Background The blood brain barrier tightly regulates the passage of molecules into the brain and becomes leaky following obstructive cholestasis. The aim of this study was to determine if increased serum bile acids observed during cholestasis permeabilize the blood brain barrier. Methods Rats underwent bile duct ligation or deoxycholic or chenodeoxycholic acid injections and blood brain barrier permeability assessed. In vitro, the permeability of rat brain microvessel endothelial cell monolayers, the expression and phosphorylation of occludin, ZO-1 and ZO-2 as well as the activity of Rac1 was assessed after treatment with plasma from cholestatic rats, or bile acid treatment, in the presence of a Rac1 inhibitor. Results Blood brain barrier permeability was increased in vivo and in vitro following bile duct ligation or treatment with bile acids. Associated with the bile acid-stimulated increase in endothelial cell monolayer permeability was elevated Rac1 activity and increased phosphorylation of occludin. Pretreatment of endothelial cell monolayers with a Rac1 inhibitor prevented the effects of bile acid treatment on occludin phosphorylation and monolayer permeability. Conclusions These data suggest that increased circulating serum bile acids may contribute to the increased permeability of the blood brain barrier seen during obstructive cholestasis via disruption of tight junctions.

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Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines, compared with normal cholangiocytes, and tissue and bile from cholangiocarcinoma patients. Specifically, there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin, respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represent the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies. [Cancer Res 2008;68(22):9184-93]

Background and objectives Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. The growth factor, progranulin, is overexpressed in a number of tumours. The study aims were to assess the expression of progranulin in cholangiocarcinoma and to determine its effects on tumour growth. Methods The expression and secretion of progranulin were evaluated in multiple cholangiocarcinoma cell lines and in clinical samples from patients with cholangiocarcinoma. The role of interleukin 6 (IL-6)-mediated signalling in the expression of progranulin was assessed using a combination of specific inhibitors and shRNA knockdown techniques. The effect of progranulin on proliferation and Akt activation and subsequent effects of FOXO1 phosphorylation were assessed in vitro. Progranulin knockdown cell lines were established, and the effects on cholangiocarcinoma growth were determined. Results Progranulin expression and secretion were upregulated in cholangiocarcinoma cell lines and tissue, which were in part via IL-6-mediated activation of the ERK1/2/RSK1/C/EBPβ pathway. Blocking any of these signalling molecules, by either pharmacological inhibitors or shRNA, prevented the IL-6-dependent activation of progranulin expression. Treatment of cholangiocarcinoma cells with recombinant progranulin increased cell proliferation in vitro by a mechanism involving Akt phosphorylation leading to phosphorylation and nuclear extrusion of FOXO1. Knockdown of progranulin expression in cholangiocarcinoma cells decreased the expression of proliferating cellular nuclear antigen, a marker of proliferative capacity, and slowed tumour growth in vivo. Conclusions Evidence is presented for a role for progranulin as a novel growth factor regulating cholangiocarcinoma growth. Specific targeting of progranulin may represent an alternative for the development of therapeutic strategies.

BackgroundAcute liver failure leads to systemic complications with one of the most dangerous being a decline in neurological function, termed hepatic encephalopathy. Neurological dysfunction is exacerbated by an increase of toxic metabolites in the brain that lead to neuroinflammation. Following various liver diseases, hepatic and circulating chemokines, such as chemokine ligand 2 (CCL2), are elevated, though their effects on the brain following acute liver injury and subsequent hepatic encephalopathy are unknown. CCL2 is known to activate microglia in other neuropathies, leading to a proinflammatory response. However, the effects of CCL2 on microglia activation and the pathogenesis of hepatic encephalopathy following acute liver injury remain to be determined.MethodsHepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) in the presence or absence of INCB 3284 dimesylate (INCB), a chemokine receptor 2 inhibitor, or C 021 dihydrochloride (C021), a chemokine receptor 4 inhibitor. Mice were monitored for neurological decline and time to coma (loss of all reflexes) was recorded. Tissue was collected at coma and used for real-time PCR, immunoblots, ELISA, or immunostaining analyses to assess the activation of microglia and consequences on pro-inflammatory cytokine expression.ResultsFollowing AOM administration, microglia activation was significantly increased in AOM-treated mice compared to controls. Concentrations of CCL2 in the liver, serum, and cortex were significantly elevated in AOM-treated mice compared to controls. Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry. Administration of INCB or C021 significantly improved the neurological outcomes of AOM-treated mice, reduced microglia activation, reduced phosphorylation of ERK1/2, and alleviated AOM-induced cytokine upregulation.ConclusionsThese findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy. Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury.

Hepatic encephalopathy (HE) is a serious neurological complication of acute and chronic liver failure. Expression of the neurosteroid/bile acid receptor TGR5 has been demonstrated in the brain and is thought to be neuroprotective. However, it is unknown how TGR5 signaling can influence the progression and associated neuroinflammation of HE. HE was induced in C57Bl/6 mice via intraperitoneal injection of azoxymethane (AOM) and tissue was collected throughout disease progression. TGR5 expression was elevated in the frontal cortex following AOM injection in mice. The cellular localization of TGR5 was found in both neurons and microglia in the cortex of C57Bl/6 mice. Central infusion of the TGR5 agonist, betulinic acid, prior to AOM injection delayed neurological decline, increased cortical cyclic adenosine monophosphate concentrations, reduced microglia activation and proliferation, and reduced proinflammatory cytokine production. Betulinic acid treatment in vitro reduced the neuronal expression of CCL2, a chemokine previously demonstrated to contribute to HE pathogenesis. Lastly, treatment of the microglia cell line EOC-20 with conditioned media from betulinic acid-treated primary neurons decreased phagocytic activity and cytokine production. Together, these data identify that activation of TGR5, which is upregulated during HE, alleviates neuroinflammation and improves outcomes of AOM-treated mice through neuron and microglia paracrine signaling.

Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca(2+)-dependent pathway involving protein kinase C, or a Ca(2+)-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH(2)-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.

Recent studies have found that vasogenic brain edema is present during hepatic encephalopathy following acute liver failure and is dependent upon increased matrix metalloproteinase 9 (MMP9) activity and downregulation of tight junction proteins. Furthermore, circulating transforming growth factor β1 (TGFβ1) is increased following liver damage and may promote endothelial cell permeability. This study aimed to assess if increased circulating TGFβ1 drives changes in tight junction protein expression and MMP9 activity following acute liver failure. Blood-brain barrier permeability was assessed in azoxymethane (AOM)-treated mice at 6, 12, and 18 hours post-injection via Evan’s blue extravasation. Monolayers of immortalized mouse brain endothelial cells (bEnd.3) were treated with recombinant TGFβ1 (rTGFβ1) and permeability to fluorescein isothiocyanate-dextran (FITC-dextran), MMP9 and claudin-5 expression were assessed. Antagonism of TGFβ1 signaling was performed in vivo to determine its role in blood-brain barrier permeability. Blood-brain barrier permeability was increased in mice at 18 hours following AOM injection. Treatment of bEnd.3 cells with rTGFβ1 led to a dose-dependent increase of MMP9 expression as well as a suppression of claudin-5 expression. These effects of rTGFβ1 on MMP9 and claudin-5 expression could be reversed following treatment with a SMAD3 inhibitor. AOM-treated mice injected with neutralizing antibodies against TGFβ demonstrated significantly reduced blood-brain barrier permeability. Blood-brain barrier permeability is induced in AOM mice via a mechanism involving the TGFβ1-driven SMAD3-dependent upregulation of MMP9 expression and decrease of claudin-5 expression. Therefore, treatment modalities aimed at reducing TGFβ1 levels or SMAD3 activity may be beneficial in promoting blood-brain barrier integrity following liver failure.