Differential effects of membrane cholesterol content on the transport activity of multidrug resistance-associated protein 2 (ABCC2) and of the bile salt export pump (ABCB11).

Rat canalicular membranes contain microdomains enriched in cholesterol and ATP-binding cassette transporters. Cholesterol is known to regulate the activity of transporters. Here, we investigated the effect of membrane cholesterol on the transport kinetics of multidrug resistance-associated protein 2 (MRP2) and of bile salt export pump (BSEP) variants and mutants. MRP2 and BSEP were expressed with baculoviruses in insect cells, followed by vesicle isolation from control and cholesterol-loaded cells (1 mM cholesterol@randomly methylated-ß-cyclodextrin) for transport assays. We found that cholesterol stimulates MRP2 transport activity for substrates of different molecular weights: estradiol-17-ß-glucuronide (E17ßG), prostaglandin E2 (PGE2), cholecystokinin 8 (CCK8), and vasopressin displayed an increase of Vmax and a variable decrease of Km. Kinetics of E17ßG showed a sigmoidal shape and a mild cooperativity in Hanes-Woolf plots in control membranes. High cholesterol content shifted E17ßG to Michaelis-Menten kinetics. PGE2/glutathione transport followed Michaelis-Menten kinetics irrespective of cholesterol. The MRP2 substrates CCK8 and vasopressin exhibited Michaelis-Menten kinetics independent of membrane cholesterol content. Transport of ochratoxin A was ATP-dependent but was neither mediated by MRP2 nor stimulated by cholesterol. Transport of the two most common BSEP variants p.444V/A showed Michaelis-Menten kinetics irrespective of membrane cholesterol, whereby cholesterol leads to an increased Vmax while Km remains unchanged. The transport activity of the BSEP mutants p.E297G and p.R432T increased at high cholesterol content but did not reach the capacity of normal BSEP. Hence, changing membrane cholesterol content modulates BSEP and MRP2 transport kinetics differently. Cholesterol increases the transport rates of BSEP and MRP2, but with the latter, may also modify the binding site as for E17ßG.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Smoothelin, a new marker to determine the origin of liver fibrogenic cells.

AIM: To explore this hypothesis that smooth muscle cells may be capable of acquiring a myofibroblastic phenotype, we have studied the expression of smoothelin in fibrotic conditions. METHODS: Normal liver tissue (n = 3) was obtained from macroscopically normal parts of hepatectomy, taken at a distance from hemangiomas. Pathological specimens included post-burn cutaneous hypertrophic scars (n = 3), fibrotic liver tissue (n = 5), cirrhotic tissue (viral and alcoholic hepatitis) (n = 5), and hepatocellular carcinomas (n = 5). Tissue samples were fixed in 10% formalin and embedded in paraffin for immunohistochemistry or were immediately frozen in liquid nitrogen-cooled isopentane for confocal microscopy analysis. Sections were stained with antibodies against smoothelin, which is expressed exclusively by smooth muscle cells, and α-smooth muscle actin, which is expressed by both smooth muscle cells and myofibroblasts. RESULTS: In hypertrophic scars, α-smooth muscle actin was detected in vascular smooth muscle cells and in numerous myofibroblasts present in and around nodules, whereas smoothelin was exclusively expressed in vascular smooth muscle cells. In the normal liver, vascular smooth muscle cells were the only cells that express α-smooth muscle actin and smoothelin. In fibrotic areas of the liver, myofibroblasts expressing α-smooth muscle actin were detected. Myofibroblasts co-expressing α-smooth muscle actin and smoothelin were observed, and their number was slightly increased in parallel with the degree of fibrosis (absent in liver with mild or moderate fibrosis; 5% to 10% positive in liver showing severe fibrosis). In cirrhotic septa, numerous myofibroblasts co-expressed α-smooth muscle actin and smoothelin (more than 50%). In hepatocellular carcinomas, the same pattern of expression for α-smooth muscle actin and smoothelin was observed in the stroma reaction surrounding the tumor and around tumoral cell plates. In all pathological liver samples, α-smooth muscle actin and smoothelin were co-expressed in vascular smooth muscle cells. CONCLUSION: During development of advanced liver fibrosis, a subpopulation of myofibroblasts expressing smoothelin may be derived from vascular smooth muscle cells, illustrating the different cellular origins of myofibroblasts.

An Arabidopsis T-DNA insertion mutant for galactokinase (AtGALK, At3g06580) hyperaccumulates free galactose and is insensitive to exogenous galactose.

Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of α-d-galactose (Gal) to α-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g(-1) FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g(-1) FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant background.

Interaction of bile salts with rat canalicular membrane vesicles: evidence for bile salt resistant microdomains.

BACKGROUND & AIMS: Canalicular phosphatidylcholine and cholesterol secretion requires the coordinate action of the ATP binding cassette transporters: the bile salt export pump (Bsep) for bile salts (BS) and the phosphatidylcholine translocator multidrug resistance protein 2 (Mdr2). After their secretion, phosphatidylcholine and BS form mixed micelles acting as acceptors for canalicular cholesterol. We have shown that the canalicular liver plasma membrane (cLPM) contains lipid raft enriched in sphingomyelin and cholesterol. As BS have detergent properties and their concentration in the canaliculus is very high, we tested the hypothesis that the canalicular membrane contains BS resistant microdomains. METHODS: Isolated cLPMs were extracted at 4°C with different BS or detergents and subjected to flotation in sucrose step gradients followed by Western blotting and lipid composition analysis. RESULTS: Incubating cLPMs with increasing taurocholate concentrations revealed the presence of BS resistant microdomains. These microdomains were found with different BS in the presence and absence of lipids and contained the raft markers reggie-1/-2 and caveolin-1 and canalicular transporters Bsep, Mrp2, and Abcg5, the latter independent of the presence of lipids. BS resistant microdomains contain mainly cholesterol, phosphatidylcholine, and phosphatidylethanolamine. Extraction of cLPMs with a mixture of different BS similar to rat bile revealed a comparable microdomain composition. CONCLUSIONS: cLPM contains BS resistant microdomains potentially protecting the cLPM against the detergent action of BS. Combination of different BS has no synergistic effect on microdomain composition.

Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices.

BACKGROUND: The debate concerning the potential remodelling and/or reversibility of cirrhotic lesions and biliary fibrosis is still open. AIMS/METHODS: In this work, we have used the precision-cut liver slice (PCLS) model, which maintains cell-cell and cell-matrix interactions to study, by immunohistochemistry, the behaviour of the different fibrogenic cells, i.e. hepatic stellate cells (HSC) and portal fibroblasts, in cultured (for 1 week) PCLS derived from normal and fibrotic human livers. RESULTS: In normal liver, before and after culture, α-smooth muscle (SM) actin was present only in the vessel walls. Platelet-derived growth factor (PDGF) receptor-ß was expressed before and after culture by portal fibroblasts, and appeared after culture in HSC. Before culture, CD 34 was not expressed in parenchyma, but appeared after culture in sinusoidal endothelial cells. In cirrhotic lesions, before culture, α-SM actin, PDGF receptor-ß and Thy-1 were expressed in septa; after culture, α-SM actin expression disappeared but the expression of the PDGF receptor-ß and Thy-1 was maintained. In cholestatic liver specimens, α-SM actin, PDGF receptor-ß and Thy-1 expression, which was present before culture in enlarged portal areas, disappeared after culture, and apoptosis was detected. In the parenchyma of both cirrhotic and cholestatic livers, the expression of the PDGF receptor-ß and of CD 34, which was not observed before culture, was present in HSC and sinusoidal endothelial cells, respectively, after culture. CONCLUSIONS: These results indicate that during remodelling of pathological tissues in cultured liver slices, the myofibroblastic cells derived from HSC or from portal fibroblasts show different behaviours, suggesting different mechanisms of activation/deactivation.

ABC-transporters are localized in caveolin-1-positive and reggie-1-negative and reggie-2-negative microdomains of the canalicular membrane in rat hepatocytes.

UNLABELLED: The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4 degrees C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. CONCLUSION: These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: "Lubrol-microdomains" and "Triton-microdomains". "Lubrol-microdomains" contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion.

Specific activation of the different fibrogenic cells in rat cultured liver slices mimicking in vivo situations.

Due to the loss of cell-cell and cell-matrix interactions, cell culture models poorly mimic the in vivo situation. Therefore, we tested the applicability of precision-cut liver slices (PCLS) to study the early activation of the two main liver fibrogenic cell subpopulations: hepatic stellate cells (HSC) and portal fibroblasts (PF). PCLS were treated with thioacetamide or acetaminophen to induce HSC activation. In PCLS culture, both were able to trigger centrolobular lesion and HSC activation as observed in vivo. However, thioacetamide also presented a toxic effect on portal tract cells. In this PCLS model of centrolobular lesion, the antioxidant N-acetylcysteine was able to prevent acetaminophen-induced injury. To induce a specific activation of PF, PCLS were treated with epidermal growth factor or beta-oestradiol. As in vivo, epidermal growth factor and beta-oestradiol induced bile duct epithelial cell proliferation accompanied by PF activation; however, beta-oestradiol also triggers sinusoidal cell proliferation. We demonstrated that treatments usually used in vivo to induce liver fibrosis allow, in cultured PCLS, the specific activation of the two main liver fibrogenic cell subpopulations, making this model very useful to study the mechanisms involved in early fibrogenic cell activation.

Fibrogenic cell fate during fibrotic tissue remodelling observed in rat and human cultured liver slices.

BACKGROUND/AIMS: Fibrotic liver remodelling was studied in culture of precision-cut liver slices (PCLS) derived from fibrotic liver. METHODS: Fibrosis was induced in rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. Human fibrotic livers were also used. PCLS were cultured for 6, 24, 48, or 72 h, and the expression of alpha-smooth muscle (SM) actin, platelet-derived growth factor (PDGF) receptor-beta, and active caspase 3 was studied by immunohistochemistry. RESULTS: Before culture, in CCl4-treated or bile duct ligated animals, fibrosis was observed around centrolobular veins, or in portal zones, respectively. In PCLS derived from CCl4-treated animals, alpha-SM actin expression disappeared after 24h in culture while PDGF receptor-beta expression decreased progressively after 48 h. These changes were observed in absence of massive apoptosis. In PCLS derived from bile duct ligated animals, both alpha-SM actin and PDGF receptor-beta expression decreased after 48 h in culture with a massive apoptosis. In PCLS derived from human fibrotic livers, alpha-SM actin expression was dramatically reduced after 48 h in culture. CONCLUSIONS: After CCl4 treatment, a proportion of myofibroblasts derived from hepatic stellate cells seems to dedifferentiate while in bile duct ligation model, myofibroblasts derived from portal fibroblasts disappear by apoptosis, underlining the relevance of this model to evaluate the mechanisms involved in fibrotic liver remodelling.

The common bile duct ligation in rat: A relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour.

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing alpha-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.

Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices.

During cholestasis, bile acids accumulate in the liver, and induce cellular alterations. Cholestasis is a major cause of liver fibrosis. We have used precision-cut liver slices (PCLS) in culture to investigate the effects of bile acids on hepatic cells. Rat PCLS were placed on an insert in a vial containing culture medium, and gently agitated on a roller platform. PCLS were treated with 100 microM taurolithocholate (TLC), taurodeoxycholate (TDC) or taurocholate (TC) for 24 or 48 h. PCLS viability was measured, and immunohistochemistry was performed with antibodies against active caspase 3, platelet-derived growth factor (PDGF) receptor-beta and ED-A fibronectin. TDC and TLC, two hydrophobic bile acids, induced hepatocyte necrosis and apoptosis, whereas TC, an hydrophilic bile acid, improved slice viability as compared with controls. Both TDC and TC induced biliary epithelial cell proliferation, together with portal fibroblast proliferation and activation, as shown by PDGF receptor-beta and ED-A fibronectin expression. TLC induced biliary epithelial cell apoptosis. Our results indicate that individual bile acids induce cell type-specific effects in a complex liver microenvironment. The fact that PCLS support biliary epithelial cell and portal fibroblast proliferation will make this model very useful for the study of the mechanisms involved in portal fibrosis.

Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved.

Fibrosis, defined as the excessive deposition of extracellular matrix in an organ, is the main complication of chronic liver damage. Its endpoint is cirrhosis, which is responsible for significant morbidity and mortality. The accumulation of extracellular matrix observed in fibrosis and cirrhosis is due to the activation of fibroblasts, which acquire a myofibroblastic phenotype. Myofibroblasts are absent from normal liver. They are produced by the activation of precursor cells, such as hepatic stellate cells and portal fibroblasts. These fibrogenic cells are distributed differently in the hepatic lobule: the hepatic stellate cells resemble pericytes and are located along the sinusoids, in the Disse space between the endothelium and the hepatocytes, whereas the portal fibroblasts are embedded in the portal tract connective tissue around portal structures (vessels and biliary structures). Differences have been reported between these two fibrogenic cell populations, in the mechanisms leading to myofibroblastic differentiation, activation and "deactivation", but confirmation is required. Second-layer cells surrounding centrolobular veins, fibroblasts present in the Glisson capsule surrounding the liver, and vascular smooth muscle cells may also express a myofibroblastic phenotype and may be involved in fibrogenesis. It is now widely accepted that the various types of lesion (e.g., lesions caused by alcohol abuse and viral hepatitis) leading to liver fibrosis involve specific fibrogenic cell subpopulations. The biological and biochemical characterisation of these cells is thus essential if we are to understand the mechanisms underlying the progressive development of excessive scarring in the liver. These cells also differ in proliferative and apoptotic capacity, at least in vitro. All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development of fibrosis/cirrhosis.

The stroma reaction myofibroblast: a key player in the control of tumor cell behavior.

The cooperation between epithelial and mesenchymal cells is essential for embryonic development and probably plays an important role in pathological phenomena such as wound healing and tumor progression. It is well known that many epithelial tumors are characterized by the local accumulation of connective tissue cells and extracellular material; this phenomenon has been called the stroma reaction. One of the cellular components of the stroma reaction is the myofibroblast, a modulated fibroblast which has acquired the capacity to neoexpress alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells, and to synthesize important amounts of collagen and other extracellular matrix components. It is now well accepted that the myofibroblast is a key cell for the connective tissue remodeling which takes place during wound healing and fibrosis development. Myofibroblasts are capable of remodeling connective tissue but also interact with epithelial cells and other connective tissue cells and may thus control such phenomena as tumor invasion and angiogenesis. In this review we discuss the mechanisms of myofibroblast evolution during fibrotic and malignant conditions and the interaction of myofibroblasts with other cells in order to control tumor progression. On this basis we suggest that the myofibroblast may represent a new important target of antitumor therapy.