Cholestan-3beta,5alpha,6beta-triol, but not 7-ketocholesterol, suppresses taurocholate-induced mucin secretion by cultured dog gallbladder epithelial cells

In order to investigate oxysterol-mediated effects on the biliary system, we studied the effects of cholestan-3beta,5alpha,6beta-triol (TriolC) and 7-ketocholesterol (7KC) on gallbladder epithelial cells. We compared their cell proliferation effects in cultured dog gallbladder epithelial cells (DGBE) to their effects in cultured human pulmonary artery endothelial cells (HPAE). Oxysterols inhibited cell proliferation in a dose-dependent fashion. Oxysterols inhibited cell growth to 50% of control at a higher dose for DGBE cells than for HPAE cells. TriolC was more cytotoxic than 7KC. We also investigated the effect of oxysterols on bile salt-induced mucin secretion by DGBE cells. TriolC suppressed mucin secretion by DGBE cells, whereas 7KC did not. These findings support the hypothesis that biliary oxysterols affect gallbladder mucosal function.     

Oxysterols from human bile induce apoptosis of canine gallbladder epithelial cells in monolayer culture

Oxysterols have been detected in various mammalian organs and blood. Biliary epithelium is exposed to high concentrations of cholesterol, and we have identified three keto-oxysterols (cholest-4-en-3-one, cholesta-4,6-dien-3-one, cholesta-3,5-dien-7-one) in human bile and gallstones. Because the effects of oxysterols on biliary physiology are not well defined, we investigated their biological effects on dog gallbladder epithelial cells. Enriched medium (culture medium containing taurocholate and lecithin and cholesterol +/- various oxysterols) was applied to confluent monolayers of dog gallbladder epithelial cells in culture. Cytotoxicity and apoptosis were studied by morphological analysis and flow cytometry. Oxysterols in the mitochondrial fraction were identified by gas chromatography/mass spectrometry, whereas release of cytochrome c from mitochondria was assayed by spectrophotometry and Western blot analysis. Compared with cells treated with culture medium or with enriched medium containing cholesterol, oxysterol-treated cells showed significantly increased apoptosis (P < 0.05). Exogenously applied oxysterols were recovered from the mitochondrial fraction. Cytochrome c release from mitochondria was increased significantly by cholest-4-en-3-one, cholesta-4,6-dien-3-one, and 5beta-cholestan-3-one (all P < 0.05). Thus oxysterols recovered from human bile and gallstones induce apoptosis of biliary epithelium via a mitochondrial-dependent pathway and may play a role in the pathogenesis of chronic inflammation and carcinogenesis in the gallbladder.     

Expression of oxysterol binding protein isoforms in opisthorchiasis-associated cholangiocarcinoma: A potential molecular marker for tumor metastasis

Oxysterols are oxygenated derivatives of cholesterol generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by inflammation-associated non-enzymatic reactions. Oxysterol binding proteins (OSBPs) are cytosolic high affinity receptors for oxysterols. We previously found that OSBPL-8 is upregulated in liver fluke (Opisthorchis viverrini)-induced hamster cholangiocarcinoma (CCA). Our aims were to determine the expression patterns of OSBP isoforms in human CCA tissues and to evaluate whether OSBPs could be used as molecular markers for the identification of blood-borne CCA metastasis. Expression levels of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 in CCA tissues were detected using qRT-PCR and immunohistochemistry. Expression of OSBPs at mRNA level in the blood of CCA patients was also investigated. We confirmed increased expression of OSBPL-8 in O. viverrini -induced hamster CCA tissues. Moreover, increased expression of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 was seen in human CCA tissues. Notably, a significant increased level of OSBPL-7 mRNA was observed in tumor compared to non-tumor liver tissue. Immunohistochemistry supported the mRNA results, in that OSBPL-7 protein was over-expressed in cancer cells and hepatocytes but not in normal biliary cells and surrounding inflammatory cells. Interestingly, in our preliminary results, significantly higher levels of OSBP2 and OSBPL-7 mRNA were seen in blood samples from CCA patients than in healthy controls. These results suggest that OSBP2 and OSBPL-7 might serve as molecular markers for the identification of CCA metastasis in the bloodstream.     

Opisthorchis viverrini excretory/secretory products induce toll-like receptor 4 upregulation and production of interleukin 6 and 8 in cholangiocyte

Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma--a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses is unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of pro-inflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma.     

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand

Introduction

Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma.

Aims

To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines.

Results

Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both.

Conclusions

1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.     

Trojan horse-like behavior of a biologically representative mixture of oxysterols

Oxysterols, 27-carbon atoms cholesterol oxidation products, are consistently detectable in minimally oxidized low density lipoproteins (oxLDLs) and accumulate in the core of fibrotic plaques. Several oxysterols of pathophysiological interest have been shown to possess many and diverse biochemical activities. In particular, 7-ketocholesterol (7K), a major cholesterol oxide both in oxLDLs and in atherosclerotic lesions, is able to lead vascular cells to apoptosis. Indeed, when 7K is added to cells of the macrophage lineage, in a concentration range actually detectable in hypercholesterolemic patients, a marked apoptotic effect was observed. However, when identical concentrations of 7K are given to the same cells in a mixture with other oxysterols, also detectable in human low density lipoprotein (LDL), cell apoptosis was dramatically reduced. Of note, identical amounts of unoxidized cholesterol did not show any significant pro-apoptotic effect. With the aim to investigate the mechanisms underlying the quenching of 7K-dependent apoptosis by the oxysterol mixture, we found that the combined oxysterol mixture counteracted the ability of 7K given alone to strongly increase the steady-state level of reactive oxygen species (ROS) in macrophages as well as the up-regulation of the pro-apoptotic factor p21 and the triggering of the mitochondria-dependent pathway of apoptosis. Competition among oxysterols, apparently at NADPH oxidase level, diminishes the macrophage ROS production and direct toxicity that is evoked by defined oxysterols, as for instance, 7-ketocholesterol.     

Characterization of regulatory volume decrease in freshly isolated mouse cholangiocytes

Cell volume regulation plays a vital role in many cell functions. Recent study indicates that both K(+) and Cl(-) channels are important for the regulatory volume decrease (RVD) of cholangiocarcinoma cells, but its physiological significance is unclear due to the tumorous nature of the cells used. This present study reports the RVD of normal mouse cholangiocytes by using freshly isolated bile duct cell clusters (BDCC). A relatively simple and practical method of measuring the cross-sectional area of BDCCs by quantitative videomicroscopy was used to indirectly measure their volumes. Mouse cholangiocytes exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, DIDS, and glibenclamide, suggesting its dependence on certain chloride channels, such as volume-activated chloride channels. It is also inhibited by barium chloride but not by tetraethylammonium chloride, indicating its dependence on certain potassium channels. However, cAMP agonists had no significant effect on the RVD of BDCCs. This indirect method described can be used to study the RVD of cholangiocytes from normal as well as genetically altered mouse livers.     

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.     

Cholestane-3β, 5α, 6β-triol Suppresses Proliferation,Migration, and Invasion of Human Prostate Cancer Cells

Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27^Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.     

Apoptosis of cholangiocytes modulated by thioredoxin of carcinogenic liver fluke

Chronic infection with the food-borne liver fluke, Opisthorchis viverrini, frequently induces cancer of the bile ducts, cholangiocarcinoma. Opisthorchiasis is endemic in Thailand, Lao PDR, Cambodia and Vietnam, where eating undercooked freshwater fish carrying the juvenile stage of this pathogen leads to human infection. Because inhibition of apoptosis facilitates carcinogenesis, this study investigated modulation by thioredoxin from O. viverrini of apoptosis of bile duct epithelial cells, cholangiocytes. Cells of a cholangiocyte line were incubated with the parasite enzyme after which they were exposed hydrogen peroxide. Oxidative stress-induced apoptosis was monitored using flow cytometry, growth in real time and imaging of living cells using laser confocal microscopy. Immunolocalization revealed liver fluke thioredoxin within cholangiocytes. Cells exposed to thioredoxin downregulated apoptotic genes in the mitogen activated protein kinases pathway and upregulated anti-apoptosis-related genes including apoptosis signaling kinase 1, caspase 9, caspase 8, caspase 3, survivin and others. Western blots of immunoprecipitates of cell lysates revealed binding of thioredoxin to apoptosis signaling kinase 1. Together the findings indicated that thioredoxin from O. viverrini inhibited oxidative stress-induced apoptosis of bile duct epithelial cells, which supports a role for this liver fluke oxidoreductase in opisthorchiasis-induced cholangiocarcinogenesis.     

Effects of bile salts on cholestan-3beta,5alpha,6beta-triol-induced apoptosis in dog gallbladder epithelial cells

Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3beta,5alpha,6beta-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14+/-11% (control) to 48+/-12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63+/-16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31+/-12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.     

Cholesterol metabolism: from lipidomics to immunology

Oxysterols, the oxidized forms of cholesterol or of its precursors, are formed in the first steps of cholesterol metabolism. Oxysterols have interested chemists, biologists, and physicians for many decades, but their exact biological relevance in vivo, other than as intermediates in bile acid biosynthesis, has long been debated. However, in the first quarter of this century, a role for side-chain oxysterols and their C-7 oxidized metabolites has been convincingly established in the immune system. 25-Hydroxycholesterol has been shown to be synthesized by macrophages in response to the activation of Toll-like receptors and to offer protection against microbial pathogens, whereas 7α,25-dihydroxycholesterol has been shown to act as a chemoattractant to lymphocytes expressing the G protein-coupled receptor Epstein-Barr virus-induced gene 2 and to be important in coordinating the action of B cells, T cells, and dendritic cells in secondary lymphoid tissue. There is a growing body of evidence that not only these two oxysterols but also many of their isomers are of importance to the proper function of the immune system. Here, we review recent findings related to the roles of oxysterols in immunology.     

Cholestane-3beta, 5alpha, 6beta-triol promotes vascular smooth muscle cells calcification

Oxysterols found in atherosclerotic plaque may be associated with vascular calcification. We investigated the effect of oxysterol cholestane-3beta, 5alpha, 6beta-triol (Triol) on in vitro calcification of rat vascular smooth muscle cells (VSMCs). In vitro calcification was induced by incubation of VSMCs with beta-glycerophosphate. Calcifying nodule formation, calcium deposition in extracellular matrix, and alkaline phosphatase (ALP) activity were measured as indices of calcification. Because apoptotic bodies can serve as nucleation sites for calcification, apoptosis of calcifying VSMCs was determined by Hoechst 33258 staining, TUNEL, and FITC-labeled annexin V/PI double staining. The calcium deposition and ALP activity in calcifying VSMCs were much higher than those in non-calcifying VSMCs. Triol increased calcifying nodule formation, calcium deposition, ALP activity, and apoptosis of nodular cells in calcifying VSMCs. As determined by 2,7-dichlorofluorescein fluorescence, Triol induced the generation of reactive oxygen species (ROS) in calcifying VSMCs dose- and time-dependently. Triol-induced increases in calcium deposition, ALP activity, apoptosis, and ROS generation were all attenuated by antioxidant vitamin C plus vitamin E (VC + VE). The results demonstrated that Triol promoted VSMCs calcification through direct increase of ALP activity and apoptosis, probably by ROS-related mechanism.     

7-Ketocholesterol and cholestane-triol increase expression of SMO and LXRα signaling pathways in a human breast cancer cell line

Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of 7-ketocholesterol (7-KC), cholestane-3β-5α-6β-triol (triol), and a mixture of 5α-cholestane-3β,6β-diol and 5α-cholestane-3β,6α-diol (diol) to promote cell death in a human breast cancer cell line (MDA-MB-231). We determined cell viability, after 24-h incubation with oxysterols. These oxysterols promoted apoptosis. At least part of the observed effects promoted by 7-KC and triol arose from an increase in the expression of the sonic hedgehog pathway mediator, smoothened. However, this increased expression was apparently independent of sonic hedgehog expression, which did not change. Moreover, these oxysterols led to increased expression of LXRα, which is involved in cellular cholesterol efflux, and the ATP-binding cassette transporters, ABCA1 and ABCG1. Diols did not affect these pathways. These results suggested that the sonic hedgehog and LXRα pathways might be involved in the apoptotic process promoted by 7-KC and triol.     

alpha-Tocopherol, but not gamma-tocopherol inhibits 7 beta-hydroxycholesterol-induced apoptosis in human U937 cells.

Oxysterols, particularly those oxidised at position 7, are toxic to cells in culture and have been shown to induce apoptosis in cell types such as vascular endothelial cells, smooth muscle cells and monocytes. The precise mechanism by which oxysterols induce apoptosis is unknown but may involve the generation of oxidative stress. In the present study we examined the ability of alpha-TOC, alpha-TOC acetate (alpha-TOCA) and gamma-TOC to protect against 7 beta-hydroxycholesterol (7 beta-OHC)-induced apoptosis of human monocytic U937 cells. 7 beta-OHC is one of the most commonly detected oxysterols in foods and its level in plasma has been positively associated with an increased risk of atherosclerosis. The present study demonstrates a significant decrease in cell membrane integrity and cellular glutathione levels when U937 cells were treated with 30 microM 7 beta-OHC. DNA fragmentation also occurred, as measured by agarose gel electrophoresis, and the number of apoptotic cells increased as assessed by nuclear morphology. Analysis by HPLC showed that there was a greater incorporation of gamma-TOC into U937 cells after a 48 h incubation, than either alpha-TOC or alpha-TOCA. However, despite the increased uptake of gamma-TOC, only alpha-TOC, and not gamma-TOC or alpha-TOCA was effective at inhibiting 7 beta-OHC-induced apoptosis in U937 cells.     

Sesqui- and diterpenes from the liverwort Gackstroemia decipiens

Six rosanes, 5 beta,11 beta-dihydroxy-ros-15-ene, 5 beta,12 beta-dihydroxy-ros-15-ene, 11 beta-hydroxy-7-oxo-rosa-5,15-diene, 1 alpha,5 beta,11 beta-trihydroxy-7-oxo-ros-15-ene, 5 beta,20-epoxy-20-hydroxy-ros-15-ene and 5 beta,20-epoxy-20-methoxy-ros-15-ene along with the enantiomer of the already reported 11 beta-hydroxy-rosa-5,15-diene and the known 5 beta-hydroxy-ros-15-ene have been isolated from the liverwort Gackstroemia decipiens. Furthermore, the sesquiterpenes 3-acetoxy-7,11-dihydroxy-farnesa-1,5,9-triene and 1 beta,10 beta-epoxy-nardosin-7,11-diene were identified. Their structures were elucidated by NMR spectroscopy.     

Effects of bile salts on cholestan-3β,5α,6β-triol-induced apoptosis in dog gallbladder epithelial cells

Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3β,5α,6β-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14±11% (control) to 48±12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63±16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31±12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.     

Lanthanum chloride suppresses oxysterol-induced ECV-304 cell apoptosis via inhibition of intracellular Ca<Superscript>2+</Superscript> concentration elevation,oxidative stress,and activation of ERK and NF-κB signaling pathways

Experimental studies have demonstrated that oral administration of lanthanum chloride (LaCl₃) inhibits the development of atherosclerosis, but the related mechanism has not been fully elucidated. Oxysterols are toxic to the vascular endothelial cells which are important in preventing the formation and progression of atheromatous plaque. In this study, we examined the effect of LaCl₃ on oxysterol cholestane-3β,5α,6β-triol (Triol)-induced apoptosis and the related mechanisms in ECV-304 cells, a presumptive endothelial cell line. Incubation with Triol resulted in apoptosis of ECV-304 cells, as determined by Hoechst 33342 staining, fluorescein isothiocyanate labeled annexin V/propidium iodide double staining, and the loss of mitochondrial membrane potential. Triol activated extracellular-signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), and inhibition of Triol-activated ERK and NF-κB signaling by specific inhibitors attenuated apoptosis induction by Triol in ECV-304 cells. Pretreatment with LaCl₃ (1 μM) for 12 h before exposure to Triol decreased Triol-mediated apoptosis as well as activation of ERK and NF-κB. In addition, Triol induced oxidative stress in ECV-304 cells, manifested by the increase of intracellular reactive oxygen species generation and malondialdehyde level, and the reduction of the content of total protein thiols and the activity of antioxidant glutathione peroxidases; LaCl₃ pretreatment significantly reversed these effects. Finally, LaCl₃ pretreatment significantly inhibited the increases of intracellular Ca²⁺ concentration induced by Triol. Our study suggests that Triol induced ECV-304 cell apoptosis, and LaCl₃ could suppress this effect probably by inhibiting intracellular Ca²⁺ concentration elevation, oxidative stress, as well as activation of ERK and NF-κB signaling pathways.     

Development of liver microtissues with functional biliary ductular network

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.     

Oxysterol-induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC- and PKA-dependent pathway

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.