSK3β was increased, while the expression of β-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3β had no significant difference among the control group and inhibitor groups. However, the expression of β-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing. The overexpression of GSK3β can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.

Structural analysis of oval-cell–mediated liver regeneration in rats

We have analyzed the architectural aspects of progenitor-cell-driven regenerative growth in rat liver by applying the 2-acetaminofluorene/partial hepatectomy experimental model. The regeneration is initiated by the proliferation of so-called oval cells. The oval cells at the proximal tips of the ductules have a more differentiated phenotype and higher proliferative rate. This preferential growth results in the formation of a seemingly random collection of small hepatocytes, called foci. These foci have no clonal origin, but possess a highly organized structure, which shows similarities to normal hepatic parenchyma. Therefore, they can easily remodel into the lobular structure. Eventually, the regenerated liver is constructed by enlarged hepatic lobules; no new lobules are formed during this process. The foci of the Solt-Farber experimental hepatocarcinogenesis model have identical morphological features; accordingly, they also represent only regenerative, not neoplastic, growth. Progenitor-cell-driven liver regeneration is a well-designed, highly organized tissue reaction, and better comprehension of the architectural events may help us to recognize this process and understand its role in physiological and pathological reactions.

HSCs play a distinct role in different phases of oval cell‐mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell-mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2-acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen-activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9 days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal-regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15 days group) contained high levels of transforming growth factor (TGF)-β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF-β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell-mediated liver regeneration.

The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.

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.

1234 PRO-APOPTOTIC MIR-34A/SIRT1/P53 PATHWAY IN NAFLD PATIENTS AND TARGETING BY URSODEOXYCHOLIC ACID IN RAT LIVER

histology was graded and staged according to the Kleiner system. Hepatic triglycerides were quantified, and qPCR/Western blotting used to assess inflammation, fibrosis and lipid metabolism. Hepatic (oval) cell response was assessed by pan-cytokeratin (panCK) and Sox9 immunohistochemistry with morphometric analysis. Results: ALIOS fed mice developed lobular inflammation/ballooning by 6m and F3 fibrosis by 12m. By 6m, 5aR1 mice fed ALIOS diet demonstrated greater steatosis histologically (median Kleiner steatosis score; 3 vs 1), and biochemically (mean hepatic triglyceride; 93.1 vs 62.4 nmol/mg, p < 0.01) than WT mice fed ALIOS diet. There was no difference in inflammation or fibrosis. 5aR1 mice had reduced carnitine palmitoyl transferase-1a mRNA expression (p < 0.05), and increased acetyl-CoA carboxylase protein expression, consistent with decreased fatty acid b-oxidation and increased de novo lipogenesis. Focal hepatocellular lesions consistent with dysplasia/hepatocellular carcinoma developed after 12m ALIOS diet in 5/10 WT mice, with none developing in 5aR1 mice. Dysplastic/hepatocellular carcinoma lesions expressed the oval cell marker Sox9. Hepatic oval cell proliferation increased after ALIOS diet, but was attenuated in 5aR1 compared to WT mice (mean surface area panCK positive; 0.62% vs. 0.94%; Sox9 positive cells per view; 60 vs. 76/field of view respectively). Conclusions: 5aR1 deficiency promotes hepatic steatosis, but protects against the development of NASH-related dysplasia/hepatocellular carcinoma and reduces hepatic oval cell proliferation. Prolonged ALIOS diet is a unique murine model for studying NASH-related oval cell proliferation and hepatocarcinogenesis in the absence of cirrhosis. These data support a role of 5aR1 in hepatocarcinogenesis associated with NASH.

Genetic Abolishment of Hepatocyte Proliferation Activates Hepatic Stem Cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

Comparative effects of gamma and microwave irradiation on anticarcinogenic effect of red chili in colon and liver of male albino rats

The comparative effect of raw red chili and red chili treated by gamma irradiation or microwave pasteuriza- tion with or without 5-fluorouracil on experimentally induced colon cancer in rats was investigated. Dimethylhydrazine was used to induce colon cancer which is histologically similar to and comparable with human neoplasm. Histopathological examination of dimethylhydrazine-treated rats demonstrated epithelial and goblet cell hyperplasia as well as preneoplastic changes of the rat colon expressed by loss of polarity of the nuclei and abnormal mitosis. The preneoplastic changes were developed to adenocarcinoma in 80% of cases. The liver showed different pathological pictures that included dysplas- tic changes, preneoplastic foci, and oval cell proliferation. We observed that red chili with or without 5-fluorouracil inhibited colon adenocarcinoma. The gamma red chili either alone or with5-fluorouracilwasthe bestgroups.The gamma irradiated red chili alone decreased epithelial and goblet cell hyperplasia and preneoplastic changes in colon and dysplastic changes, oval cell proliferation, and Kupffer cell hyperplasia in the liver. The gamma irradiated red chili with 5-fluorouracil inhibited the preneoplastic changes in colon. It was concluded that the sanitization of red chili by gamma irradiation either alone or with 5-fluorouracil was more beneficial from the human health point of view than that by microwave.

Histopathological Effects of 2,4-Dichlorophenoxyacetic Acid on Sprague-Dawley Rats with Special Reference to its Possible Carcinogenicity

This study investigated the toxic effects of 2,4-dichlorophenoxyacetic acid on male Sprague-Dawley rats. In which, 30 rats were divided into three even groups. First group received orally normal saline (3 ml/kg b.w.) and kept as normal control in parallel to treated groups (5 rats each), second group received orally 2,4-dichlorophenoxyacetic acid (30 mg/kg b.w.) for 2 months and third group received orally 2,4-dichlorophenoxyacetic acid (30 mg/kg b.w.) for 6 months. At the end of each treatment, specimens from the liver, kidneys, lungs, testicles, epididymis and brain were obtained and subjected for histopathological examination. After 2 months of treatment with 2,4-dichlorophenoxyacetic acid, the overall changes were milder and ranged from vascular to necrobiotic findings. On the other hand, changes seen after 6 months were severe particularly in the liver and kidney. In the liver, there were focal areas of necrosis and preneoplastic changes in the form eosinophilic preneoplastic foci, oval cell proliferation and spongiotic pericytoma. Kidneys showed glomerular swelling, thickening of the glomerular basement membrane and proliferation of the renal tubular epithelium. Lesions in the other organs included necrosis and sloughing of bronchiolar epithelial lining and mononuclear cellular infiltration in the lungs, necrosis of seminiferous tubular epithelium in the testicles and neuronal degeneration in the brain. In conclusion, 2,4-dichlorophenoxyacetic acid induced deleterious pathological effects on the vital organs including preneoplastic changes in the liver in Sprague-Dawley rats.

Nuclear receptor CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation in mouse liver

The liver is endowed with the ability to regenerate hepatocytes in response to injury. When this regeneration ability is impaired during liver injury, oval cells, which are considered to be postnatal hepatic progenitors, proliferate and differentiate into hepatocytes. Here we have demonstrated that 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) activates the nuclear receptor constitutive active/androstane receptor (CAR), resulting in proliferation of oval cells in mouse liver. Activation of CAR by DDC was shown by hepatic nuclear CAR accumulation and cytochrome P450 (CYP)2B10 mRNA induction after feeding a 0.1% DDC-containing diet to Car+/+ mice. After being fed the DDC diet, Car+/+, but not Car−/− mice, developed severe liver injury and an A6 antibody-stained ductular reaction in an area around the portal tract. Oval cell proliferation was confirmed by laser capture microdissection and real-time PCR; mRNAs for the two oval cell markers epithelial cell adhesion molecule and TROP2 were specifically induced in the periportal region of DDC diet-fed Car+/+, but not Car−/− mice. Although rates of both hepatocyte growth and death were initially enhanced only in DDC diet-fed Car+/+ mice, growth was attenuated when oval cells proliferated, whereas death continued unabated. DDC-induced liver injury, which differs from other CAR activators such as phenobarbital, occurred in the periportal region where cells developed hypertrophy, accumulated porphyrin crystals and inflammation developed, all in association with the proliferation of oval cells. Thus, CAR provides an excellent experimental model for further investigations into its roles in liver regeneration, as well as the development of diseases such as hepatocellular carcinoma.

The role of canonical Wnt signaling pathway in the proliferation and differentiation of rat hepatic oval cell

Objective To investigate the role of canonical Wnt signaling pathway on the proliferation and differentiation of hepatic oval cells in vitro.Methods WB­F344 cells were treated with recombinant Wnt3a(20,40,80,160,200 ng/ml) in the serum­free medium for 24 hours.Cell proliferation was measured by Brdu incorporation analysis，un­treated WB­F344 cells were taken as control.After treated with Wnt3a(160 ng/ml) for 24 hours，the subcellular localization and protein expression of β­catenin in WB­F344 cells were examined by immunofluorescence staining and Western­blot analysis.CyclinD1 mRNA expression was determined by semi­quantitative RT­PCR. The mRNA levels of some phenotypic markers，including AFP，CK­19，ALB and two hepatic nuclear factors(HNF­4α，HNF­6) were determined by RT­PCR.CK­19 and AFP protein expression were examined by Western­blot analysis.Results We found that Wnt3a promoted proliferation of WB­F344 cells.Stimulating of WB­F344 cells with recombinant Wnt3a resulted in the accumulation of the transcriptional activator β­catenin，together with its translocation into the nucleus，and the upregulation of typical Wnt target genes cyclin D1.After 3 days of Wnt3a treatment in the absence of any serum，these cells retained their bipotential to express several specific hepatocyte and cholangiocyte phenotypic markers，such as AFP，CK­19 following activation of canonical Wnt signaling pathway.Conclusions The canonical Wnt signaling pathway promotes proliferation and self­renewal of rat hepatic oval cells.

EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells

Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Metflx/flx and Met−/− oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Metflx/flx cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Metflx/flx and Met−/− oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in normal, untransformed oval cells, c-Met and EGFR represent critical molecular players to control proliferation and survival that function independent of one another.

RETRACTED: Effect of sublethal dose of mercury toxicity on liver cells and tissue of yellowfin seabream

The aim of the present study was to provide baseline data on the prevalence of histopathological liver lesions in Acanthopagrus latus under experimental mercury exposure. Experimental study was at seawater recirculatory tanks. Mercury concentrations were determined using a standard cold vapour atomic absorption. Histopathological analyses were done in tissue processor and the slides were stained with haematoxylin and counterstained with eosin. There were many liver lesions including enlarged and lateral nuclei, nuclear degeneration and vacuolation; oncotic, apoptic, focal, massive, centrilobular and periportal necrosis; atrophy, lipidosis, hydropic and cloudy swelling, oval cell proliferation; bile stagnation, dilation of sinusoid, intracellular oedema and dark granules in both field and laboratory conditions. In conclusion the present investigation indicated that mercury is a toxic substance in yellowfin seabream and the sublethal concentrations of mercury may cause several changes in the histological indices of the studied fish, and we can use these changes as biomarkers of mercury detection.

Inhibition of Notch signaling affects hepatic oval cell response in rat model of 2AAF-PH

Background and aimsActivation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade.MethodsThe established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver.ResultsNotch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21Waf1/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α.ConclusionTaken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional repair of the liver by oval cells.

Proliferation of Hepatic Oval Cells via Cyclooxygenase-2 and Extracellular Matrix Protein Signaling during Liver Regeneration Following 2-AAF/Partial Hepatectomy in Rats

Background/AimsIn the 2-acetylaminofluorene (2-AAF)/70% partial hepatectomy (PHx) model, the mechanism underlying the differentiation of activated hepatic oval cells (HOCs) into hepatocytes and bile ductile cells is unclear. We investigated the role of cyclooxygenase-2 (COX-2) in HOCs and the relationship between COX-2 and extracellular matrix proteins in cellular proliferation.MethodsReverse transcription-polymerase chain reaction, immunohistochemical staining, and Western blotting were used to assess COX-2 expression. The co-localization of COX-2 with Thy1, c-Met, epithelial cell adhesion molecule, and α-smooth muscle actin was also examined. Additionally, we investigated whether connective tissue growth factor (CTGF), fibronectin (FN), extracellular signal-regulated kinase 1/2 (P-ERK1/2), and AKT were expressed in HOCs.ResultsThe expression of COX-2, prostaglandin E2 receptors, and c-Met was upregulated in HOCs. However, HOCs treated with the COX-2 inhibitor NS398 showed decreased COX-2, CTGF, FN, and AKT expression, whereas P-ERK1/2 was unaffected. Additionally, NS398 inhibited HOC proliferation, but not the proliferation of HOCs cultured on FN-coated dishes. Furthermore, the proliferative response of HOCs treated with NS398 was reversed by hepatic growth factor treatment.ConclusionsThese results suggest that HOC proliferation is mediated through COX-2, extracellular FN expression, and AKT activation. Thus, COX-2 plays an important role in HOC proliferation following acute injury.

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.

Genome-wide expression profiling of hepatic oval cells after partial hepatectomy in rats

To examine the changes of biological activities in hepatic oval cells (HOCs) elicited by 70% partial hepatectomy (PH) and understand the relationship between this cell and liver regeneration (LR), this study isolated and obtained the high purity HOCs (≥95%) from rat regenerating livers, and then monitored gene expression profiling of rat hepatic oval cells following surgical operation. Results showed that there were LR-related 1059 genes. These genes were grossly classified into three groups using a fold change cut-off threshold of three-fold: up-regulation, down-regulation and up/down regulation. Analyses of gene expression patterns combined with gene functional categorization suggested that genes in the categories “nucleic acid metabolism” and “cell cycle” were dominated by up-regulated expression. Genes in the functional groups “cell metabolism” and “oxidation reduction” were significantly enriched in expression pattern characterized by down-regulation. According to above mentioned results, the synchronized induction of DNA replication and cell proliferation-involved genes suggested that the peak of oval cell proliferation might occur between 30 and 36 h post-PH. The amino acid transformation-involved genes were down-regulated at the early phase of LR, which perhaps trigger the storage of those amino acids essential for protein synthesis. Reduced oxidative-reduction activity at early phase might be related to negative influence of surgical operation on its detoxification capacity. Conclusively, the genome-wide transcriptional analysis of oval cells would contribute to our understanding of the nature of LR at cell level.