Thèses sur le sujet « Biliary epithelium »

Liver injury stimulates hepatocyte proliferation, regenerating the liver through self-replication. In cases where there is severe, repetitive, parenchymal damage, as seen in human chronic liver disease, hepatocyte mediated regeneration becomes impaired. In this setting it is currently unclear whether endogenous biliary epithelial cells can repopulate the hepatocyte compartment. This thesis therefore aimed to address this point by lineage tracing the main two liver epithelia populations on a background of impaired hepatocyte regeneration. To impair regeneration, an Itgb1 transgene was specifically deleted, conditionally, from the hepatocyte epithelium. Long-term loss of β1-Integrin alone or with additional injury caused an epithelial ductular reaction of biliary origin. Alongside β1-Integrin ablation, the hepatocyte epithelium was also labelled with a heritable ROSA26LSLtdTomato reporter. Impaired hepatocyte regeneration mediated by β1- integrin ablation resulted in 25% of hepatocytes becoming tdTomato negative (non-hepatocyte derived). To verify that the non-hepatocyte mediated regeneration was originating from the biliary epithelium, anti-Itgb1 RNAi was administrated to K19CreERT LSLtdTomato mice. Resulting in tdTomato positive hepatocytes that had differentiated from the labelled tdTomato positive biliary epithelial cells. In summary, this thesis demonstrates that hepatocyte β1-Integrin ablation combined with toxic damage causes marked ductular reactions and results in a substantial regeneration of functional hepatocytes from the biliary epithelium.

Cystic fibrosis-associated liver disease (CFLD) is a chronic cholangiopathy that negatively impacts the quality of life and survival of patients with Cystic Fibrosis (CF). CF is a disease of secretory epithelia caused by genetically-determined defective function of CFTR (cystic fibrosis conductance regulator), a cAMP-activated Cl- channel that, in the liver, is uniquely expressed in the biliary epithelium. The pathogenesis of CFLD is thought to be related to the ductal cholestasis caused by the defective channel function of CFTR. However, a number of evidence indicate that, in association with the genetic defect, other pathogenetic factors concur to determine the liver phenotype. During the first year of my doctorate we have demonstrated that in CFTR-defective biliary epithelium, exposed to bacterial-derived endotoxins, TLR4/NF-kB-dependent innate immune responses are increased, indicating a loss of “endotoxin tolerance”. The current standard of care for CFLD is limited to the administration of choleretic agents. Based on our novel interpretation of the pathogenesis of CFLD, a rational therapeutic approach to the management of CFLD should be to target the exaggerated inflammatory response of the biliary epithelium. Nuclear receptors (NRs), ligand-activated transcription factors that regulate several intracellular metabolic functions by controlling the expression of specific target genes, are now emerging as important regulators of inflammation in several conditions. In this work we sought to investigate the ability of NRs to modulate the TLR4/NF-kB-dependent innate immune responses in CF biliary epithelia. Our data indicate that cholangiocytes express several subtypes of NRs, including Peroxisome Proliferator Activated Receptor (PPAR) subgroup α, γ and β/δ, Liver X Receptors (LXR) subgroup β, Farnesoid X Receptor (FXR) and Vitamin D Receptor (VDR). Among these NRs, PPAR-γ was barely expressed in wild type (WT) cholangiocytes , but its expression, both at the gene and protein levels, was strongly and significantly more expressed in CF cells. In spite of its increased expression in the nucleus, PPAR-γ was not transcriptionally active in CF cholangiocytes; in fact the basal level of expression of PPAR-γ target genes (Acaa1b, Angptl4 and Hmgcs2) was similar to WT cholangiocytes. On the other hand, treatment of CF cholangiocytes with the PPAR-γ agonist pioglitazone significantly increased the expression of its target genes, indicating that the transcriptional activity of nuclear PPAR-γ could be readily activated in the presence of proper agonists. Consistent with the higher expression level of the receptor, the increase in PPAR-γ target genes stimulated by pioglitazone was higher in CF than in WT cholangiocytes. The observation that PPAR-γ, while inactive at baseline levels, was able to respond to exogenous stimulation, indicates that a defect in the availability of endogenous PPAR-γ activators might impair PPAR-γ function in CF and increase the receptor expression as counter-regulatory mechanism. Thus, to study the availability of endogenous PPAR-γ activators in CF, we performed lipidomic analysis of extracts from cultured cholangiocytes, and quantified the major ω-3 and ω-6 polyunsaturated fatty acids (PUFAs), i.e. the precursors of the active form of PPAR-γ ligands. We detected an increased ratio between arachidonic acid and docosahexaenoic acid (AA/DHA) and between arachidonic acid and linoleic acid (AA/LA) in CF cells compared to their controls. LA, the precursor of AA, is the main source of pro-inflammatory mediators, while DHA is an important precursor of anti-inflammatory eicosanoids that are able to activate PPAR-γ. This finding might explain, at least in part, the increased receptor expression in CF biliary cells. Having shown that CF cells express higher amounts of PPAR-γ, that can be activated by exogenous ligands, but it is not transcriptionally active because of the lack of endogenous ligands, we next tested if exogenous activation of PPAR-γ by synthetic ligands, might decrease the TLR4/NF-kB-driven inflammation in CFTR-defective biliary epithelium. We found that in Cftr-KO cells, in the presence of pioglitazone, nuclear translocation of p65/NF-kB, as well as its transcriptional activity, were significantly inhibited, both before and after stimulation with bacterial LPS. Moreover, the gene expression and protein secretion of several NF-kB-dependent pro-inflammatory cytokines, such as LIX (LPS-induced CXC chemokine), MCP-1 (monocyte chemotactic protein-1), MIP-2 (macrophage inflammatory protein 2), G-CSF (granulocyte colony-stimulating factor) and KC (keratinocyte chemo-attractant) were significantly inhibited both at baseline and after LPS stimulation. The anti-inflammatory effect of pioglitazone was the result of a direct activation of PPAR-γ receptor, as shown by reversal of pioglitazone-induced protection in the presence of the PPAR-γ antagonist GW9662. Finally, to understand the mechanism by which ligand-activated PPAR-γ blocks inflammation in CF cholangiocytes we analyzed the effect of pioglitazone on NF-kB activation pathway steps, and found that pioglitazone up-regulated IkB-α, a negative regulator of NF-kB. In conclusion, our results indicate that a decreased PPAR-γ function caused by a decreased availability of PPAR-γ endogenous agonists might be responsible for the chronic inflammatory state of CFTR-defective cholangiocytes. Stimulation of PPAR-γ signaling by the agonist pioglitazone is able to decrease NF-kB-dependent inflammatory responses by increasing IkB-α, thus may represent a novel strategy to limit inflammation in CFLD. Studies in mice models of liver damage in CFLD have been planned and may represent a proof of concept for anti-inflammatory treatment in CF.

El purinoma está formado por la red de proteínas de membrana que median el efecto biológico final de la adenosina (Ado) extracelular y sus metabolitos (ATP, ADP y AMP). Sus componentes actúan de forma sinérgica y concertada para iniciar, mantener y terminar la señal purinérgica. Los elementos que conforman esta red son: los receptores purinérgicos P1 de adenosina, los receptores purinérgicos P2 para ATP y ADP, las ATP/ADPasas (nucleotidasas) responsables de la degradación extracelular del ATP y ADP, y los transportadores de nucleósidos equilibrativos (ENTs) y concentrativos (CNTs) encargados del reciclaje de la adenosina. Los colangiocitos son células epiteliales que conforman el tracto biliar intra y extrahepático y se distribuyen en una red tridimensional de ductos interconectados. Su principal función es la de sensar y modificar la composición de la bilis proveniente de los cálculos biliares. Pero también participan junto a los hepatocitos en la detoxificación de xenobióticos. A un cuando los principales mecanismos efectores de estas funciones están directamente relacionados con el ATP extracelular y activación de los receptores P2, nada ha sido descrito para el resto de los componentes del purinoma. En los últimos años, el interés en el estudio de la fisiología de los colangiocitos ha aumentado significativamente debido al incremento progresivo en la incidencia de las enfermedades hepáticas colestásicas y del cáncer de tracto biliar a nivel mundial. El colangiocarcinoma (CC) proveniente de la transformación neoplásica de los colangiocitos, es el principal cáncer de conducto biliar. Debido a que es un cáncer de detección tardía, la principal opción para el tratamiento es la quimioterapia. Entre los agentes quimioterapéuticos más estudiados para su uso en monoterapia se encuentran derivados de uracilo como el 5‐Fluoruracilo (5‐FU), o derivados de nucleósidos como la citidina (gemcitabina), o de la uridina (capecitabina); también se utilizan agentes como el Cisplatino. Estos fármacos se utilizan también en combinaciones para aumentar la efectividad de la terapia. A pesar de que en otros modelos epiteliales humanos se ha demostrado que el transporte de los quimioterápicos, análogos de nucleósidos, al interior celular se hace por medio de NT, en la actualidad no existe ninguna evidencia ni de los mecanismos de entrada, ni de las entidades que median estos procesos en CC o en modelos no tumorales de colangiocitos. Los resultados principales de esta tesis muestran la presencia funcional de nuevos elementos del purinoma tanto en colangiocitos de rata, como en líneas celulares humanas de CC. En el modelo fisiológico de colangiocitos, describimos un crosstalk específico entre los transportadores de adenosina (CNT3 y CNT2), el receptor de alta afinidad para adenosina (A2A) y el receptor de ATP (P2Y2). Estos resultados indican que los receptores A2A y P2Y2, a través de un mecanismo dependiente de la vía intracelular dependiente de cAMP/PKA/ERK/CREB, están controlando la actividad de CNT3 y CNT2 y por lo tanto la recaptación de adenosina. Adicionalmente, confirmamos por primera vez que la entrada de la adenosina es diferencialmente modulada por los principales estímulos secretores del colangiocito (secretina y ATP). En el segundo capítulo, describimos los resultados del estudio extensivo de la expresión y actividad de los transportadores de nucleósidos en 8 biopsias humanas con diagnóstico confirmado de CC y en 8 líneas celulares humanas de CC, para la evaluación de cambios en la expresión luego de la exposición a diferentes régimenes de tratamientos con fármacos utilizados en la terapia del CC. Para complementar estos resultados también analizamos la expresión basal de 40 genes involucrados en la captación, metabolización y salida de estos fármacos. Los principales resultados revelaron que la expresión de MRP3 y OATP3 podrían correlacionar con la respuesta a la quimioterapia del CC.
The purinoma is the molecular network that mediates the final effects of the extracellular adenosine and its metabolites. The components of this network are: P1 receptors for adenosine, P2 receptors for ATP/ADP, the ATP/ADPasas responsible for extracellular degradation of ATP, and the equilibrative (ENTs) and concentrative (CNTs) nucleoside tranporters responsible for the recycling of adenosine. Cholangiocytes are epithelial cells that form the intra and extrahepatic biliary tract. In recent years, interest in the study of the physiology of cholangiocytes has increased signicantly due to the progressive increment in the incidence of cholestatic liver diseases and cholangiocarcinoma (CC) worldwide. The nucleoside‐derivatives drugs; 5‐Fluorouracil and gemcitabine, are the principal therapy indicated for the treatment of CC. Although in other human epithelial models have been shown that transport of nucleosideanalogues drugs into the cell is done by means of the nucleoside transporters, at the moment there is no evidence of the uptake mechanisms, nor the proteins that mediate these processes in CC or in non‐tumor models of the biliary epithelia. The principal results in this thesis showed the presence of new elements of the purinome in both rat cholangiocytes and in human CC cell lines. In the physiologic model of cholangiocytes we found a specific crosstalk between the adenosine transporters (CNT3 and CNT2), the adenosine A2A receptor and the P2Y2 receptor. Our key findings indicated that A2A and P2Y2 receptors, which trough a mechanism dependent of the cAMP/PKA/ERK/CREB axis, are controlling the activity of CNT3 and CNT2 and thus the cellular uptake of adenosine. Additionally, for the first time we confirmed that adenosine transport is differentially modulated by the main secretory stimuli described in cholangiocytes (secretin and ATP). We also performed an extensive study of the expression and activity of the nucleoside transporters in 8 cell lines and in 8 human biopsies of CC, evaluating changes after the exposure to different cancer drugs. We also analyzed the basal expression of 40 genes involved in the uptake, metabolism and extrusion of these drugs. The principal results of this approach revealed that the expression of MRP3 and OATP3 would possibly correlate with the response to the chemotherapy in CC.

The biliary epithelium is organized as a single layer of biliary epithelial cells (BECs) lining the biliary tree. BECs have three major biological functions: protection, secretion and proliferation. These functions may all be controlled by bile salts, the main constituents of bile. We therefore determined if human gallbladder-derived BECs exhibited bile salt transport activities that affect their secretory functions. We could show that the apical bile acid sodium- dependent transporter (ASBT) is expressed and functional in primary cultures of human BECs. Once transported inside BECs, bile salts stimulate chloride and mucin secretion through intracellular pathways linked to calcium. In BECs, the secretion process is however mainly elicited by membrane receptors that signal through the intracellular second messenger cAMP. We could show in a subsequent study that bile salts potentiate cAMP-regulated secretion in BECs by stimulating adenylyl cyclase activity through PKC α and δ. One of the most potent bile secretagogues is the vasoactive intestinal peptide, which exerts its effects through the vasoactive intestinal peptide receptor-1 (VPAC1). We have shown that VPAC1 is expressed in all major cell types participating in bile formation in humans (i.e. hepatocytes, intrahepatic and gallbladder biliary epithelial cells). Moreover, VPAC1 displays a gradient of expression along the human biliary tree, the gallbladder showing the highest level of expression. Interestingly, we could show that bile salts regulate VPAC1 expression in BECs through the farnesoid X receptor (FXR), indicating that bile salts may also regulate BECs secretion through transcriptional control. The secretory activities of BECs may be seen as a protective mechanism avoiding excessive exposure to toxics, such as endotoxins. We have shown that bile salts may also exert protection by controlling the expression of the antimicrobial peptide cathelicidin. Bile salts induced cathelicidin expression through two distinct nuclear receptors, FXR and the vitamin D nuclear receptor (VDR). Taken together, our results indicate that bile salts control BECs biological functions through post-traductional and transcriptional control. The involvement of VDR in the control of BECs biology may be of particular interest because VDR hepatic expression is restricted to non-parenchymal liver cells, such as BECs or resident hepatic macrophages (i.e. Kupffer cells).

Senescence and its associated secretory phenotype have been investigated in several vanishing bile duct syndromes. The current study evaluated the presence of senescent biliary epithelial cells (BEC) in acute cellular rejection of human liver allografts to ascertain whether senescent cells contribute to human disease progression in liver transplantation. There was a significant correlation between senescent BEC and grade of rejection. Furthermore there was a significant correlation between grade of rejection and BEC undergoing epithelial to mesenchymal transition (EMT). There was never any overlap between senescence and EMT markers in BEC. Further investigation of the association between senescence and EMT in vitro using both primary and immortalised human BEC exposed to oxidative stress showed, for the first time, that TGF-­b2 is part of the Senescence Associated Secretory Phenotype (SASP) in liver disease. Blockade of TGF-­b signalling by inhibition of the TGFbR, prevented any of the oxidative stress-­induced changes in BEC. Blockade of integrin aVb6 integrin also showed a variable ability to prevent TGF-­b mediated changes in BEC. HGF and its mimetic, 1K1, were able to prevent oxidative stress induced EMT in BEC. Furthermore 1K1 showed a smaller induction of autophagy than HGF and was able to prevent up regulation of senescence markers. The paradigm of oxidative stress-­induced senescence leading to EMT was assessed in the current study, with identification of powerful therapeutic agents able to prevent these changes. This suggests that premature cellular ageing (senescence) in acute liver disease could be pharmacologically prevented from occurring. Inhibition of senescence prevents disease promotion by the effects of the SASP, blocking the progression to chronic disease; this may well apply to all organ systems.

博士
國立陽明大學
臨床醫學研究所
89
Biliary atresia (BA) is the most puzzled disease in the field of pediatric surgery. Clinical manifestations are neonatal jaundice, direct hyperbilirubinemia, passage of clay stool and obliteration of extrahepatic bile duct. This disease was incurable until Dr. Kasai, a Japanese surgeon, reported his portoenterostomy in 1957, which was the procedure of excision of extrahepatic fibrotic bile duct to liver hilum and roux-en-y portojejunostomy. During the past four decades, many investigations have been made to figure out the etiologies of BA and the predictors for post-Kasai outcome. There are many hypotheses about the etiology, including congenital ductal plate malformation, viral infection, immunological/inflammatory system disorder and defect in fetal/perinatal circulation. There are also many predictors for post-Kasai outcome, including age at Kasai operation, diameter of bile duct at hilum, severity of liver fibrosis and the presentation of some inflammatory mediators on hepatocytes and intrahepatic bile ductule epithelia. In this study, we try to further study the etiology and clinical predictors of BA. Most studies on BA focused on the extrahepatic bile duct and intrahepatic bile duct and ductule, very few studies discussed about bile canaliculi, which is the most cephalic part of bile secretory apparatus. The hepatocyte is known as a polarized epithelial cell, composed of sinusoid, basolateral and bile canalicular domains. Previous studies used monoclonal antibodies (9B2), shown to react with the antigens on the bile canalicular domain of human hepatocytes, and the human hepatoma cell lines as in vitro model to study the biliary polarity of human hepatocytes. In Hep G2 and HuH-7, known as well differentiated hepatoma cell lines, the 9B2 antigen could be located on the bile canaliculus structure; whereas in HA22T/VGH and SK-HEP-1, known as poorly differentiated lines, no 9B2 antigen located on the bile canaliculus structure. In this study, we hypothesize the bile canaliculus of human fetal hepatocytes is progressively developed and the under-developed status could be found in some cases of BA. Congenital malformation could be one of the causative factors of BA in the cases with under-development of bile canaliculi while the postnatal insults should be responsible for the cases without under-development of bile canaliculi. When comparing the expression of 9B2 antigens in the livers of 20-weeks fetuses, premature babies, term neonates and children by means of semiquantitative analysis of immunohistochemistry staining and quantitative analysis of flow cytometry, we find that bile canaliculi are well developed in term neonates, under-developed in premature babies and even less developed in 20 week fetuses. We also find the fact that obstructive jaundice rather than hepatitis will induce 9B2 antigen expression stronger by examining the livers of choledochal cysts and neonatal hepatitis. In the livers of BA patients, some cases have much stronger 9B2 expression than term neonates, some cases have about the same expressive intensity as term neonates but a few cases have the intensity as premature babies. We also find that the more intensive expression of 9B2 antigens, the higher failure rate of Kasai operation. Based on the results, we suggest the intensity of 9B2 expression in the pre-Kasai livers could reflect the severity and duration of extrahepatic obstruction before Kasai operation. In the cases showing premature development of bile canaliculi, congenital malformation may be a causative factor of BA. The disorder of immunological system had been proposed to be one of the etiologies in BA. Many reports indicated that apoptosis induced by Fas/Fas ligand (FasL) system may play a role on various types of hepatitis and some animal studies indicated ligation of extrahepatic bile duct would cause hepatocytes apoptosis by Fas/FasL system. So, we question whether the Fas/FasL system is involved in the persistent liver damage of BA by examining the expression of FasL proteins and FasL mRNA on the livers of BA patients. We find that positive expression of FasL protein and mRNA is noted on some intrahepatic bile ductule epithelia in some BA patients. In these cases, apoptosis of intrahepatic bile ductule epithelia is noted and larger amount of apoptosis of infiltrating lymphocytes as compared with the cases without expression of FasL on bile ductule epithelia. We also note that the post-Kasai outcome in the cases with positive FasL expression had a significantly higher failure rate. This result suggests intrahepatic bile ductule epithelia could secret FasL to attack the infiltrating lymphocytes and result in apoptotic suicide/fratricide and these leads to persistent destruction of intrahepatic bile ductule after Kasai-operation. Although Fas/ FasL system should not be the initiating cause of BA, it may play a significant role in the pathogenesis of BA. Very interestingly, in the cases of BA with under- development of 9B2, there is neither expression of FasL on intrahepatic bile ductile epithelia nor the phenomenon of apoptosis. From our study, we conclude that the etiology of BA is multiple and congenital malformation and post-natal disorder of immunological system may play a role in the pathogenesis. Both the expression intensity of 9B2 and FasL on bile ductule epithelia could serve as the prognostic factors of Kasai operation.