Inflammasome Is Activated in the Liver of Cholestatic Patients and Aggravates Hepatic Injury in Bile Duct-Ligated Mouse.

BACKGROUND & AIMS: Inflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study. METHODS: Gene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1-/- (Casp1-/-) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids. RESULTS: Caspase-1, NLRP1, NLRP3 and IL-1ß were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1ß (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1-/- BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1-/- BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1-/- BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1-/- BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300µM TCA), neither IL-1ß induction nor procaspase-1 cleavage were detected. CONCLUSIONS: The inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.

Expression of the type 3 InsP3 receptor is a final common event in the development of hepatocellular carcinoma.

BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.

Cenicriviroc, a cytokine receptor antagonist, potentiates all-trans retinoic acid in reducing liver injury in cholestatic rodents.

BACKGROUND & AIMS: Cholestatic liver injury is mediated by bile acid-induced inflammatory responses. We hypothesized that superior therapeutic effects might be achieved by combining treatments that reduce the bile acid pool size with one that blocks inflammation. METHODS: Bile duct-ligated (BDL) rats and Mdr2(Abcb4)-/- mice were treated with all-trans retinoic acid (atRA), a potent inhibitor of bile acid synthesis, 5 mg/kg/d by gavage, or Cenicriviroc (CVC), a known antagonist of CCR2 and CCR5, 50 mg/kg/d alone or in combination for 14 days and 1 month respectively. RESULTS: All-trans retinoic acid alone reduced bile acid pool size and liver necrosis in BDL rats. However, the combination with CVC further reduced liver to body weight ratio, bile acid pool size, plasma liver enzyme, bilirubin, liver necrosis and fibrosis when compared to the atRA treatment. The assessment of hepatic hydroxyproline content further confirmed the reduced liver injury concurrent with reduction of pro-inflammatory cytokines emphasizing the synergistic effects of these two agents. Profiling of hepatic inflammatory cells revealed that combination therapy reduced neutrophils and T cells but not macrophages. The superior therapeutic effects of combination treatment were also confirmed in Mdr2-/- mice where a significant reduction in plasma liver enzymes, bilirubin, liver fibrosis, bile duct proliferation and hepatic infiltration of neutrophils and T cells and expression of cytokines were found. CONCLUSIONS: Multitargeted therapy is an important paradigm for treating cholestatic liver injury. The combination of CVC with atRA or other FXR activators may warrant a clinical trial in patients with cholestatic liver disease.

Nonalcoholic fatty liver disease impairs expression of the type II inositol 1,4,5-trisphosphate receptor.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It may result in several types of liver problems, including impaired liver regeneration (LR), but the mechanism for this is unknown. Because LR depends on calcium signaling, we examined the effects of NAFLD on expression of the type II inositol 1,4,5-trisphosphate receptor (ITPR2), the principle calcium release channel in hepatocytes. ITPR2 promoter activity was measured in Huh7 and HepG2 cells. ITPR2 and c-Jun protein levels were evaluated in Huh7 cells, in liver tissue from a rat model of NAFLD, and in liver biopsy specimens of patients with simple steatosis and nonalcoholic steatohepatitis (NASH). LR was assessed in wild-type and Itpr2 knockout (Itpr2-/- ) mice following 67% hepatectomy. Cell proliferation was examined in ITPR2-knockout HepG2 cells generated by the CRISPR/Cas9 system. c-Jun dose dependently decreased activity of the human ITPR2 promoter. c-Jun expression was increased and ITPR2 was decreased in fat-loaded Huh7 cells and in livers of rats fed a high-fat, high-fructose diet. Overexpression of c-Jun reduced protein and mRNA expression of ITPR2 in Huh7 cells, whereas knockdown of c-Jun prevented the decrease of ITPR2 in fat-loaded Huh7 cells. ITPR2 expression was decreased and c-Jun was increased in liver biopsies of patients with steatosis and NASH compared to controls. ITPR2-knockout cells exhibited less nuclear calcium signaling and cell proliferation than control cells. LR assessed by Ki-67 and proliferating cell nuclear antigen was markedly decreased in Itpr2-/- mice. Conclusion: Fatty liver induces a c-Jun-mediated decrease in ITPR2 in hepatocytes. This may account for the impaired LR that occurs in NAFLD. (Hepatology 2018;67:560-574).

Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response.

Mechanisms of bile acid-induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies. We examined how BAs initiate liver injury using isolated liver cells from humans and mice and in-vivo mouse models. At pathophysiologic concentrations, BAs induced proinflammatory cytokine expression in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes. These hepatocyte-specific cytokines stimulated neutrophil chemotaxis. Inflammatory injury was mitigated in Ccl2-/- mice treated with BA or after bile duct ligation, where less hepatic infiltration of neutrophils was detected. Neutrophils in periportal areas of livers from cholestatic patients also correlated with elevations in their serum aminotransferases. This liver-specific inflammatory response required BA entry into hepatocytes via basolateral transporter Ntcp. Pathophysiologic levels of BAs induced markers of ER stress and mitochondrial damage in mouse hepatocytes. Chemokine induction by BAs was reduced in hepatocytes from Tlr9-/- mice, while liver injury was diminished both in conventional and hepatocyte-specific Tlr9-/- mice, confirming a role for Tlr9 in BA-induced liver injury. These findings reveal potentially novel mechanisms whereby BAs elicit a hepatocyte-specific cytokine-induced inflammatory liver injury that involves innate immunity and point to likely novel pathways for treating cholestatic liver disease.

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Na(+) /H(+) exchanger regulatory factor 1 knockout mice have an attenuated hepatic inflammatory response and are protected from cholestatic liver injury.

UNLABELLED: The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice. CONCLUSION: NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases.

Glucocerebrosidase 2 gene deletion rescues type 1 Gaucher disease.

The inherited deficiency of the lysosomal glucocerebrosidase (GBA) due to mutations in the GBA gene results in Gaucher disease (GD). A vast majority of patients present with nonneuronopathic, type 1 GD (GD1). GBA deficiency causes the accumulation of two key sphingolipids, glucosylceramide (GL-1) and glucosylsphingosine (LysoGL-1), classically noted within the lysosomes of mononuclear phagocytes. How metabolites of GL-1 or LysoGL-1 produced by extralysosomal glucocerebrosidase GBA2 contribute to the GD1 pathophysiology is not known. We recently recapitulated hepatosplenomegaly, cytopenia, hypercytokinemia, and the bone-formation defect of human GD1 through conditional deletion of Gba in Mx1-Cre(+):GD1 mice. Here we show that the deletion of Gba2 significantly rescues the GD1 clinical phenotype, despite enhanced elevations in GL-1 and LysoGL-1. Most notably, the reduced bone volume and bone formation rate are normalized. These results suggest that metabolism of GL-1 or LysoGL-1 into downstream bioactive lipids is a major contributor to the bone-formation defect. Direct testing revealed a strong inhibition of osteoblast viability by nanomolar concentrations of sphingosine, but not of ceramide. These findings are consistent with toxicity of high circulating sphingosine levels in GD1 patients, which decline upon enzyme-replacement therapy; serum ceramide levels remain unchanged. Together, complementary results from mice and humans affected with GD1 not only pinpoint sphingosine as being an osteoblast toxin, but also set forth Gba2 as a viable therapeutic target for the development of inhibitors to ameliorate certain disabling consequences of GD1.

Altered expression and function of canalicular transporters during early development of cholestatic liver injury in Abcb4-deficient mice.

Deficiency of ABCB4 is associated with several forms of cholestasis in humans. Abcb4(-/-) mice also develop cholestasis, but it remains uncertain what role other canalicular transporters play in the development of this disease. We examined the expression of these transporters in Abcb4(-/-) mice compared with their wild-type littermate controls at ages of 10 days and 3, 6, and 12 wk. Elevated plasma bile acid levels were already detected at 10 days and at all ages thereafter in Abcb4(-/-) mice. The expression of Bsep, Mrp2, Atp8b1, Abcg5, and Abcg8 liver proteins did not change at 10 days, but Bsep, Mrp2, and Atp8b1 were reduced, whereas Abcg5 and Abcg8 expression were increased in Abcb4(-/-) mice at all later ages. Lower bile acid concentrations were also detected in the bile of 6-wk-old Abcb4(-/-) mice. Immunofluorescence labeling revealed distorted canalicular architecture in the liver tissue by 12 wk in Abcb4(-/-) mice. Whereas Bsep and Mrp2 remained associated with the apical membrane, Atp8b1 was now localized in discrete punctuate structures adjacent to the canalicular membrane in these mice. Expression of Bsep mRNA was increased in the livers of 10-day-old Abcb4(-/-) mice, whereas Ost-α was decreased. By 12 wk, Bsep, Mrp2, and Abcg5 mRNA were all increased, whereas Ost-α and Ntcp were reduced. These findings indicate that canalicular transporters that determine the formation of bile are altered early in the development of cholestasis in Abcb4(-/-) mice and may contribute to the pathogenesis of cholestasis in this disorder.

All-trans-retinoic acid improves cholestasis in α-naphthylisothiocyanate-treated rats and Mdr2-/- mice.

Chronic cholestasis results in liver injury and eventually liver failure. Although ursodeoxycholic acid (UDCA) showed limited benefits in primary biliary cirrhosis, there is an urgent need to develop alternative therapy for chronic cholestatic disorders. Previous studies from our laboratory demonstrated that all-trans-retinoic acid (atRA) is a potent suppressor of CYP7A1, the rate-limiting enzyme in bile acid synthesis. atRA also repressed the expression of tumor growth factor-ß and collagen 1A1 in activated primary human stellate cells and LX2 cells. When administered together with UDCA to bile duct-ligated rats, this combined therapy significantly reduced the bile acid pool size and improved liver conditions. To further examine whether atRA alone or in combination with UDCA has greater beneficial effects than UDCA treatment alone, we assessed this treatment in two additional chronic cholestatic rodent models: α-naphthylisothiocyanate (ANIT)-treated rats and the Mdr2(-/-) (Abcb4(-/-)) knockout mouse. atRA alone significantly reduced bile duct proliferation, inflammation, and hydroxyproline levels in ANIT-treated rats, whereas the combination of atRA and UDCA significantly reduced plasma bile salt level compared with UDCA treatment. atRA alone or in combination with UDCA significantly reduced plasma levels of alkaline phosphatase and bile salts in 12-week-old Mdr2(-/-) mice. Reduced bile duct proliferation and inflammation were also observed in the livers of these mice. Together, atRA alone or in combination with UDCA significantly reduced the severity of liver injury in these two animal models, further supporting the combination treatment of atRA and UDCA as a potential new therapy for patients with chronic cholestatic liver disease who have not responded fully to UDCA.

The insulin receptor translocates to the nucleus to regulate cell proliferation in liver.

UNLABELLED: Insulin's metabolic effects in the liver are widely appreciated, but insulin's ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca(2+) signals within the nucleus regulate cell proliferation, we investigated whether insulin's mitogenic effects result from activation of Ca(2+)-signaling pathways by IRs within the nucleus. Insulin-induced increases in Ca(2+) and cell proliferation depended upon clathrin- and caveolin-dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,-trisphosphate (InsP3) in the nucleus, whereas insulin's metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus. CONCLUSION: These findings provide evidence that insulin's mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3 -dependent Ca(2+)-signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease.

Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice.

BACKGROUND & AIMS: Oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown. METHODS: We administered oltipraz to mice for 5 days prior to bile duct ligation (BDL) for 3 days. Liver histology, liver function markers, bile flow rates and hepatic expression of profibrogenic genes were evaluated. RESULTS: Mice pretreated with oltipraz prior to BDL demonstrated higher levels of serum aminotransferases and more severe liver damage than in control mice. Higher bile flow and glutathione secretion rates were observed in unoperated mice treated with oltipraz than in control mice, suggesting that liver necrosis in oltipraz-treated BDL mice may be related partially to increased bile-acid independent flow and biliary pressure. Oltipraz treatment in BDL mice enhanced α-smooth muscle actin expression, consistent with activation of hepatic stellate cells and portal fibroblasts. Matrix metalloproteinases (Mmp) 9 and 13 and tissue inhibitors of metalloproteinases (Timp) 1 and 2 levels were increased in the oltipraz-treated BDL group, suggesting that the secondary phase of liver injury induced by oltipraz might be due to excessive Mmp and Timp secretions, which induce remodeling of the extracellular matrix. CONCLUSIONS: Oltipraz treatment exacerbates the severity of liver injury following BDL and should be avoided as therapy for extrahepatic cholestatic disorders due to bile duct obstruction.

Adult sea lamprey tolerates biliary atresia by altering bile salt composition and renal excretion.

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia, as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the postmetamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic, with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (∼10 µM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ∼35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72-hour period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts, and BSP, were predominantly excreted by way of the kidney, with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney, with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. (HEPATOLOGY 2013;57:2418-2426).

Nuclear factor-E2-related factor 2 is a major determinant of bile acid homeostasis in the liver and intestine.

The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2(-/-) mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2(-/-) compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ostα) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2(-/-) mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL.

Ostα depletion protects liver from oral bile acid load.

Bile acid homeostasis is tightly maintained through interactions between the liver, intestine, and kidney. During cholestasis, the liver is incapable of properly clearing bile acids from the circulation, and alternative excretory pathways are utilized. In obstructive cholestasis, urinary elimination is often increased, and this pathway is further enhanced after bile duct ligation in mice that are genetically deficient in the heteromeric, basolateral organic solute transporter alpha-beta (Ostα-Ostß). In this study, we examined renal and intestinal function in Ostα-deficient and wild-type mice in a model of bile acid overload. After 1% cholic acid feeding, Ostα-deficient mice had significantly lower serum ALT levels compared with wild-type controls, indicating partial protection from liver injury. Urinary clearance of bile acids, but not clearance of [(3)H]inulin, was significantly higher in cholic acid-fed Ostα-deficient mice compared with wild-type mice but was not sufficient to account for the protection. Fecal excretion of bile acids over the 5 days of cholic acid feeding was responsible for almost all of the bile acid loss in Ostα-deficient mice, suggesting that intestinal losses of bile acids accounted for the protection from liver injury. Thus fecal loss of bile acids after bile acid overload reduced the need for the kidney to filter and excrete the excess bile acids. In conclusion, Ostα-deficient mice efficiently eliminate excess bile acids via the feces. Inhibition of intestinal bile acid absorption might be an effective therapeutic target in early stages of cholestasis when bile acids are still excreted into bile.

Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells.

UNLABELLED: Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration-approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate-buffered saline (PBS), UDCA, all-trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor ß1 (Tgf-ß1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, α-smooth muscle actin (α-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor α, and interleukin-ß1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX-2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and α-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-ß1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases.

Confocal endomicroscopic examination of malignant biliary strictures and histologic correlation with lymphatics.

BACKGROUND AND AIMS: Current methods to diagnose malignant biliary strictures are of low sensitivity. Confocal endomicroscopy is a new approach that may improve the diagnosis of indeterminate biliary strictures. The purpose of this study was to evaluate indeterminate biliary strictures using probe-based confocal laser endomicroscopy and to understand the histologic basis for the confocal images. METHODS: Fourteen patients with indeterminate biliary strictures underwent endoscopic retrograde cholangiopancreatography with examination of their common bile duct with fluorescein-aided probe-based confocal laser endomicroscopy. Standard brushings and biopsies were performed. In parallel, rat bile ducts were examined either with conventional staining and light microscopy or with multiphoton microscopy. RESULTS: Earlier published criteria were used to evaluate possible malignancy in the confocal images obtained in the 14 patients. None of the individual criteria were found to be specific enough for malignancy, but a normal-appearing reticular pattern without other putative markers of malignancy was observed in all normal patients. Multiphoton reconstructions of intact rat bile ducts revealed that the reticular pattern seen in normal tissue was in the same focal plane but was smaller than blood vessels. Special stains identified the smaller structures in this network as lymphatics. CONCLUSIONS: Our limited series suggests that a negative confocal imaging study of the biliary tree can be used to rule out carcinoma, but there are frequent false positives using individual earlier published criteria. An abnormal reticular network, which may reflect changes in lymphatics, was never seen in benign strictures. Better correlation with known histologic structures may lead to improved accuracy of diagnoses.

Needle-based confocal laser endomicroscopy to assess liver histology in vivo.

BACKGROUND: Confocal endomicroscopy enables histopathology of the GI lumen to be observed in vivo. Recent prototypes of a confocal miniprobe are thin enough to be introduced through a needle. OBJECTIVE: To evaluate the ability of needle-based confocal laser endomicroscopy to distinguish between normal and cirrhotic liver tissue in vivo in a rat model. DESIGN: Feasibility study, nonsurvival animal experiments. SETTING: Academic research facility. INTERVENTION: Three normal control and 4 cirrhotic rats were examined under general anesthesia. The liver was exposed by laparotomy and imaged by using 3 different prototypes of confocal miniprobes, with 0, 50, and 100 µm working distances. Images were acquired on the surface of the liver capsule and through a 19-gauge needle inserted into the liver parenchyma. Real-time sequences were recorded after intravenous injection of fluorescein. Biopsy specimens were taken for standard histopathology. MAIN OUTCOME MEASUREMENTS: Confocal microscopic images of the surface and interior of livers in normal and cirrhotic rats. RESULTS: Miniprobes with 50 or 100 µm working distances identified cords of hepatocytes radiating toward central venules in normal livers and distorted hepatic architecture in cirrhotic livers when the miniprobes were either placed on the liver capsule or inserted into the parenchyma. The miniprobe with a 0 µm working distance identified a novel reticular pattern on the liver surface that was detected only in cirrhosis. Like the 2 longer-working-distance probes, this probe also identified cords of hepatocytes radiating toward central venules in normal livers and distorted hepatic architecture in cirrhotic livers, but this occurred only when the probe was inserted into the parenchyma. LIMITATIONS: Data were assessed in an experimental animal setting, confocal imaging was performed invasively during laparotomy, only 1 model of cirrhosis was used, and no noncirrhotic liver diseases were examined. CONCLUSION: Needle-based confocal laser endomicroscopy provides sufficient detail to distinguish normal from cirrhotic livers in a rat model. This innovative, minimally invasive technique has the potential to provide real-time identification of liver histology during EUS or natural-orifice transluminal endoscopic surgery procedures.

Role of breast cancer resistance protein in the adaptive response to cholestasis.

Breast cancer resistance protein (Bcrp) is a member of the ATP-binding cassette membrane transporter family, which is expressed apically in liver, kidney, and intestine epithelium. Recent reports suggest that in addition to xenobiotics, porphyrins, and food toxins, Bcrp can also transport bile acids and, therefore, may participate in the adaptive response to cholestasis. Bile duct ligation (BDL), an experimental model of obstructive cholestasis, was performed in male wild-type (WT) and Bcrp knockout (KO) mice. An initial time course of 3, 7, and 14 days of BDL in WT mice revealed that Bcrp expression was significantly reduced in liver but increased in ileum by 7 days. Subsequent experiments using 7-day BDL in WT and Bcrp KO mice demonstrated that there was no difference in liver necrosis, serum glutamic pyruvate aminotransferase, bilirubin, or bile acid levels in serum, hepatic tissue, bile, urine, or feces between the two groups. Protein expression levels for liver organic solute transporter (Ost) α and multidrug resistance protein 1 and kidney multidrug resistance-associated protein (Mrp) 2, Mrp3, and Mrp4 were significantly greater in the sham Bcrp KO versus sham WT mice. The expression of Mrp2 and Mrp4 in KO kidneys was further increased after BDL. In contrast, the adaptive response of transporters to BDL in the liver was similar in KO and WT BDL mice, including Ostα and Ostß expression, which increased in liver and kidney but decreased in the ileum. These findings suggest that Bcrp does not have a significant role in the adaptive response to cholestasis in the liver but may be more important for solute export in the kidney and intestine.

Regulation of multidrug resistance-associated protein 2 by calcium signaling in mouse liver.

UNLABELLED: Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. CONCLUSION: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane.