Oval Cell Proliferation Research Articles

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

The ability of the bone marrow cells to differentiate into liver, pancreas, and other tissues led to the speculation that these cells might be the source of adult stem cells found in these organs. The present study analyzed whether the bone marrow cells are a source of hepatic oval cells involved in rat liver regeneration induced by 2-acetylaminofluorene (2-AAF) and 70% partial hepatectomy (PHx). Three groups of mutant F344 dipeptidyl peptidase IV-deficient (DPPIV(-)) rats were required for the study. Groups A and B received the mitotic inhibitor monocrotaline, followed by male F344 (DPPIV(+)) bone marrow transplantation. Next, group A received PHx only, while group B received the 2-AAF/PHx required for the oval cell activation. The last group C was used to analyze the effects of monocrotaline on transplanted bone marrow cells. These rats underwent transplantation with bone marrow cells and were then treated with monocrotaline. Subsequently, the animals were treated with 2-AAF/PHx. In group A, DPPIV(+) hepatocytes were found in the liver. Group B showed that approximately 20% of the oval cell population expressed both donor marker (DPPIV) and alpha-fetoprotein, and some differentiated into hepatocytes. In contrast, animals in group C failed to significantly induce oval cells with the donor DPPIV antigen. In addition, X/Y-chromosome analysis revealed that fusion was not contributing to differentiation of donor-derived oval cells. Our results suggest that under certain physiologic conditions, a portion of hepatic stem cells might arise from the bone marrow and can differentiate into hepatocytes.

Opposing roles of gp130-mediated STAT-3 and ERK-1/2 signaling in liver progenitor cell migration and proliferation

Gp130-mediated IL-6 signaling may play a role in oval cell proliferation in vivo. Levels of IL-6 are elevated in livers of mice treated with a choline-deficient ethionine-supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL-6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL-6 signaling on oval cell-mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6(+ve) liver progenitor oval cells (LPCs) in CDE-treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE-treated mouse strains that depict models of gp130-mediated hyperactive ERK-1/2 signaling (gp130(deltaSTAT)), hyperactive STAT-3 signaling (gp130(Y757F) and Socs-3(-/deltaAlb)) or active ERK-1/2 as well as active STAT-3 signaling (wild-type). The A6(+ve) LPC numbers were increased with IL-6 treatment in vivo. The gp130(Y757F) mice displayed increased A6(+ve) LPCs numbers compared with wild-type and gp130(deltaSTAT) mice. Numbers of A6(+ve) LPCs were also increased in the livers of CDE treated Socs-3(-/deltaAlb) mice compared with their control counterparts. Lastly, inhibition of ERK-1/2 activation in cultured oval cells increased hyper IL-6-induced cell growth. For the first time, we have dissected the gp130-mediated signaling pathways, which influence liver progenitor oval cell proliferation. Hyperactive STAT-3 signaling results in enhanced oval cell numbers, whereas ERK-1/2 activation suppresses oval cell proliferation.

Blocking SCF/c‐Kit signal did not inhibit the proliferation of cultured liver progenitor cells

Oval cells are the putative liver stem cells that proliferate during hepatocarcinogenesis and chemically-induced severe liver injury. Antigens traditionally associated with haematopoietic cells, such as c-Kit, have been reported to be expressed by oval cells. Previous studies suggested that stem cell factor (SCF) and c-Kit were critical to oval cell development. However, the role of SCF/c-Kit signals in oval cell proliferation still remains unclear. Recently, we reported the establishment of oval cell-derived liver epithelial progenitor cells (LEPCs). In this work, we showed LEPCs co-expressed c-Kit and its ligand SCF. The involvement of SCF/c-Kit signals in LEPCs proliferation was investigated either by exposing LEPCs to c-Kit inhibitors (STI571 and AG1296), SCF, anti-SCF neutralized antibody or by using small interfering RNA to knock-down c-Kit expression. Our data demonstrate that blocking SCF/c-Kit signal did not inhibit the proliferation of LEPCs, which suggest SCF/c-Kit is not necessary for the proliferation of oval cells, at least for the cultured oval cell counterpart LEPCs.

Giant Cell Tumor in the Cuboid

Giant cell tumors are uncommon in the hand or foot. Although several case reports with giant cell tumor in these locations are described, there have only been a few reported cases of this tumor type located in the cuboid of the foot. In this report, we present a case with giant cell tumor in the cuboid. A 19-year-old Japanese man was admitted with a three-month history of aching pain in his right foot. Radiographs demonstrated an osteolytic lesion in the cuboid. Magnetic resonance (MR) images showed that the lesion had homogeneously iso-signal intensity in T1-weighted images, and relatively high signal intensity in T2-weighted images. A biopsy specimen exhibited a proliferation of mononuclear round or oval cells and multinucleated giant cells, indicating a diagnosis of giant cell tumor. Curettage with cautery using 50°C high-temperature saline for 15 minutes followed by an artificial bone graft were performed. The patient has remained asymptomatic with no signs of local recurrence or pulmonary dissemination seven years after surgery.Giant cell tumors typically occur in the epiphyseal/metaphyseal region of the long tubular bone but are uncommon in the hand or foot [1,2]. In English literature, several case reports with giant cell tumor in the hand or foot are certainly described, but there have only been a few reported cases of this tumor type located in the cuboid of the foot [2,3,4,5,6]. To our knowledge, no case of this type has been described in the Japanese literature. In this report, we present a 19-year-old Japanese man with giant cell tumor in the cuboid treated with curettage and cautery using high-temperature saline followed by an artificial bone graft. Radiological, histological differential diagnoses, and biological behavior are also discussed.

Low selection of preneoplastic hepatocytes after treatment with the 2-acetylaminofluorene diet-partial hepatectomy regimen in the liver of hepatocarcinogenesis-resistant DRH strain rats

In hepatocarcinogenesis-resistant DRH rats, preneoplastic hepatocytic lesions are smaller than those of usual rats during carcinogenesis. When preneoplastic hepatocytes from DRH and Donryu (original strain of DRH) were reciprocally transplanted into the livers of DRH and Donryu treated with 2-acetylaminofluorene (2-AAF) diet/two-thirds hepatectomy (PH), the Donryu cells formed small colonies within the DRH liver, whereas the DRH cells formed large colonies within the Donryu liver. The DRH liver showed less degree of oval cell proliferation after treatment with 2-AAF and PH, and DRH hepatocytes were more resistant to the growth-inhibitory effect of 2-AAF after PH. Furthermore, DRH hepatocytes were generally resistant to cytotoxicity of hepatotoxins. The tissue environment of the DRH liver, therefore, is less effective for selective growth of preneoplastic hepatocytes during the carcinogen treatment, which is probably a major cause of the hepatocarcinogenesis-resistance in DRH rats.

Function and significance of gap junction intercellular communication in rat liver for hepatic oval cell proliferation in vivo

Function and significance of gap junction intercellular communication in rat liver for hepatic oval cell proliferation <i>in vivo</i>

Fibronectin and laminin induce expression of islet cell markers in hepatic oval cells in culture

Hepatic oval cells (OC) are considered to be facultative liver stem cells and, because they may undergo differentiation into a variety of cell lineages, they might have the potential to be used in cellular therapy. Signals delivered by extracellular matrix (ECM) proteins take part in cellular differentiation in cooperation with signals from growth factors; indeed, some ECM proteins, such as laminin (LAM) and fibronectin (FN), have been shown to contribute to beta-cell differentiation and islet development, respectively. Since no previous studies have investigated the effect of ECM proteins on the expression of islet cell markers by cultured OC, the purpose of the present study was to evaluate whether FN and LAM modulate the expression of genes related to the endocrine pancreas in these liver cells. OC proliferation was induced in Wistar rats by prolonged treatment with 2-acetoaminofluorene/allyl alcohol and confirmed by reverse transcription/polymerase chain reaction and hepatic immunocytochemical and histopathological analyses. OC isolation was performed by Ficoll gradient and magnetic-activated cell sorting. OC were cultured for 1 and 2 months under several conditions with specific growth factors, over a FN or LAM substrate or in high glucose, nicotinamide and fetal calf serum. OC cultured on FN substrate expressed Pdx-1, Pax-6, insulin 2 and glucagon. LAM also induced the expression of Pdx-1, insulin 1 and insulin 2, glucagon, somatostatin and GLUT-2. Our results suggest that these ECM proteins can be used in protocols of OC transdifferentiation aimed at reducing the period necessary for complete transdifferentiation.

Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice

Liver regeneration following chronic injury is associated with inflammation, the proliferation of liver progenitor (oval) cells and fibrosis. Previous studies identified interferon-gamma as a key mediator of oval cell proliferation. Interferon-gamma is known to regulate Th1 cell activities during immune challenge. Therefore, we hypothesised that progenitor cell-mediated regeneration is associated with a Th1 immune response. C57Bl/6 (normal Th1 response) and BALB/c mice (deficient in Th1 signalling) were placed on a carcinogenic diet to induce liver injury, progenitor cell proliferation and fibrosis. Serum transaminases and mortality were elevated in BALB/c mice fed the diet. Proliferation of liver progenitor cells was significantly attenuated in BALB/c animals. The pattern of cytokine expression and inflammation differed between strains. Liver fibrosis and hepatic stellate cell activation were significantly inhibited in BALB/c mice compared to C57Bl/6. In addition, interferon-gamma knockout mice also showed reduced fibrosis compared to wild type. These findings are in contrast to published results, in which interferon-gamma is shown to be anti-fibrogenic. Our data demonstrate that the hepatic progenitor cell response to a CDE diet is inhibited in mice lacking Th1 immune signalling and further show that this inhibition is associated with reduced liver fibrosis.

Plasticity of Hepatic Cell Differentiation: Bipotential Adult Mouse Liver Clonal Cell Lines Competent to Differentiate In Vitro and In Vivo

In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells (cholangiocytes). The persistence of such progenitor cells in adult mouse liver is still debated. In damaged liver of adult murine animals, when hepatocyte proliferation is compromised, bipotential oval cells emerge, probably from bile ducts, proliferate, and differentiate to regenerate the liver. However, treatment to elicit oval cell proliferation is not necessary to obtain bipotential stem cells from adult mouse liver. Here, we have isolated bipotential clonal cell lines from healthy liver of 8-10-week-old C57BL/6 mice. Primary cultures established from hepatocyte-enriched suspensions were characterized by time-lapse image acquisition, immunocytology, and RNA transcript analysis. Although hepatocytes dedifferentiated with loss of apical polarity and other hepatocyte markers, they rapidly activated expression of bile duct/oval cell markers. Reversibility of these processes was achieved in part by culture under dilute Matrigel or by aging of confluent cultures. Cell lines were obtained at high frequency from mass cultures, from isolated colonies, and by primary cloning of the hepatocyte-enriched suspension. Cells of the clonal cell lines do not grow in soft agar and are nontumorigenic, and they express cytokeratin 19, A6 antigen, and alpha6 integrin, as well as a large panel of hepatocyte functions. Furthermore, they can participate in liver regeneration in albumin-urokinase-type plasminogen activator/severe combined immune-deficient mice, where they differentiate in clusters of hepatocytes and occasionally bile ducts. These results demonstrate the existence, in normal adult mouse liver, of a significant pool of clonogenic cells that are (or can become) bipotential.

Expression of AFP and Rev-Erb A/Rev-Erb B and N-CoR in fetal rat liver, liver injury and liver regeneration

BackgroundAlpha-fetoprotein (AFP) expression can resume in the adult liver under pathophysiological conditions. Orphan nuclear receptors were supposed to regulate AFP gene expression, in vitro. We were interested to study the expression of AFP and orphan nuclear receptors, in vivo.ResultsThe expression of AFP gene and orphan nuclear receptors in the liver was examined in different rat models: (a) fetal liver (b) liver regeneration [partial hepatectomy (PH) with and without 2-acetyl-aminofluren treatment (2-AAF)], (c) acute liver damage [treatment with CCl4] and (d) acute phase reaction [treatment with turpentine oil]. After PH of 2-AAF treated rats, clusters of AFP positive cells occurred in the periportal region. In the Northern blot analysis, a positive hybridization signal for the full-length AFP-RNA was observed only in liver samples from 2-AAF treated rats after PH. In real-time PCR analysis, the full-length AFP-RNA was highly up regulated in the fetal liver (maximum at day 14: 21,500 fold); after PH of 2-AAF treated rats, the full-length AFP-RNA was also up regulated up to 400 fold (day 7 after PH). The orphan nuclear receptors were down regulated at nearly each time points in all models, also at time point of up regulation of the AFP gene.ConclusionExpression of "fetal" AFP could be demonstrated during liver development and during proliferation of the so-called oval cells. Changes of expression of orphan nuclear receptors, however, did not correlate with AFP expression. Other regulatory pathways were possibly involved in controlling AFP expression, in vivo.

Lack of evidence that bone marrow cells contribute to cholangiocyte repopulation during experimental cholestatic ductal hyperplasia

Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear. Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated. Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7. In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.

A 26-Week Carcinogenicity Study of 2-Amino-3-Methylimidazo[4,5-f]Quinoline in rasH2 Mice

To evaluate the carcinogenic susceptibility of rasH2 mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 7-week-old rasH2 mice and their wild-type littermates (non-Tg mice) of both the sexes were fed a diet containing 0 or 300 ppm IQ for 26 weeks. Microscopical examinations revealed that the proliferative lesions of the forestomach, including squamous cell hyperplasias, papillomas, and carcinomas, were frequently encountered in male and female rasH2 mice fed with IQ. In non-Tg mice, no significant differences in the incidence of forestomach lesions were observed between the 0 ppm and 300 ppm groups. Histopathological changes such as periportal hepatocellular hypertrophy and oval cell proliferation in the liver were more apparent in female rasH2 and non-Tg mice than in males, and the incidence of hepatocellular altered foci significantly increased in female rasH2 mice in the 300 ppm group as compared to that in the 0 ppm group. These results suggest that the carcinogenic potential of IQ can be detected in rasH2 mice by a 26-week, short-term carcinogenicity test.

The role of p28GANK in rat oval cells activation and proliferation

Human gankyrin gene product (p28GANK) is a novel oncogenic protein ubiquitously overexpressed in hepatocellular carcinoma and also plays a role in cell cycle progression in normal hepatocytes and liver regeneration. However, little is known about the physiological role of p28GANK in the liver oval cell activation and proliferation. We investigated the possible involvement of p28GANK in oval cell-mediated liver regeneration and cell cycle progression. We examined the different p28GANK expression in 2-acetylaminofuorene/partial heptectomy (2-AAF/PH) rats, as a model of oval cell activation, and PH rats by Western blot and immunohistochemistry. Oval cells isolated from 2-AAF/PH rat model were cultured in our study. p28GANK expression was examined in the oval cells after mitogenic stimulation. In 2-AAF/PH rats, p28GANK was expressed in the activated oval cells and located in the nucleus. p28GANK protein expression was increased in 2-AFF/PH rats after hepatectomy lasting for 96 h when retinoblastoma maintained hyperphosphorylation status at Ser-795. The isolated oval cells express AFP, OV6, CK19, CD34, CD45, c-kit and albumin. After epidermal growth factor stimulation, p28GANK protein was up-regulated in oval cells from 24 to 72 h, which coincided with increased expression of CyclinD1, CDK4 and decreased of Rb protein. p28GANK expression was increased in oval cell-mediated liver regeneration and oval cells after mitogenic stimulation. Thus, p28GANK may play a role in oval cell-mediated liver regeneration and liver oval cell cycle progression.

Chronic liver disease is triggered by taurine transporter knockout in the mouse

Taurine is an abundant organic osmolyte with antioxidant and immunomodulatory properties. Its role in the pathogenesis of chronic liver disease is unknown. The liver phenotype was studied in taurine transporter knockout (taut-/-) mice. Hepatic taurine levels were ~21, 15 and 6 mumol/g liver wet weight in adult wild-type, heterozygous (taut+/-) and homozygous (taut-/-) mice, respectively. Immunoelectronmicroscopy revealed an almost complete depletion of taurine in Kupffer and sinusoidal endothelial cells, but not in parenchymal cells of (taut-/-) mice. Compared with wild-type mice, (taut-/-) and (taut+/-) mice developed moderate unspecific hepatitis and liver fibrosis with increased frequency of neoplastic lesions beyond 1 year of age. Liver disease in (taut-/-) mice was characterized by hepatocyte apoptosis, activation of the CD95 system, elevated plasma TNF-alpha levels, hepatic stellate cell and oval cell proliferation, and severe mitochondrial abnormalities in liver parenchymal cells. Mitochondrial dysfunction was suggested by a significantly lower respiratory control ratio in isolated mitochondria from (taut-/-) mice. Taut knockout had no effect on taurine-conjugated bile acids in bile; however, the relative amount of cholate-conjugates acid was decreased at the expense of 7-keto-cholate-conjugates. In conclusion, taurine deficiency due to defective taurine transport triggers chronic liver disease, which may involve mitochondrial dysfunction.

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation

Oval cells are hepatocytic precursors that proliferate in late-stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor-beta (TGF-beta) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF-beta levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF-beta signaling in vivo in oval cells, we analyzed livers of rats fed a choline-deficient, ethionine-supplemented (CDE) diet for phospho-Smad2. Phospho-Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF-beta signaling and proliferation in oval cells and hepatocytes, we examined TGF-beta signaling in vitro. TGF-beta caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF-beta-induced growth inhibition may result from the absence of Smad6 in these cells. Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF-beta-induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation.

Hematopoietic mobilization in mice increases the presence of bone marrow–derived hepatocytes via in vivo cell fusion†

The mechanisms for in vivo production of bone marrow-derived hepatocytes (BMDHs) remain largely unclear. We investigated whether granulocyte colony-stimulating factor (G-CSF)-mediated mobilization of hematopoietic cells increases the phenomenon. Recurrent liver injury in mice expressing green fluorescent protein (EGFP) in all hematopoietic-derived cells was produced by 3 months of carbon tetrachloride (CCL4) injections. Histologically, there were necrotic foci with histiocyte-rich infiltrates, but little oval cell proliferation. Subsequently, some animals were mobilized with G-CSF for 1, 2, or 3 weeks. Animals were sacrificed 1 month after growth factor treatment. BMDH percentages were lower than previously reported, though G-CSF mobilization significantly augmented BMDH production in injured livers. BMDHs originating from in vivo fusion were evaluated by transplanting female EGFP+ cells into male mice. Binucleated, EGFP+ hepatocytes with one Y chromosome, indicating fusion, were identified. In conclusion, (1) mobilization of hematopoietic cells increases BMDH production and (2) as with the FAH-null model, the first model demonstrating hematopoietic/hepatocyte fusion, recurring CCl4-induced injury has macrophage-rich infiltrates, a blunted oval cell response, and a predominantly in vivo fusion process for circulating cell engraftment into the liver. These findings open the possibility of using hematopoietic growth factors to treat nonhematopoietic degenerative diseases.

Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxygenase 2 inhibitor

Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M(2)-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 microM Prostaglandin E(2) partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.

Hepatocyte growth factor accelerates the proliferation of hepatic oval cells and possibly promotes the differentiation in a 2‐acetylaminofluorene/partial hepatectomy model in rats

Hepatocyte growth factor (HGF) is the primary agent promoting the proliferation of mature hepatocytes. The purpose of the present paper was to clarify the effects of HGF on the proliferation and differentiation of hepatic oval cells using a 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model in rats. Recombinant human HGF (0.2 mg/day) was administered to 2-AAF/PH rats for 7 days using osmotic pumps intraperitoneally implanted in conjunction with hepatectomy (day zero). Periportal basophilic areas consisting of oval cells were significantly enlarged by treatment with HGF on day 8. In control animals, expression of alpha-fetoprotein (AFP) in the liver was gradually upregulated, leading a marked increase on day 12. In HGF-treated rats, AFP expression was stimulated at an earlier date and decreased to an undetectable level on day 12. Conversely, expression of albumin transcripts, which was stimulated by HGF-treatment at a later date, continued to increase even after HGF administration ceased, leading to an extremely high level on day 12. Moreover, treatment with HGF also stimulated the expression of hepatocyte nuclear factor-1alpha and -4alpha at an early date. These results indicate that, besides the proliferation of hepatic oval cells, HGF possibly promotes the differentiation to hepatocytes in vivo, suggesting that recombinant human HGF accelerates the regeneration of severely damaged livers, a situation in which the proliferation of mature hepatocytes is impaired.

TWEAK induces liver progenitor cell proliferation

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.

Dual effects of adenovirus-mediated thrombopoietin gene transfer on hepatic oval cell proliferation and platelet counts

Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt–Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days ( P < 0.05) and reached its plateau at 9 days ( P < 0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred ( P < 0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred ( P < 0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells.