Oval Cell Proliferation Research Articles

A Protocol for the Isolation of Oval Cells without Preconditioning.

Oval cells (OCs) is the name of hepatic progenitor cells (HPCs) in rodents. They are a small population of cells in the liver with the remarkable ability to proliferate and regenerate hepatocytes and cholangiocytes in response to acute liver damage. Isolating OCs generally requires a pretreatment with special diets, chemicals, and/or surgery to induce hepatic damage and OC proliferation in mice. Unfortunately, these pretreatments are not only painful for the mice but also increase the cost of the assays, and the effects on the different organs as well as on various liver cells are still unclear. Therefore, the search for a protocol to obtain OCs without prior liver damage is mandatory. In our study, we present a protocol to isolate murine OCs from healthy liver (HL-OCs) and compare them with OCs isolated from mice pretreated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC-OCs). Our results demonstrated that cells derived from untreated mice exhibited similar behavior to those from treated mice in terms of surface marker expression, proliferation, and differentiation capacity. Therefore, given the impracticability of isolating human cells with prior hepatotoxic treatment, our model holds promise for enabling the isolation of progenitor cells from human tissue in the future. This advancement could prove invaluable for translational medicine in the understanding and treatment of liver diseases.

Toxicity Profile of eBAT, a Bispecific Ligand-Targeted Toxin Directed to EGFR and uPAR, in Mice and a Clinical Dog Model.

EGFR-targeted therapies are efficacious, but toxicity is common and can be severe. Urokinase type plasminogen activator receptor (uPAR)-targeted drugs are only emerging, so neither their efficacy nor toxicity is fully established. Recombinant eBAT was created by combining cytokines EGF and uPA on the same single-chain molecule with truncated Pseudomonas toxin. Its purpose was to simultaneously target tumors and their vasculature in the tumor microenvironment. In prior studies on mice and dogs, the drug proved efficacious. Here, we report the safety of eBAT in normal wildtype, uPAR knockout, and immunoreplete and immunodeficient tumor-bearing mice, as well as in dogs with spontaneous sarcoma that more closely mirror human cancer onset. In immunocompetent mice, tumor-bearing mice, uPAR knockout mice, and mice receiving species-optimized eBAT, toxicities were mild and self-limiting. Likewise, in dogs with life-threatening sarcoma given dosages found to be biologically active, eBAT was well tolerated. In mice receiving higher doses, eBAT was associated with dose-dependent evidence of liver injury, including portal biliary hyperplasia, oval cell proliferation, lymphoplasmacytic inflammation, periportal hepatocellular microvesicular change, hemorrhage, necrosis, and apoptosis. The results support continuing the clinical development of eBAT as a therapeutic agent for individuals with sarcoma and other cancers.

MFGE-8 identified in fetal mesenchymal-stromal-cell-derived exosomes ameliorates acute hepatic failure pathology

Liver transplantation is the gold-standard therapy for acute hepatic failure (AHF) with limitations related to organ shortage and life-long immunosuppressive therapy. Cell therapy emerges as a promising alternative to transplantation. We have previously shown that IL-10 and Annexin-A1 released by amniotic fluid human mesenchymal stromal cells (AF-MSCs) and their hepatocyte progenitor-like (HPL) or hepatocyte-like (HPL) cells induce liver repair and downregulate systemic inflammation in a CCl4-AHF mouse model. Herein, we demonstrate that exosomes (EXO) derived from these cells improve liver phenotype in CCl4-induced mice and promote oval cell proliferation. LC-MS/MS proteomic analysis identified MEFG-8 in EXO cargo that facilitates rescue of AHF by suppressing PI3K signaling. Administration of recombinant MFGE-8 protein also reduced liver damage in CCl4-induced mice. Clinically, MEFG-8 expression was decreased in liver biopsies from AHF patients. Collectively, our study provides proof-of-concept for an innovative, cell-free, less immunogenic, and non-toxic alternative strategy for AHF.

GLI1 -Rearranged Enteric Tumor : Expanding the Spectrum of Gastrointestinal Neoplasms With GLI1 Gene Fusions.

GLI1 encodes a transcription factor that targets cell cycle regulators affecting stem cell proliferation. GLI1 gene fusions were initially described in pericytomas with a t[7;12] translocation and more recently in gastric plexiform fibromyxomas and gastroblastomas. This study describes the clinicopathologic, immunohistochemical, and molecular features of three intestinal-based neoplasms harboring GLI1 gene fusions. We studied three unique mesenchymal small bowel tumors. Paraffin embedded tumor tissues from these cases and 62 additional tumor samples that included a plexiform fibromyxoma were sequenced using a targeted RNAseq method to detect fusion events. The study patients included two women and one man who were 52, 80, and 22 years of age at the time of diagnosis. The tumors involved the submucosa and muscularis propria of the duodenum, jejunum, and ileum. All 3 tumors contained a proliferation of monotonous oval or spindle cells with scattered, somewhat dilated vessels. Two cases showed epithelioid structures such as glands, tubules, or nests. Immunohistochemical analysis revealed cytokeratin expression in the epithelioid components of both tumors displaying these features, and variable numbers of mesenchymal cells. Diffuse CD56 positivity was seen in the mesenchymal component of 2 tumors and desmin and smooth muscle actin staining in the other tumor. Immunostains for S-100 protein, DOG-1, and CD117 were negative in all cases. GLI1 fusions with different partner genes were detected in all tumors, and in the plexiform fibromyxoma, used as a control. Validation by fluorescence in situ hybridization was performed. None of the tumors have recurred or metastasize after surgery. We describe novel GLI1 fusions in 3 mesenchymal neoplasms of the small intestine, including 2 with biphenotypic features. Thus far, all cases have pursued indolent clinical courses. We propose the term " GLI1 -rearranged enteric tumor" to encompass this group of unique neoplasms of the small intestine that harbor GLI1 gene fusions and expand the spectrum of gastrointestinal neoplasms with these alterations.

Troglitazone inhibits hepatic oval cell proliferation by inducing cell cycle arrest through Hippo/YAP pathway regulation.

Hepatic oval cells have strong proliferation and differentiation capabilities and are activated when chronic liver injury occurs or when liver function is severely impaired. Peroxisome proliferation-activated receptors (PPARs) are ligand-dependent, sequence-specific nuclear transcription factors. PPARγ is closely related to liver diseases (such as liver cancer, liver fibrosis and non-alcoholic fatty liver disease). As the main effector downstream of the Hippo signaling pathway, YAP can activate the hepatic progenitor cell program, and different expression or activity levels of YAP can determine different liver cell fates. We found that troglitazone (TRO), a classic PPARγ activator, can inhibit the growth of hepatic oval cells, and flow cytometry results showed that TRO inhibited the growth of WB-F344 cells by arresting the cells in the G0/1 phase. Western blot results also confirmed changes in G0/1 phase-related protein expression. Further experiments showed that PPARγ agonists induced hepatic oval cell proliferation inhibition and cell cycle G0/1 phase arrest through the Hippo/YAP pathway. Our experiment demonstrated, for the first time, the relationship between PPARγ and the Hippo/YAP pathway in liver oval cells and revealed that PPARγ acts as a negative regulator of liver regeneration by inhibiting the proliferation of oval cells.

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies

BackgroundCurrently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver-targeted genotoxic carcinogens (Group B) were evaluated.ResultsIn the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses.Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3).ConclusionEvaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies.

AGGRESSIVE CENTRAL GIANT CELL LESION IN THE YOUNG PATIENT: A CASE REPORT

The giant cell central lesion (LCCG) is considered to be rare and benign, and its etiology is uncertain. Clinically and radiographically, it can be differentiated between aggressive and nonaggressive lesions. A female patient with leukoderma, 13 years old, presented with discrete asymmetry in the right hemiface and increased volume in the anterior region of the mandible, of indurated consistency, and painless palpation. In the imaging examinations, the presence of a radiolucent, multilocular, well-delimited image in the anterior region of the mandible crossing the midline, with tooth dislocation was observed. Histopathology revealed a loose, richly vascularized fibrous connective tissue fragment exhibiting intense proliferation of spindle and oval cells with large numbers of nonspecific multinucleated giant cells. Hemorrhagic areas and deposits of brownish pigmentation were compatible with hemosiderin. The diagnosis was central giant cell lesion. The conservative drug therapy was performed, which was not successful, and the surgical intervention was performed. Postoperative control is 6 months, without recurrence.

JUVENILE OSSIFYING FIBROMA IN CHILDREN: A CASE REPORT

Juvenile ossifying fibroma is a benign fibro-osseous neoplasm of mesenchymal origin that affects young individuals and has an aggressive clinical behavior with high rates of recurrence. A 5-year-old female patient presented intraosseous growth, approximately 1.5 cm in size, normochromic, hard in consistency, sessile, with painful symptoms and 1-month of evolution. Imaging examination revealed well-delimited hypodense lesion and buccal and lingual cortical expansion. The patient had already performed an incisional biopsy with ossifying fibroma diagnosis. Then, an excisional biopsy was accomplished in association with local bone curettage. Microscopic examination showed fusiform and oval mesenchymal cell proliferation. There was deposition of numerous bone trabeculae with varying degrees of maturation, exhibiting prominent osteoblastic paving. After 1 year, the patient is still on regular follow-up, with no signs of recurrence.

Mammalian Target of Rapamycin Complex 2 Signaling Is Required for Liver Regeneration in a Cholestatic Liver Injury Murine Model

Cholestatic liver injury may lead to a series of hepatobiliary syndromes, which can progress to cirrhosis and impaired liver regeneration, eventually resulting in liver-related death. Mammalian target of rapamycin complex 2 (mTORC2) is a major regulator of liver metabolism and tumor development. However, the role of mTORC2 signaling in cholestatic liver injury has not been characterized to date. In this study, we generated liver-specific Rictor knockout mice to block the mTORC2 signaling pathway. Mice were treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce cholestatic liver injury. DDC feeding induced cholestatic liver injury and ductular reaction as well as activation of the mTORC2/Akt signaling pathway in wild-type mice. Loss of mTORC2 led to significantly decreased oval cell expansion after DDC feeding. Mechanistically, this phenotype was independent of mTORC1/fatty acid synthase cascade (Fasn) or yes-associated protein (Yap) signaling. Notch pathway was instead strongly inhibited during DDC-induced cholestatic liver injury in liver-specific Rictor knockout mice. Furthermore, mTORC2 deficiency in adult hepatocytes did not inhibit ductular reaction in this cholestatic live injury mouse model. Our results indicated that mTORC2 signaling effectively regulates liver regeneration by inducing oval cell proliferation. Liver progenitor cells or bile duct cells, rather than mature hepatocytes, would be the major source of ductular reaction in DDC-induced cholestatic liver injury.

SESAME OIL MITIGATES INITIATION STAGE OF DIETHYNITROSAMINE HEPATOCARCINOGENESIS IN RATS

Diethylnitrosamine (DEN) induced hepatocarcinogenesis in experimental animals through triggering reactive oxygen species (ROS) release and subsequent induction of oxidative stress dependant liver damage. This study was conducted to estimate the protective role of sesame oil (SO) in the initial phase of DEN induced hepatocarcinogenesis. Forty five male Wistar rats were randomly divided into five groups groups (n = 9 each). In the first group (control), rats were orally administrated normal saline. Rats of second group (DEN) were intraperitoneally (i.p) injected with a single dose of (200mg/kg body weight, DEN) at the 8thday of the experiment. The third, fourth, fifth groups orally administrated SO at a dose (2.5, 5, 10 mL/kg b.w), respectively 1 week before i.p injection of DEN and continued for 4 successive weeks. DEN- induced hepatotoxicity as detected by normocytic normochromic anemia with marked increase in white blood cells and significant increase in hepatic damage enzymatic markers (alanine transaminase (ALT), aspartate aminotransferase (AST), γ-glutamyl Transferase (γGT) and alkaline phosphatase (ALP)) with significant decrease in serum total protein. Hepatic malondialdehyde (MDA) was increased significantly while hepatic antioxidant biomarkers superoxide dismutase (SOD), catalase (CAT) and hepatic reduced glutathione (GSH) were significantly decreased. Histological examination of hepatic tissue of DEN treated rats proved centrolobular necrosis associated with bile duct and oval cell proliferation. This was accompanied with over expression of CYP2E1and down regulation in BAX gene expression in liver. Administration of SO minimized the harmful effects of DEN on hematological, biochemical, antioxidant and histopathological parameter as well as on gene expression. The degree of improvement was in dose dependant manner. Our findings revealed that SO supplementation can mitigate the toxic effects of DEN via their potent antioxidant and free radical-scavenging activities.Key words: diethylnitrosamine, sesame oil, antioxidant, gene expression, rats

Angiomyofibroblastoma mimicking an inguinal hernia: a challenging diagnosis in a male patient.

IntroductionAngiomyofibroblastoma is a rare benign myofibroblastic neoplasm which mainly occurs in the soft tissues of the pelvi-perineal region of females.AimTo present an unusual case of angiomyofibroblastoma mimicking an inguinal hernia in a 62-year-old male.Material and methodsThe patient was hospitalized with an irreducible, painless inguinal mass and surgical intervention for inguinal hernia was decided. The well-defined nodular mass was sent for histological examination.ResultsUnder microscope, proliferation of spindle and oval cells around thin-walled vessels was observed, being intermingled with mature adipocytes. We did not identify necrosis, haemorrhage, cytologic atypia or mitotic figures. The tumour cells displayed positivity for desmin, vimentin, CD34, oestrogen and progesterone receptors, a low Ki67 index and unusual nuclear positivity for c-theta (PKCθ). They were negative for smooth muscle actin (SMA), S100, CD44, maspin, synaptophysin, DOG1 and CD117. The case was diagnosed as angiomyofibroblastoma, the main challenge being the differential diagnosis with aggressive angiomyxoma, which can present a similar histologic aspect and immunophenotype and recurs more frequently. No recurrences were observed 8 months after the surgery.ConclusionsAngiomyofibroblastoma should be included in the differential diagnosis of inguinal hernia. This is the fourteenth case of angiomyofibroblastoma diagnosed in males.

HBV induces different responses of the hepatocytes and oval cells during HBV-related hepatic cirrhosis

Although hepatitis B virus (HBV)-related cirrhosis and hepatocellular carcinoma (HCC) cause a sever health problem worldwide, the underlying mechanisms are still elusive. This study aimed to investigate the responses of different cell types isolated from HBV transgenic mice. A cross-sectional set of hepatocytes and oval cells were obtained from HBV transgenic and control mice. Flow cytometry, immunohistochemistry and microarray were applied to investigate the cell biology of the hepatocytes and oval cells. Our results showed that HBV induced the proliferation of both cell oval cells and hepatocytes, and induced cell death of HBV hepatocytes while had minimal effects on oval cells. Further molecular and pathways analysis identified some genes and signaling pathways may be responsible for the different responses between oval cells and hepatocytes. In addition, analyses of selectively ten genes by IHC staining in human samples were consistent with microarray data. In summary, HBV transgenic mice is a useful model for studying the biological behaviors of oval cells affected by HBV and HBV-cirrhosis. Also, our results help better understand the mechanisms of HBV induced cirrhosis, and provide novel progenitor markers or prognostic/therapeutic markers.

Participation of liver stem cells in cholangiocarcinogenesis after aflatoxin B1 exposure of glutathione S-transferase A3 knockout mice.

Aflatoxin B1, arguably the most potent human carcinogen, induces liver cancer in humans, rats, trout, ducks, and so on, but adult mice are totally resistant. This resistance is because of a detoxifying enzyme, mouse glutathione S-transferase A3, which binds to and inactivates aflatoxin B1 epoxide, preventing the epoxide from binding to DNA and causing mutations. Glutathione S-transferase A3 or its analog has not been detected in any of the sensitive species, including humans. The generation of a glutathione S-transferase A3 knockout (represented as KO or -/-) mice has allowed us to study the induction of liver cancer in mice by aflatoxin B1. In contrast to the induction of hepatocellular carcinomas in other species, aflatoxin B1 induces cholangiocarcinomas in GSTA3-/- mice. In other species and in knockout mice, the induction of liver cancer is preceded by extensive proliferation of small oval cells, providing additional evidence that oval cells are bipolar stem cells and may give rise to either hepatocellular carcinoma or cholangiocarcinoma depending on the nature of the hepatocarcinogen and the species of animal. The recent development of mouse oval cell lines in our laboratory from aflatoxin B1-treated GSTA3-/- mice should provide a new venue for study of the properties and potential of putative mouse liver stem cells.

Pharmacological Mobilization of Endogenous Bone Marrow Stem Cells Promotes Liver Regeneration after Extensive Liver Resection in Rats

Rapid regeneration of the remnant liver is critical for preventing liver failure and promoting recovery after extensive liver resection. Numerous studies have demonstrated the involvement of bone marrow-derived stem cells in liver regeneration and the potential benefits of bone marrow stem cell therapy. To avoid the preparation of stem cells, we proposed in this study to mobilize endogenous bone marrow stem cells pharmacologically with a combination of AMD3100 (A), an antagonist of CXCR4 and low-dose FK506 (F). Here we show that AF combination therapy significantly increased lineage negative (Lin-) CD34+ and Lin-CD133+ stem cells in peripheral blood and enhanced recruitment of CD133+ cells into the remnant liver in a rat model of 85% partial hepatectomy. Recruiting CD133+ stem cells in the remnant liver was associated with increased proliferation of hepatic oval cells and paralleled the increased SDF-1, CXCR4 and HGF expression. Importantly, AF combination therapy increased the number of Ki67 positive hepatocytes and BrdU incorporation in the remnant liver and improved serum levels of albumin. Our results demonstrate that pharmacological mobilization of endogenous bone marrow stem cells with AF combination therapy can enhance endogenous stem cell mobilization to promote liver regeneration and improve liver function after extensive hepatectomy.

Interleukins-17 and 27 promote liver regeneration by sequentially inducing progenitor cell expansion and differentiation.

Liver progenitor cells (LPCs)/ductular reactions (DRs) are associated with inflammation and implicated in the pathogenesis of chronic liver diseases. However, how inflammation regulates LPCs/DRs remains largely unknown. Identification of inflammatory processes that involve LPC activation and expansion represent a key step in understanding the pathogenesis of liver diseases. In the current study, we found that diverse types of chronic liver diseases are associated with elevation of infiltrated interleukin (IL)‐17‐positive (+) cells and cytokeratin 19 (CK19)+ LPCs, and both cell types colocalized and their numbers positively correlated with each other. The role of IL‐17 in the induction of LPCs was examined in a mouse model fed a choline‐deficient and ethionine‐supplemented (CDE) diet. Feeding of wild‐type mice with the CDE diet markedly elevated CK19+Ki67+ proliferating LPCs and hepatic inflammation. Disruption of the IL‐17 gene or IL‐27 receptor, alpha subunit (WSX‐1) gene abolished CDE diet‐induced LPC expansion and inflammation. In vitro treatment with IL‐17 promoted proliferation of bipotential murine oval liver cells (a liver progenitor cell line) and markedly up‐regulated IL‐27 expression in macrophages. Treatment with IL‐27 favored the differentiation of bipotential murine oval liver cells and freshly isolated LPCs into hepatocytes. Conclusion: The current data provide evidence for a collaborative role between IL‐17 and IL‐27 in promoting LPC expansion and differentiation, respectively, thereby contributing to liver regeneration. (Hepatology Communications 2018;2:329‐343)

Phylogenetic analyses of the hepatic architecture in vertebrates.

The mammalian liver has a structural and functional unit called the liver lobule, in the periphery of which the portal triad consisting of the portal vein, bile duct and hepatic artery is developed. This type of hepatic architecture is detectable in many other vertebrates, including amphibians and birds, whereas intrahepatic bile ducts run independently of portal vein distribution in actinopterygians such as the salmon and tilapia. It remains to be clarified how the hepatic architectures are phylogenetically developed among vertebrates. The present study morphologically and immunohistochemically analyzed the hepatic structures of various vertebrates, including as many classes and subclasses as possible, with reference to intrahepatic bile duct distribution. The livers of vertebrates belonging to the Agnatha, Chondrichthyes, Amphibia, Aves, Mammalia, and Actinopterygii before Elopomorpha, had the portal triad-type architecture. The Anguilliformes livers developed both periportal bile ducts and non-periportal bile ducts. The Otocephala and Euteleostei livers had independent configuration of bile ducts and portal veins. Pancreatic tissues penetrated the liver parenchyma along portal veins in the Euteleostei. The liver of the lungfish, which shares the same origin with amphibians, did not have the portal triad-type architecture. Teleostei and lungfish livers had ductular development in the liver parenchyma similar to oval cell proliferation in injured mammalian livers. Euteleostei livers had penetration of significant numbers of independent portal veins from their intestines, suggesting that each liver lobe might receive a different blood supply. The hepatic architectures of the portal triad-type changed to non-portal triad-type architecture along the evolution of the Actinopterygii. The hepatic architecture of the lungfish resembles that of the Actinopterygii after Elopomorpha in intrahepatic biliary configuration, which may be an example of convergent evolution.

Autophagy regulates proliferation and biliary differentiation of hepatic oval cells via the MAPK/ERK signaling pathway.

Hepatic oval cells (HOCs) are thought to possess self‑renewal ability and a bipotential capacity for differentiation, which allows them to differentiate into hepatocytes and cholangiocytes. Autophagy serves an important role in self‑renewal and differentiation of stem cells; however, how autophagy contributes to proliferation and differentiation of hepatic progenitor cells has yet to be elucidated. In the present study, autophagy was regulated by rapamycin (Rapa) and chloroquine (Chlo) administration. The results demonstrated that Chlo‑treated HOCs exhibited decreased autophagic activity alongside a decreased tendency to proliferate, as determined by Cell Counting Kit‑8. In addition, activation of autophagy by Rapa enhanced the biliary differentiation of HOCs. Furthermore, increased phosphorylated (p)‑extracellular signal‑regulated kinase (ERK)/p‑p38 expression was observed following the induction of autophagy, thus indicating that the mitogen‑activated protein kinase (MAPK)/ERK signaling pathway was activated by autophagy to exert effects on the stimulation of HOC proliferation and differentiation. In conclusion, the present study demonstrated that autophagy regulates proliferation and biliary differentiation of HOCs via the MAPK/ERK signaling pathway. These results suggest a role for autophagy in stimulating the proliferation and differentiation of HOCs.

Dual role of protein tyrosine phosphatase 1B in the progression and reversion of non-alcoholic steatohepatitis

ObjectivesNon-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH). MethodsNASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/choline-deficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2–7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice. ResultsPTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were up-regulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated. ConclusionsPTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell proliferation during NAFLD.

Inhibitory effects of HNF4\u03b1 on migration/maltransformation of hepatic progenitors: HNF4\u03b1-overexpressing hepatic progenitors for liver repopulation

BackgroundAlthough they are expandable in vitro, hepatic progenitors are immature cells and share many immunomarkers with hepatocellular carcinoma, raising potential concerns regarding maltransformation after transplantation. This study investigated the effects of hepatic nuclear factor (HNF) 4α on the proliferation, migration, and maltransformation of hepatic progenitors and determined the feasibility of using these manipulated cells for transplantation.MethodsThe effects of HNF4α on rat hepatic progenitors (i.e. hepatic oval cells) were analyzed by HNF4α overexpression and HNF4α shRNA. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice injured by carbon chloride (CCl4) were then transplanted with control, HNF4α-overexpressing or HNF4α-suppressing hepatic oval cells. Finally, the engraftment of these cells in the recipient liver was analyzed.ResultsRat hepatic progenitors (i.e. hepatic oval cells) expressed HNF4α, although less than that in hepatocytes. When HNF4α was overexpressed in these cells, the proliferation and migration of hepatic oval cells were reduced; but when HNF4α was suppressed by shRNA, the proliferation and migration, and even anchorage-independent growth, of these cells were accelerated. RNA microarray and gene functional analysis revealed that suppressing HNF4α not only impaired many biosynthesis and metabolism pathways of hepatocytes but also increased pathways for cancer. When transplanted into CCl4-injured NOD/SCID mice, few HNF4α-suppressing hepatic oval cells localized into the liver, while control cells and HNF4α-overexpressing cells engrafted into the liver and differentiated into albumin-positive hepatocytes. Interestingly, the hepatocytes derived from HNF4α-overexpressing cells were less migrative and expressed less c-Myc than the cells derived from control cells.ConclusionHNF4α constrains proliferation, migration, and maltransformation of hepatic progenitors, and HNF4α-overexpressing hepatic progenitors serve as an optimal candidate for cell transplantation.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.