A pendant proton shuttle on [Fe4N(CO)12]- alters product selectivity in formate vs. H2 production via the hydride [H-Fe4N(CO)12].

Proton relays are known to increase reaction rates for H2 evolution and lower overpotentials in electrocatalytic reactions. In this report we describe two electrocatalysts, [Fe4N(CO)11(PPh3)]- (1-) which has no proton relay, and hydroxyl-containing [Fe4N(CO)11(Ph2P(CH2)2OH)]- (2-). Solid state structures indicate that these phosphine-substituted clusters are direct analogs of [Fe4N(CO)12]- where one CO ligand has been replaced by a phosphine. We show that the proton relay changes the selectivity of reactions: CO2 is reduced selectively to formate by 1- in the absence of a relay, and protons are reduced to H2 under a CO2 atmosphere by 2-. These results implicate a hydride intermediate in the mechanism of the reactions and demonstrate the importance of controlling proton delivery to control product selectivity. Thermochemical measurements performed using infrared spectroelectrochemistry provided pKa and hydricity values for [HFe4N(CO)11(PPh3)]-, which are 23.7, and 45.5 kcal mol-1, respectively. The pKa of the hydroxyl group in 2- was determined to fall between 29 and 41, and this suggests that the proximity of the proton relay to the active catalytic site plays a significant role in the product selectivity observed, since the acidity alone does not account for the observed results. More generally, this work emphasizes the importance of substrate delivery kinetics in determining the selectivity of CO2 reduction reactions that proceed through metal-hydride intermediates

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

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

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

Synthetic conjugates of ursodeoxycholic acid inhibit cystogenesis in experimental models of polycystic liver disease

Background and aims: polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of symptomatic biliary cysts. Current surgical and pharmacological approaches are ineffective, and liver transplantation represents the only curative option. Ursodeoxycholic acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6is) have arisen as promising therapeutic strategies, but with partial benefits. Approach and results: here, we tested an approach based on the design, synthesis, and validation of a family of UDCA synthetic conjugates with selective HDAC6i capacity (UDCA-HDAC6i). Four UDCA-HDAC6i conjugates presented selective HDAC6i activity, UDCA-HDAC6i #1 being the most promising candidate. UDCA orientation within the UDCA-HDAC6i structure was determinant for HDAC6i activity and selectivity. Treatment of polycystic rats with UDCA-HDAC6i #1 reduced their hepatomegaly and cystogenesis, increased UDCA concentration, and inhibited HDAC6 activity in liver. In cystic cholangiocytes UDCA-HDAC6i #1 restored primary cilium length and exhibited potent antiproliferative activity. UDCA-HDAC6i #1 was actively transported into cells through BA and organic cation transporters. Conclusions: these UDCA-HDAC6i conjugates open a therapeutic avenue for PLDs