Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafish model of Alagille Syndrome.

BACKGROUND AND AIMS: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. APPROACH AND RESULTS: Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

The Role of microRNAs in Cholangiocarcinoma.

Cholangiocarcinoma (CCA), an aggressive malignancy, is typically diagnosed at an advanced stage. It is associated with dismal 5-year postoperative survival rates, generating an urgent need for prognostic and diagnostic biomarkers. MicroRNAs (miRNAs) are a class of non-coding RNAs that are associated with cancer regulation, including modulation of cell cycle progression, apoptosis, metastasis, angiogenesis, autophagy, therapy resistance, and epithelial-mesenchymal transition. Several miRNAs have been found to be dysregulated in CCA and are associated with CCA-related risk factors. Accumulating studies have indicated that the expression of altered miRNAs could act as oncogenic or suppressor miRNAs in the development and progression of CCA and contribute to clinical diagnosis and prognosis prediction as potential biomarkers. Furthermore, miRNAs and their target genes also contribute to targeted therapy development and aid in the determination of drug resistance mechanisms. This review aims to summarize the roles of miRNAs in the pathogenesis of CCA, their potential use as biomarkers of diagnosis and prognosis, and their utilization as novel therapeutic targets in CCA.

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Regional Differences in Human Biliary Tissues and Corresponding In Vitro-Derived Organoids.

BACKGROUND AND AIMS: Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. APPROACH AND RESULTS: Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. CONCLUSIONS: Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

Multidimensional imaging of liver injury repair in mice reveals fundamental role of the ductular reaction.

Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells that give rise to new hepatocytes, recent lineage-tracing analyses have challenged this notion, thereby leaving the pro-regenerative role of the ductular reaction enigmatic. Here, we show that the expanded and remodelled intrahepatic biliary epithelia in the ductular reaction constituted functional and complementary bile-excreting conduit systems in injured parenchyma where hepatocyte bile canalicular networks were lost. The canalicular collapse was an incipient defect commonly associated with hepatocyte injury irrespective of cholestatic statuses, and could sufficiently provoke the ductular reaction when artificially induced. We propose a unifying model for the induction of the ductular reaction, where compensatory biliary epithelial tissue remodeling ensures bile-excreting network homeostasis.

Bim is an independent prognostic marker in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver malignant tumor and has a poor prognosis. The prognostic factors associated with outcome remain poorly defined. In this study, we investigated the role of an important cell apoptosis initiator, Bcl-2 interacting mediator of cell death (Bim), by evaluating its expression and association with other clinicopathological features in ICCs. We analyzed 56 cases of ICC with clinical follow-up. The expression of Bim in ICC cells and other cellular components was evaluated by immunohistochemistry. Bim expression was considered up-regulated if Bim was detected in 10% or more of tumor cells. Of the 56 ICC samples, 19 (34%) had high Bim expression level, 15 (27%) were completely negative, and 22 (39%) were classified as low Bim expression (<10% positivity). Patients who had tumors with high Bim level had significantly longer overall survival than did those with low or no staining (median survival, 7.6 versus 2.6 years; hazard ratio, 0.40; P = .006). High Bim expression was also correlated with low Ki-67 index, and more importantly, none of the tumors with high Bim expression had lymph node metastases at the time of surgery. Our study demonstrates that Bim is an important and independent prognostic factor in ICC. Tumors with high Bim expression are associated with better prognosis through inhibiting tumor cell proliferation and metastatic ability. The development of new agents directly or indirectly targeting Bim may provide promising anticancer treatments.

Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease.

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium.

Adaptive remodeling of the biliary architecture underlies liver homeostasis.

UNLABELLED: Serving as the center for metabolism and detoxification, the liver is inherently susceptible to a wide variety of damage imposed by toxins or chemicals. Induction of cell populations with biliary epithelial phenotypes, which include progenitor-like cells and are referred to as liver progenitor cells, is often observed in histopathological examination of various liver diseases in both human patients and animal models and has been implicated in regeneration. However, the tissue dynamics underlying this phenomenon remains largely unclear. We have developed a simple imaging technique to reveal the global and fine-scale architecture of the biliary tract spreading in the mouse liver. Using this novel method, we show that the emergence and expansion of liver progenitor cells actually reflect structural transformation of the intrahepatic biliary tree in mouse liver injury models. The biliary branches expanded their area gradually and contiguously along with the course of chronic injury. Relevant regulatory signals known to be involved in liver progenitor cell regulation, including fibroblast growth factor 7 and tumor necrosis factor-like weak inducer of apoptosis, can modulate the dynamics of the biliary epithelium in different ways. Importantly, the structural transformations of the biliary tree were diverse and corresponded well with the parenchymal injury patterns. That is, when chronic hepatocyte damage was induced in the pericentral area, the biliary branches exhibited an extended structure from the periportal area with apparent tropism toward the distant injured area. CONCLUSION: The hepatobiliary system possesses a unique and unprecedented structural flexibility and can remodel dynamically and adaptively in response to various injury conditions; this type of tissue plasticity should constitute an essential component to maintain liver homeostasis.

Intrahepatic bile duct regeneration in mice does not require Hnf6 or Notch signaling through Rbpj.

The potential for intrahepatic bile duct (IHBD) regeneration in patients with bile duct insufficiency diseases is poorly understood. Notch signaling and Hnf6 have each been shown to be important for the morphogenesis of IHBDs in mice. One congenital pediatric liver disease characterized by reduced numbers of IHBDs, Alagille syndrome, is associated with mutations in Notch signaling components. Therefore, we investigated whether liver cell plasticity could contribute to IHBD regeneration in mice with disruptions in Notch signaling and Hnf6. We studied a mouse model of bile duct insufficiency with liver epithelial cell-specific deficiencies in Hnf6 and Rbpj, a mediator of canonical Notch signaling. Albumin-Cre Hnf6(flox/flox)Rbpj(flox/flox) mice initially developed no peripheral bile ducts. The evolving postnatal liver phenotype was analyzed using IHBD resin casting, immunostaining, and serum chemistry. With age, Albumin-Cre Hnf6(flox/flox)Rbpj(flox/flox) mice mounted a ductular reaction extending through the hepatic tissue and then regenerated communicating peripheral IHBD branches. Rbpj and Hnf6 were determined to remain absent from biliary epithelial cells constituting the ductular reaction and the regenerated peripheral IHBDs. We report the expression of Sox9, a marker of biliary epithelial cells, in cells expressing hepatocyte markers. Tissue analysis indicates that reactive ductules did not arise directly from preexisting hilar IHBDs. We conclude that liver cell plasticity is competent for regeneration of IHBDs independent of Notch signaling via Rbpj and Hnf6.

Role of stem cell factor and granulocyte colony-stimulating factor in remodeling during liver regeneration.

UNLABELLED: Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte macrophage colony-stimulating factors (GM-CSFs), and stem cell factors (SCFs) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblotting and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time polymerase chain reaction (PCR). There was a significant, stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100 calcium-binding protein A4 (S100A4) and miR-181b, after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCFs and GM-CSFs in response to transforming growth factor-beta (TGF-ß). When SMCCs were incubated with TGF-ß plus anti-SCF+GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF+GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) messenger RNA as well as miR-181b expression, along with a reduction of metalloproteinase inhibitor 3. Levels of MMP-2, MMP-9, and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. CONCLUSION: Our data suggest that altered expression of SCF+GM-CSF after PH can contribute to biliary remodeling (e.g., post-transplantation) by functional deregulation of the activity of key signaling intermediates involved in cell expansion and multipotent differentiation.

Autophagy may precede cellular senescence of bile ductular cells in ductular reaction in primary biliary cirrhosis.

BACKGROUND AND AIM: Recent studies disclosed that autophagy facilitates the process of senescence. Given that cellular senescence is involved in the pathophysiology of ductular reaction (DR) in primary biliary cirrhosis (PBC), we examined an involvement of autophagy in DRs in PBC and control livers. METHODS: We examined immunohistochemically the expression of microtubule-associated proteins light chain 3ß (LC3) as autophagy marker, p62/sequestosome-1 (p62) as autophagy-related marker in bile ductular cells in livers taken from the patients with PBC (n = 42), and control livers (n = 100). The expression of senescent markers (p16(INK4a) and p21(WAF1/Cip1)) in bile ductular cells and their correlation with autophagy was also evaluated. RESULTS: The expression of LC3 was seen in coarse vesicles in the cytoplasm of bile ductular cells and significantly more frequently in PBC of both early and advanced stages when compared to control livers (p < 0.01). The expression of p62 was seen as intracytoplasmic aggregates and significantly more frequently in PBC when compared to control livers (p < 0.05). The expression of LC3 and p62 significantly correlated with each other (p < 0.01). The expression of LC3 and p62 significantly correlated with the expression of p16(INK4a), p21(WAF1/Cip1) (p < 0.05). CONCLUSIONS: Autophagy is frequently seen in bile ductular cells in DRs in PBC. Since cellular senescence of bile ductular cells is rather frequent in the advanced stage of PBC, autophagy may precede cellular senescence of bile ductular cells in DRs in PBC.

Ductular reactions in human liver: diversity at the interface.

Interest in hepatic ductular reactions (DRs) has risen in recent years because of a greater appreciation of their potential roles in regeneration, fibrogenesis, and carcinogenesis. However, confusion exists because there is significant, but often unappreciated diversity at the tissue, cellular, and subcellular levels in DRs of different diseases and stages of disease. DRs are encountered in virtually all liver disorders in which there is organ-wide liver damage and cell loss, but are also present in focal lesions such as focal nodular hyperplasia and adenoma. Moreover, diverse DR phenotypes can be present within any single disease entity, and are shaped by the etiology and evolution of the disease. Although much remains to be clarified, recent studies suggest that the diversity of appearances of the DRs are likely to reflect the differing signals at the anatomic, cellular, and molecular levels driving the proliferative response. These appear to determine the relative proportions of transit-amplifying cells, the degree of hepatocytic or cholangiocytic differentiation, and their relationships with stromal, vascular, and inflammatory components. The molecular signaling pathways governing these regenerative fate decisions closely replicate those found in human and other vertebrate embryos and more generally in stem cell niches throughout the body. Like the latter, complex interactions with matrix as well as mesenchymal and inflammatory cells, vessels, and innervation are likely to be of fundamental importance. Embracing systems/tissue biological approaches to exploring DRs, in addition to more traditional cellular and molecular biological techniques, will further enhance our understanding and, thereby, we believe potentiate new therapeutic possibilities.

Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth.

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin → melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth.

RhGH attenuates ischemia injury of intrahepatic bile ducts relating to liver transplantation.

BACKGROUND: To study the effect of rhGH administration on intrahepatic cholangiocytes relating to liver transplantation with ischemia of hepatic artery, and ultimately, clarify pathologic mechanism of the injury. METHODS: Rat orthotopic autologous liver transplantation was performed first. Three hours later, the rats were grouped as followed: HAL (hepatic artery ligation) group; HAL + rhGH (hepatic artery ligation followed by rhGH administration) group; CON (without hepatic artery ligation) group. Specimen was collected after 7 d. ALT and ALP of serum were measured. The pathologic changes of bile ducts of liver tissue were observed. The number of bile ducts and blood vessels in portal area were counted. Immunochemistry for VEGF, VEGFR-2, VEGFR-3, GHR, and IGF-1R of intrahepatic cholangiocytes was performed. Cholangiocytes apoptosis was evaluated by TUNEL analysis. Cholangiocytes proliferation was evaluated by PCNA immunolabeling. RESULTS: ALT and ALP of HAL + rhGH group were significantly ameliorated compared with untreated animals (P < 0.05). ALT and ALP of HAL group were significantly higher compared with CON group (P < 0.05). In HAL group, the main injury of bile ducts was not reversible, whereas it was reversible in CON and rhGH groups. In HAL group, the number of bile ducts in portal area decreased, while the number of bile ducts not accompanying blood vessels increased (P < 0.05). In rhGH group, the number of bile ducts in portal area increased, while the number of bile ducts accompanying blood vessels increased compared with HAL group (P < 0.05). The expression of VEGF, VEGFR-2, VEGFR-3, GHR, and IGF-1R was significantly lower in HAL group than in CON group (P < 0.05). Following administration of rhGH to HAL rats, the expression of VEGF, VEGFR-2, VEGFR-3, IGF-1R, and GHR was significantly higher (P < 0.05). Administration of rhGH prevented increase in cholangiocytes apoptosis induced by HAL (P < 0.05). Administration of rhGH promoted increase in cholangiocytes proliferation held by HAL (P < 0.05). CONCLUSIONS: Administration of rhGH appears to attenuate ischemia injury of intrahepatic bile ducts relating to liver transplantation. This function is partly related to the capacity that rhGH inhibits the apoptosis of intrahepatic cholangiocytes and prompts the proliferation and angiogenesis by increasing the expression of VEGF, VEGFR2, VEGFR3, GHR, and IGF1-R.

Role of vascular endothelial growth factor in protection of intrahepatic cholangiocytes mediated by hypoxic preconditioning after liver transplantation in rats.

OBJECTIVE: To investigate the effect on intrahepatic cholangiocytes mediated by hypoxic preconditioning (HP) after liver transplantation and the role of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: This experiment was based on a model of rat orthotopic liver autotransplantation. Sprague-Dawley rats were randomly divided into 3 groups: normal control, autotransplantation (AT), and HP. The HP group was subjected to 8% oxygen atmosphere for 90 minutes before surgery. At 6, 12, 24, and 48 hours after autotransplantation, the rats were killed for testing .Serum total bilirubin, direct bilirubin, and alkaline phosphatase concentrations were determined. The microstructure of cholangiocytes and the ultramicrostructure of cholangioles were determined. Immunohistochemistry was used to detect the expression of VEGF and the proliferation rate of cholangiocytes. RESULTS: Total bilirubin, direct bilirubin, and alkaline phosphatase concentrations in the AT group increased considerably more than in the HP group during the entire interval (P < .05). Light microscopy demonstrated that the microstructure of cholangiocytes in the AT group was damaged more seriously than in the HP group. At transmission electron microscopy, the ultramicrostructure of cholangioles was changed more obviously than in the HP group. The expression of VEGF on cholangiocytes and the proliferation rate of cholangiocytes were higher in the HP group than in the AT group over the entire experiment (P < .05). CONCLUSION: Hypoxic preconditioning has a protective effect on cholangiocytes after liver autotransplantation. The mechanism may be related to HP-induced overexpression of VEGF on cholangiocytes.

Rapamycin inhibits cholangiocyte regeneration by blocking interleukin-6-induced activation of signal transducer and activator of transcription 3 after liver transplantation.

Cholangiocyte proliferation is necessary for biliary recovery from cold ischemia and reperfusion injury (CIRI), but there are few studies on its intracellular mechanism. In this process, the role of rapamycin, a new immunosuppressant used in liver transplantation, is still unknown. In order to determine whether rapamycin can depress cholangiocyte regeneration by inhibiting signal transducer and activator of transcription 3 (STAT3) activation, rapamycin (0.05 mg/kg) was administered to rats for 3 days before orthotopic liver transplantation. The results indicated that cholangiocytes responded to extended cold preservation (12 hours) with severe bile duct injures, marked activation of the interleukin-6 (IL-6)/STAT3 signal pathway, and increased expression of cyclin D1 until 7 days after transplantation, and this was followed by compensatory cholangiocyte regeneration. However, rapamycin treatment inhibited STAT3 activation and resulted in decreased cholangiocyte proliferation and delayed biliary recovery after liver transplantation. On the other hand, rapamycin showed no effect on the expression of IL-6. We conclude that the IL-6/STAT3 signal pathway is involved in initiating cholangiocytes to regenerate and repair CIRI. Rapamycin represses cholangiocyte regeneration by inhibiting STAT3 activation, which might have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation. Insights gained from this study will be helpful in designing therapy using rapamycin in clinical patients after liver transplantation.

Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.

BACKGROUND: Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis. MATERIALS AND METHODS: Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined. RESULTS: The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis. CONCLUSIONS: DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.

Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Adult mouse intrahepatic biliary epithelial cells induced in vitro to become insulin-producing cells.

Transdifferentiation of cells from a patient's own liver into pancreatic beta-cells could be useful for beta-cell replacement. We hypothesized that intrahepatic biliary epithelial cells (IHBECs) could become a new source of insulin-producing cells. IHBECs isolated from adult mice were expanded using our novel culture method termed, collagen-embedded floating culture method (CEFCM). With CEFCM, IHBECs formed three-dimensional ductal cysts and rapidly expanded their number by about 15-fold within 2 weeks. Over 90% of cells were positive for cytokeratin 7 and 19. At day 14, IHBECs were transfected with adenoviral (Ad)- pancreas duodenum homeobox 1 (Pdx-1), NeuroD or Pdx-1/VP16. After 7 additional days in serum- and insulin-free differentiation medium (DM), cell phenotypes were determined by RT-PCR, immunostaining and ELISA for insulin. In DM control IHBECs started to express some endocrine progenitor genes (Neurog3, NeuroD, Nkx6.1, and Pdx-1) but lacked insulin gene (Ins) mRNA. Transduced expression of PDX-1, NEUROD or PDX-1/VP16 led to expression of not only INS but also GLUT2 and prohormone convertase 1 and 2. About 3% of 4000 cells counted in PDX-1/VP16 transduced cultures stained strongly for C-peptide suggesting that a subpopulation may have the capacity for differentiation. Transduced cells released insulin (Ad-PDX-1 0.08+/-0.05, Ad-NEUROD 0.33+/-0.09, Ad-PDX-1/VP16 0.37+/-0.14 ng/1x10(5) cells after 48 h in culture). IHBECs can be markedly expanded, and then with molecular manipulation a subpopulation of these cells can differentiate towards a beta-cell phenotype. This approach may lead to a new source of beta-cells that can be used for transplantation in diabetes.

Inflammatory cytokines suppress NAD(P)H:quinone oxidoreductase-1 and induce oxidative stress in cholangiocarcinoma cells.

PURPOSE: The aim of this study was to evaluate the effect of inflammation on NAD(P)H: quinone oxidoreductase-1 (NQO1), the xenobiotic metabolizing and antioxidant enzyme protecting cells against electrophiles and reactive oxygen species in biliary cancer (cholangiocarcinoma) cells. METHODS: Human cholangiocarcinoma cell line, KKU-OCA17 and HeLa Chang liver cells were treated with inflammatory cytokine combinations (interferon-gamma, interleukin-1beta and tumor necrosis factor-alpha) for 48 h before NQO1 activity was assayed. Oxidant status was examined by assays of formation of nitric oxide (NO) and superoxide, and glutathione (GSH) levels. Expression of NQO1 was assessed by a reverse transcription and polymerase chain reaction. Effects of S-nitroso-glutathione (GSNO) were examined if the effects of inflammatory cytokines could be mimicked by nitric oxide donor. RESULTS: NQO1 activity in KKU-OCA17 and HeLa Chang liver cells was suppressed by cytokine combination. Cytokines induced formation of NO and suppression of redox ratios of GSH and glutathione disulfide (GSSG). GSNO produced the similar effects as cytokines on KKU-OCA17, in contrast, GSNO induced increase of NQO1 activity in HeLa Chang liver cells. The treatment of cytokines or GSNO suppressed expression of NQO1 in KKU-OCA17 and HeLa Chang liver cells. CONCLUSIONS: Inflammatory cytokines induced oxidative stress and this is associated with suppression of NQO1, whereas may contribute to differential susceptibility of biliary epithelial cells to chemical-induced cytotoxicity and carcinogenesis.