R. Jiménez-Agüero

Inhibition of metalloprotease hyperactivity in cystic cholangiocytes halts the development of polycystic liver diseases

Objective Polycystic liver diseases (PCLDs) are genetic disorders characterised by progressive bile duct dilatation and/or cyst development. Their pathogenesis is a consequence of hyperproliferation, hypersecretion and microRNA alterations in cholangiocytes. Here we evaluate the role of matrix metalloproteases (MMPs) in the hepatic cystogenesis of PCLDs. Design Metalloprotease activity was measured by microfluorimetric assays in normal and polycystic cholangiocyte cultures from humans and rats, and gene expression by real time quantitative PCR. The role of cytokines, oestrogens and growth factors present in the cystic fluid of PCLD patients was evaluated for MMP activity. The MMP inhibitor marimastat was examined for cystic expansion in vitro and in polycystic kidney (PCK) rats. Results Polycystic human and rat cholangiocytes displayed increased MMP activity, which was associated with increased mRNA levels of different MMPs. Interleukin (IL)-6 and IL-8, and 17β-oestradiol, all stimulated MMP activity in human cholangiocytes. The presence of antibodies against IL-6 and/or IL-8 receptor/s inhibited baseline MMP hyperactivity of polycystic human cholangiocytes but had no effect on normal human cholangiocytes. MMP-3 was overexpressed in cystic cholangiocytes from PCLD human and PCK rat livers by immunohistochemistry. Marimastat reduced MMP hyperactivity of polycystic human and rat cholangiocytes and blocked the cystic expansion of PCK cholangiocytes cultured in three-dimensions. Chronic treatment of 8-week-old PCK rats with marimastat inhibited hepatic cystogenesis and fibrosis. Conclusions PCLDs are associated with cholangiocyte MMP hyperactivity resulting from autocrine/paracrine stimulation by IL-6 and IL-8. Inhibition of this MMP hyperactivity with marimastat decreased hepatic cystogenesis in vitro and in an animal model of PCLD, offering a potential therapeutic tool.

Ursodeoxycholic acid inhibits hepatic cystogenesis in experimental models of polycystic liver disease

BACKGROUND & AIMS Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive biliary cystogenesis. Current therapies show short-term and/or modest beneficial effects. Cystic cholangiocytes hyperproliferate as a consequence of diminished intracellular calcium levels ([Ca(2+)]i). Here, the therapeutic value of ursodeoxycholic acid (UDCA) was investigated. METHODS Effect of UDCA was examined in vitro and in polycystic (PCK) rats. Hepatic cystogenesis and fibrosis, and the bile acid (BA) content were evaluated from the liver, bile, serum, and kidneys by HPLC-MS/MS. RESULTS Chronic treatment of PCK rats with UDCA inhibits hepatic cystogenesis and fibrosis, and improves their motor behaviour. As compared to wild-type animals, PCK rats show increased BA concentration ([BA]) in liver, similar hepatic Cyp7a1 mRNA levels, and diminished [BA] in bile. Likewise, [BA] is increased in cystic fluid of PLD patients compared to their matched serum levels. In PCK rats, UDCA decreases the intrahepatic accumulation of cytotoxic BA, normalizes their diminished [BA] in bile, increases the BA secretion in bile and diminishes the increased [BA] in kidneys. In vitro, UDCA inhibits the hyperproliferation of polycystic human cholangiocytes via a PI3K/AKT/MEK/ERK1/2-dependent mechanism without affecting apoptosis. Finally, the presence of glycodeoxycholic acid promotes the proliferation of polycystic human cholangiocytes, which is inhibited by both UDCA and tauro-UDCA. CONCLUSIONS UDCA was able to halt the liver disease of a rat model of PLD through inhibiting cystic cholangiocyte hyperproliferation and decreasing the levels of cytotoxic BA species in the liver, which suggests the use of UDCA as a potential therapeutic tool for PLD patients.

PNPLA3 p.I148M variant is associated with greater reduction of liver fat content after bariatric surgery.

BACKGROUND Obesity is the major trigger of nonalcoholic fatty liver disease (NAFLD). NAFLD is further favored by the patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M, transmembrane 6 superfamily member 2 (TM6SF2) p.E167K, and membrane-bound O-acyltransferase domain containing 7 (MBOAT7) rs641738 variants. OBJECTIVES To investigate the relationship between the PNPLA3, TM6SF2, and MBOAT7 genotypes and the outcomes of bariatric surgery. SETTING University hospital. METHODS Prospectively we monitored 84 obese individuals (body mass index 35-64 kg/m2) scheduled for bariatric surgery. The PNPLA3 p.I148M, TM6SF2 p.E167K, and MBOAT7 rs641738 variants were genotyped using restriction fragment length polymorphism analysis and TaqMan assays. Hepatic steatosis was determined before surgery using analysis of liver biopsy samples and a novel magnetic resonance imaging-based equation. One year later, steatosis was reevaluated by magnetic resonance imaging. RESULTS The presence of the PNPLA3 allle [M] was associated with increased hepatic triglyceride content (P = .03), steatosis detected by magnetic resonance imaging (P = 0.04), and decreased serum glucose concentrations (P = .04). Neither variant TM6SF2 nor MBOAT7 increased hepatic steatosis (all P>.05); however, the MBOAT7 polymorphism was associated with increased triglyceride, total cholesterol, low density lipoprotein, and serum glucose levels (all P<.05). Patients carrying the prosteatotic PNPLA3 allele [M] lost more weight (P<.01) and liver fat (P = .04) one year after surgery, as compared to individuals having the common genotype. The PNPLA3 genotype and initial grade of steatosis, but not the TM6SF2 or MBOAT7 variants, were independent predictors of NAFLD improvement (P = .03 and P<.01, respectively). CONCLUSION In obese patients, the presence of the PNPLA3 p.I148M allele might be associated with greater improvement of hepatic steatosis after bariatric surgery in comparison to carriers of PNPLA3 wild-type alleles.

MiRNA-506 promotes primary biliary cholangitis-like features in cholangiocytes and immune activation

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR‐506 is overexpressed in PBC cholangiocytes and directly targets both Cl–/ HCO3− anion exchanger 2 and type III inositol 1,4,5‐trisphosphate receptor, leading to cholestasis. Here, the regulation of miR‐506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin‐8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR‐506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR‐506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR‐506 (1) induced dedifferentiation with down‐regulation of biliary and epithelial markers together with up‐regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase‐3‐dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR‐506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. Conclusion: Different proinflammatory cytokines enhance the expression of miR‐506 in biliary epithelial cells; miR‐506 induces PBC‐like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (Hepatology 2018;67:1420‐1440)

SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma

BACKGROUND & AIMS Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.

Molecular Mechanisms of Cholangiocarcinogenesis: New Potential Targets for Therapy

Cholangiocarcinoma (CCA) is a heterogeneous group of dysplastic disorders affecting the biliary epithelium. It is the second most common primary liver tumor which accounts for around 3% of all gastrointestinal cancers. CCA is very deadly due to its aggressiveness, late diagnosis and high chemoresistance. The incidence is increasing worldwide and the therapeutic options are very limited. Radiotherapy, chemotherapy, surgery and/or liver transplantation may be indicated in patients who meet certain criteria, but chances of success are low. There is therefore increasing interest in understanding the molecular mechanisms involved in the pathogenesis of this cancer type and in identifying new targets for therapy. Current strategies are based on targeting key signaling pathways involved in proliferation, survival, apoptosis and migration. In this review, the most relevant molecular mechanisms involved in the pathogenesis of CCA are discussed and the main preclinical and clinical studies are highlighted. Moreover, future directions in basic and clinical research are indicated.

Non-parenchymal TREM-2 protects the liver from immune-mediated hepatocellular damage

Objective Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury. Design TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2-/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments. Results TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2-/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses. Conclusion Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression

BACKGROUND AND AIMS Cholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. METHODS FXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. RESULTS FXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. CONCLUSION Activation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

Metabolomic‐based noninvasive serum test to diagnose nonalcoholic steatohepatitis: Results from discovery and validation cohorts

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease worldwide and includes a broad spectrum of histologic phenotypes, ranging from simple hepatic steatosis or nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). While liver biopsy is the reference gold standard for NAFLD diagnosis and staging, it has limitations due to its sampling variability, invasive nature, and high cost. Thus, there is a need for noninvasive biomarkers that are robust, reliable, and cost effective. In this study, we measured 540 lipids and amino acids in serum samples from biopsy‐proven subjects with normal liver (NL), NAFL, and NASH. Using logistic regression analysis, we identified two panels of triglycerides that could first discriminate between NAFLD and NL and second between NASH and NAFL. These noninvasive tests were compared to blinded histology as a reference standard. We performed these tests in an original cohort of 467 patients with NAFLD (90 NL, 246 NAFL, and 131 NASH) that was subsequently validated in a separate cohort of 192 patients (7 NL, 109 NAFL, 76 NASH). The diagnostic performances of the validated tests showed an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.88 ± 0.05, 0.94, and 0.57, respectively, for the discrimination between NAFLD and NL and 0.79 ± 0.04, 0.70, and 0.81, respectively, for the discrimination between NASH and NAFL. When the analysis was performed excluding patients with glucose levels >136 mg/dL, the area under the receiver operating characteristic curve for the discrimination between NASH and NAFL increased to 0.81 ± 0.04 with sensitivity and specificity of 0.73 and 0.80, respectively. Conclusion: The assessed noninvasive lipidomic serum tests distinguish between NAFLD and NL and between NASH and NAFL with high accuracy. (Hepatology Communications 2018;2:807‐820)