Cholangiocyte anion exchange and biliary bicarbonate excretion

Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. The excretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3- exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis

Extracellular Vesicles in Hepatobiliary Malignancies

Primary hepatobiliary malignancies include a heterogeneous group of cancers with dismal prognosis, among which hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatoblastoma (HB) stand out. These tumors mainly arise from the malignant transformation of hepatocytes, cholangiocytes (bile duct epithelial cells) or hepatoblasts (embryonic liver progenitor cells), respectively. Early diagnosis, prognosis prediction and effective therapies are still a utopia for these diseases. Extracellular vesicles (EVs) are small membrane-enclosed spheres secreted by cells and present in biological fluids. They contain multiple types of biomolecules, such as proteins, RNA, DNA, metabolites and lipids, which make them a potential source of biomarkers as well as regulators of human pathobiology. In this review, the role of EVs in the pathogenesis of hepatobiliary cancers and their potential usefulness as disease biomarkers are highlighted. Moreover, the therapeutic value of EV regulation is discussed and future directions on basic and clinical research are indicated

Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes

AE2 (SLC4A2) is the member of the Na(+)-independent anion exchanger (AE) family putatively involved in the secretion of bicarbonate to bile. In humans, three variants of AE2 mRNA have been described: the full-length transcript AE2a (expressed from the upstream promoter in most tissues), and alternative transcripts AE2b(1) and AE2b(2) (driven from alternate promoter sequences in a tissue-restricted manner, mainly in liver and kidney). These transcripts would result in AE protein isoforms with short N-terminal differences. To ascertain their translation, functionality, and membrane sorting, we constructed expression vectors encoding each AE2 isoform fused to GFP at the C-terminus. Transfected HEK293 cells showed expression of functional GFP-tagged AE2 proteins, all three isoforms displaying comparable AE activities. Primary rat hepatocytes transfected with expression vectors and repolarized in a collagen-sandwich configuration showed a microtubule-dependent apical sorting of each AE2 isoform. This shared apical sorting is liver-cell specific, as sorting of AE2 isoforms was basolateral in control experiments on polarized kidney MDCK cells. Hepatocytic apical targeting of AE2 isoforms suggests that they all may participate in the canalicular secretion of bicarbonate to bile

The search for novel diagnostic and prognostic biomarkers in cholangiocarcinoma

[EN] The poor prognosis of cholangiocarcinoma (CCA) is in part due to late diagnosis, which is currently achieved by a combination of clinical, radiological and histological approaches. Available biomarkers determined in serum and biopsy samples to assist in CCA diagnosis are not sufficiently sensitive and specific. Therefore, the identification of new biomarkers, preferably those obtained by minimally invasive methods, such as liquid biopsy, is important. The development of innovative technologies has permitted to identify a significant number of genetic, epigenetic, proteomic and metabolomic CCA features with potential clinical usefulness in early diagnosis, prognosis or prediction of treatment response. Potential new candidates must be rigorously evaluated prior to entering routine clinical application. Unfortunately, to date, no such biomarker has achieved validation for these purposes. This review is an up-to-date of currently used biomarkers and the candidates with promising characteristics that could be included in the clinical practice in the next future. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen

Validation and optimization of AFP-based biomarker panels for early HCC detection in Latin America and Europe

Background: HCC is a major cause of cancer death worldwide. Serum biomarkers such as alpha-fetoprotein (AFP), protein induced by vitamin K absence-II, and the Gender, Age, AFP-L3, AFP, Des-gamma-carboxy prothrombin (GALAD) score have been recommended for HCC surveillance. However, inconsistent recommendations in international guidelines limit their clinical utility.Methods: In this multicenter study, over 2000 patient samples were collected in 6 Latin American and 2 European countries. The performance of the GALAD score was validated in cirrhotic cases, and optimized versions were tested for early-stage HCC and prediagnostic HCC detection.Results: The GALAD score could distinguish between HCC and cirrhosis in Latin American patients with an AUC of 0.76, sensitivity of 70%, and specificity of 83% at the conventional cutoff value of −0.63. In a European cohort, GALAD had an AUC of 0.69, sensitivity of 66%, and specificity of 72%. Optimizing the score in the 2 large multicenter cohorts revealed that AFP-L3 contributed minimally to early-stage HCC detection. Thus, we developed a modified GALAD score without AFP-L3, the ASAP (age, sex, AFP, and protein induced by vitamin K absence-II), which showed promise for early-stage HCC detection upon validation. The ASAP score also identified patients with cirrhosis at high risk for advanced-stage HCC up to 15 months before diagnosis (p < 0.0001) and differentiated HCC from hemangiomas, with a specificity of 100% at 71% sensitivity.Conclusion: Our comprehensive analysis of large sample cohorts validates the GALAD score’s utility in Latin American, Spanish, and Dutch patients for early-stage HCC detection. The optimized GALAD without AFP-L3, the ASAP score, is a good alternative and shows greater promise for HCC prediction

Current and novel therapeutic opportunities for systemic therapy in biliary cancer

none24Biliary tract cancers (BTCs) are a group of rare and aggressive malignancies that arise in the biliary tree within and outside the liver. Beyond surgical resection, which is beneficial for only a small proportion of patients, current strategies for treating patients with BTCs include chemotherapy, as a single agent or combination regimens, in the adjuvant and palliative setting. Increased characterisation of the molecular landscape of these tumours has facilitated the identification of molecular vulnerabilities, such as IDH mutations and FGFR fusions, that can be exploited for the treatment of BTC patients. Beyond targeted therapies, active research avenues explore the development of novel therapeutics that target the crosstalk between cancer and stroma, the cellular pathways involved in the regulation of cell death, the chemoresistance phenotype and the dysregulation of RNA. In this review, we discuss the therapeutic opportunities currently available in the management of BTC patients, and explore the strategies that can support the implementation of precision oncology in BTCs, including novel molecular targets, liquid biopsies and patient-derived predictive tools.openMarin J.J.G.; Prete M.G.; Lamarca A.; Tavolari S.; Landa-Magdalena A.; Brandi G.; Segatto O.; Vogel A.; Macias R.I.R.; Rodrigues P.M.; Casta A.L.; Mertens J.; Rodrigues C.M.P.; Fernandez-Barrena M.G.; Da Silva Ruivo A.; Marzioni M.; Mentrasti G.; Acedo P.; Munoz-Garrido P.; Cardinale V.; Banales J.M.; Valle J.W.; Bridgewater J.; Braconi C.Marin, J. J. G.; Prete, M. G.; Lamarca, A.; Tavolari, S.; Landa-Magdalena, A.; Brandi, G.; Segatto, O.; Vogel, A.; Macias, R. I. R.; Rodrigues, P. M.; Casta, A. L.; Mertens, J.; Rodrigues, C. M. P.; Fernandez-Barrena, M. G.; Da Silva Ruivo, A.; Marzioni, M.; Mentrasti, G.; Acedo, P.; Munoz-Garrido, P.; Cardinale, V.; Banales, J. M.; Valle, J. W.; Bridgewater, J.; Braconi, C

A TGFβ-ECM-Integrin signalling axis drives structural reconfiguration of the bile duct to promote polycystic liver disease

: The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-β (TGFβ) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFβ signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery

Oral Methylthioadenosine Administration Attenuates Fibrosis and Chronic Liver Disease Progression in Mdr2−/− Mice

BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis

Dual Pharmacological Targeting of HDACs and PDE5 Inhibits Liver Disease Progression in a Mouse Model of Biliary Inflammation and Fibrosis

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor β (TGFβ). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strateg