Dearomatization of Unactivated Arenes via Catalytic Hydroalkylation.

The dearomative cyclization of linear amides to complex spirocyclic butyrolactams has been enabled by photoredox catalysis through a reductive radical-polar crossover mechanism. This mechanism operates with precision on unactivated aromatic substrates to give a wide range of 1,4-hydroalkylation products. This method utilizes a simple organic catalyst/reductant pair to deliver products in a highly flexible manner with respect to substitution, and the products can be further functionalized under simple conditions to afford a collection of motifs. The mechanistic analysis performed here outlines the salient features of this strategy, which were applied to prepare a collection of complex scaffolds including the anticonvulsive agent gabapentin.

Dearomatization through Photoredox Hydroarylation: Discovery of a Radical-Polar Crossover Strategy.

Indole dearomatization has been achieved via radical hydroarylation. Under mild photoredox conditions, a range of indole derivatives undergo hydroarylation to form 2-arylindoline products. Mechanistically, radical termination occurs primarily via stepwise reduction/protonation, with a small contribution from concerted hydrogen atom transfer. This mechanistic understanding prompted the extension of this reactivity to benzenoid dearomatization. This work formed the foundation of our program, which utilizes reductive radical-polar crossover to drive highly selective dearomatization pathways.

Hydroarylation of Arenes via Reductive Radical-Polar Crossover.

A photocatalytic system for the dearomative hydroarylation of benzene derivatives has been developed. Using a combination of an organic photoredox catalyst and an amine reductant, this process operates through a reductive radical-polar crossover mechanism where aryl halide reduction triggers a regioselective radical cyclization event, followed by anion formation and quenching to produce a range of complex spirocyclic cyclohexadienes. This light-driven protocol functions at room temperature in a green solvent system (aq. MeCN) without the need for precious metal-based catalysts or reagents or the generation of stoichiometric metal byproducts.

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell-derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)-/- mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2-/- mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2-/- mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Regulation of adipose tissue inflammation by adenosine 2A receptor in obese mice.

Adenosine 2A receptor (A2AR) exerts anti-inflammatory effects. However, the role of A2AR in obesity-associated adipose tissue inflammation remains to be elucidated. The present study examined the expression of A2AR in adipose tissue of mice with diet-induced obesity and determined the effect of A2AR disruption on the status of obesity-associated adipose tissue inflammation. WT C57BL/6J mice and A2AR-disrupted mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and adipose tissue inflammation. In vitro, bone marrow-derived macrophages from A2AR-disrupted mice and WT control mice were treated with palmitate and examined for macrophage proinflammatory activation. Compared with that of low-fat diet (LFD)-fed WT mice, A2AR expression in adipose tissue of HFD-fed WT mice was increased significantly and was present predominantly in adipose tissue macrophages. The increase in adipose tissue A2AR expression in HFD-fed mice was accompanied with increased phosphorylation states of c-Jun N-terminal kinase 1 p46 and nuclear factor kappa B p65 and mRNA levels of interleukin (Il)-1beta, Il6 and tumor necrosis factor alpha. In A2AR-disrupted mice, HFD feeding induced significant increases in adipose tissue inflammation, indicated by enhanced proinflammatory signaling and increased proinflammatory cytokine expression, and adipose tissue insulin resistance, indicated by a decrease in insulin-stimulated Akt phosphorylation relative to those in WT mice. Lastly, A2AR disruption enhanced palmitate-induced macrophage proinflammatory activation. Taken together, these results suggest that A2AR plays a protective role in obesity-associated adipose tissue inflammation, which is attributable to, in large part, A2AR suppression of macrophage proinflammatory activation.

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice.

BACKGROUND & AIMS: Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. METHODS: Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. RESULTS: Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. CONCLUSIONS: Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Knockout of microRNA-21 attenuates alcoholic hepatitis through the VHL/NF-κB signaling pathway in hepatic stellate cells.

microRNA-21 (miRNA) is one of the most abundant miRNAs in chronic liver injuries including alcoholic liver injury. Previous studies have demonstrated that miR-21 plays a role in inflammation in the liver and functions in hepatic stellate cells (HSCs), which reside in the perisinusoidal space between sinusoidal endothelial cells and hepatocytes and regulate sinusoidal circulation. HSCs integrate cytokine-mediated inflammatory responses in the sinusoids and relay them to the liver parenchyma. Here, we showed that the activation of Von Hippel-Lindau (VHL) expression, by miR-21 knockout in vivo and anti-miR-21 or VHL overexpression in vitro, suppressed the production of proinflammatory cytokines, such as interleukin (IL)-6, monocyte chemoattractant protein-1, and IL-1ß, in human HSCs during alcoholic liver injury. Sequence and functional analyses confirmed that miR-21 directly targeted the 3'-untranslated region of VHL. Immunofluorescence and real-time PCR analysis revealed that miR-21 depletion blocked NF-κB activation in human HSCs both in cultured HSCs as well as HSCs isolated from alcohol-related liver disease mice liver by laser capture microdissection. We also showed that conditioned medium from anti-miR-21-transfected HSCs suppressed human monocyte-derived THP-1 cell migration. Taken together, our study indicates that depletion of miR-21 may downregulate cytokine production in HSCs and macrophage chemotaxis during alcoholic liver injury and that the targeting of miR-21 may have therapeutic potential for preventing the progression of alcoholic liver diseases. NEW & NOTEWORTHY This study demonstrates that silencing microRNA-21 can inhibit cytokine production and inflammatory responses in human hepatic stellate cells during alcoholic liver injury and that the targeting of microR-21 in hepatic stellate cells may have therapeutic potential for prevention and treatment of alcoholic liver diseases.

Regulation of Cellular Senescence by miR-34a in Alcoholic Liver Injury.

Alcoholic liver disease remains a major cause of liver-related morbidity and mortality, which ranges from alcoholic steatohepatitis to fibrosis/cirrhosis and hepatocellular carcinoma, and the related mechanisms are understood poorly. In this study, we aimed to investigate the role of miR-34a in alcohol-induced cellular senescence and liver fibrosis. We found that hepatic miR-34a expression was upregulated in ethanol-fed mice and heavy drinkers with steatohepatitis compared with respective controls. Mice treated with miR-34a Vivo-Morpholino developed less severe liver fibrosis than wild-type mice after 5 weeks of ethanol feeding. Further mechanism exploration showed that inhibition of miR-34a increased cellular senescence of hepatic stellate cells (HSCs) in ethanol-fed mice, although it decreased senescence in total liver and hepatocytes, which was verified by the changes of senescence-associated ß-galactosidase and gene expression. Furthermore, enhanced cellular senescence was observed in liver tissues from steatohepatitis patients compared with healthy controls. In addition, the expression of transforming growth factor-ß1, drosophila mothers against decapentaplegic protein 2 (Smad2), and Smad3 was decreased after inhibition of miR-34a in ethanol-fed mice. Our in vitro experiments showed that silencing of miR-34a partially blocked activation of HSCs by lipopolysaccharide and enhanced senescence of HSCs. Furthermore, inhibition of miR-34a decreased lipopolysaccharide-induced fibrotic gene expression in cultured hepatocytes. In conclusion, our data suggest that miR-34a functions as a profibrotic factor that promotes alcohol-induced liver fibrosis by reducing HSC senescence and increasing the senescence of hepatocytes.

The let-7/Lin28 axis regulates activation of hepatic stellate cells in alcoholic liver injury.

The let-7/Lin28 axis is associated with the regulation of key cellular regulatory genes known as microRNAs in various human disorders and cancer development. This study evaluated the role of the let-7/Lin28 axis in regulating a mesenchymal phenotype of hepatic stellate cells in alcoholic liver injury. We identified that ethanol feeding significantly down-regulated several members of the let-7 family in mouse liver, including let-7a and let-7b. Similarly, the treatment of human hepatic stellate cells (HSCs) with lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß) significantly decreased the expressions of let-7a and let-7b. Conversely, overexpression of let-7a and let-7b suppressed the myofibroblastic activation of cultured human HSCs induced by LPS and TGF-ß, as evidenced by repressed ACTA2 (α-actin 2), COL1A1 (collagen 1A1), TIMP1 (TIMP metallopeptidase inhibitor 1), and FN1 (fibronectin 1); this supports the notion that HSC activation is controlled by let-7. A combination of bioinformatics, dual-luciferase reporter assay, and Western blot analysis revealed that Lin28B and high-mobility group AT-hook (HMGA2) were the direct targets of let-7a and let-7b. Furthermore, Lin28B deficiency increased the expression of let-7a/let-7b as well as reduced HSC activation and liver fibrosis in mice with alcoholic liver injury. This feedback regulation of let-7 by Lin28B is verified in hepatic stellate cells isolated by laser capture microdissection from the model. The identification of the let-7/Lin28 axis as an important regulator of HSC activation as well as its upstream modulators and down-stream targets will provide insights into the involvement of altered microRNA expression in contributing to the pathogenesis of alcoholic liver fibrosis and novel therapeutic approaches for human alcoholic liver diseases.

Knockdown of Hepatic Gonadotropin-Releasing Hormone by Vivo-Morpholino Decreases Liver Fibrosis in Multidrug Resistance Gene 2 Knockout Mice by Down-Regulation of miR-200b.

Hepatic fibrosis occurs during the progression of primary sclerosing cholangitis (PSC) and is characterized by accumulation of extracellular matrix proteins. Proliferating cholangiocytes and activated hepatic stellate cells (HSCs) participate in the promotion of liver fibrosis during cholestasis. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and the biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype (GnRHR1) expressed by cholangiocytes and HSCs. Previously, we demonstrated that administration of GnRH to normal rats increased intrahepatic biliary mass (IBDM) and hepatic fibrosis. Also, miR-200b is associated with the progression of hepatic fibrosis; however, the role of the GnRH/GnRHR1/miR-200b axis in the development of hepatic fibrosis in PSC is unknown. Herein, using the mouse model of PSC (multidrug resistance gene 2 knockout), the hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo and in vitro administration of GnRH increased the expression of miR-200b and fibrosis markers. The GnRH/GnRHR1 axis and miR-200b were up-regulated in human PSC samples. Cetrorelix, a GnRHR1 antagonist, inhibited the expression of fibrotic genes in vitro and decreased IBDM and hepatic fibrosis in vivo. Inhibition of miR-200b decreased the expression of fibrosis genes in vitro in cholangiocyte and HSC lines. Targeting the GnRH/GnRHR1/miR-200b axis may be key for the management of hepatic fibrosis during the progression of PSC.

Forkhead box A2 regulates biliary heterogeneity and senescence during cholestatic liver injury in mice‡.

Biliary-committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real-time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2-/- ) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post-BDL showed significant changes compared to vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2-/- mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. CONCLUSION: The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary-committed progenitor cells during cholestatic liver injury. (Hepatology 2017;65:544-559).

Lin28 and let-7: roles and regulation in liver diseases.

The diagnosis and treatment of liver disease remain a major health concern worldwide because of the diverse etiologies of this disease. For this reason, new therapeutic targets are greatly needed to halt the progression of this damaging disease. Upon initiation of liver injury by viral infection, autoimmune disease or toxin, and/or hepatitis, chronic disease may develop, which can progress to cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma, liver failure, or death. The Lin28/lethal-7 (let-7) molecular switch has emerged as a central regulator of multiorgan injuries and cancer development. Lin28 is a stem cell marker vital to initiation or maintenance of a stem cell phenotype. Lin28 has not been extensively studied in the liver, despite its ability to induce tissue regeneration via reprogramming of oxidative enzymes in other tissues and its involvement with numerous upstream regulators and downstream targets in liver disease. Theoretically, overexpression of Lin28 in certain forms of liver disease could be a potential treatment that aids in liver regeneration. Alternatively, Lin28 has been implicated numerous times in the progression of diverse cancer types and is associated with increased severity of disease. In this case, Lin28 could be a potential inhibitory target to prevent malignant transformation in the liver. This review seeks to characterize the role of Lin28 in liver disease.

The time horizon matters: results of an exploratory study varying the timeframe in time trade-off and standard gamble utility elicitation.

INTRODUCTION: The purpose of this study was to examine whether the time horizon of time trade-off (TTO) and standard gamble (SG) utility assessment influences utility scores and discrimination between health states. METHODS: In two phases, UK general population participants rated three osteoarthritis health states in TTO and SG procedures with two time horizons: (1) 10-year and (2) a time horizon derived from self-reported additional life expectancy (ALE). The two time horizons were compared in terms of mean utilities and discrimination among health states. RESULTS: In Phase 1, the 10-year tasks were completed by 80 participants, 35 of whom also completed utility assessment with the ALE. In Phase 2, all 101 participants completed procedures with both time horizons. Utility scores tended to be lower with the ALE than the 10-year, a difference that was statistically significant for two health states with SG in Phase 1 (P < 0.05), two health states with TTO in Phase 2 (P < 0.01), and one health state with SG in Phase 2 (P < 0.001). In Phase 1, rates of discrimination between mild and moderate osteoarthritis health states were significantly higher with the ALE than the 10-year (TTO: P = 0.03; SG: P = 0.001). This pattern of discrimination was similar in Phase 2. DISCUSSION: Results suggest that the time horizon could influence utility scores and discrimination among health states. When designing utility evaluations, researchers should carefully consider the time horizon so that the value of health states is accurately represented in cost-utility models.

Role of stem cells during diabetic liver injury.

Diabetes mellitus is one of the most severe endocrine metabolic disorders in the world that has serious medical consequences with substantial impacts on the quality of life. Type 2 diabetes is one of the main causes of diabetic liver diseases with the most common being non-alcoholic fatty liver disease. Several factors that may explain the mechanisms related to pathological and functional changes of diabetic liver injury include: insulin resistance, oxidative stress and endoplasmic reticulum stress. The realization that these factors are important in hepatocyte damage and lack of donor livers has led to studies concentrating on the role of stem cells (SCs) in the prevention and treatment of liver injury. Possible avenues that the application of SCs may improve liver injury include but are not limited to: the ability to differentiate into pancreatic ß-cells (insulin producing cells), the contribution for hepatocyte regeneration, regulation of lipogenesis, glucogenesis and anti-inflammatory actions. Once further studies are performed to explore the underlying protective mechanisms of SCs and the advantages and disadvantages of its application, there will be a greater understand of the mechanism and therapeutic potential. In this review, we summarize the findings regarding the role of SCs in diabetic liver diseases.

Functional role of cellular senescence in biliary injury.

Cellular senescence is a state of irreversible cell cycle arrest that has been involved in many gastrointestinal diseases, including human cholestatic liver disorders. Senescence may play a role in biliary atresia, primary sclerosing cholangitis, cellular rejection, and primary biliary cirrhosis, four liver diseases affecting cholangiocytes and the biliary system. In this review, we examine proposed mechanisms of senescence-related biliary diseases, including hypotheses associated with the senescence-associated phenotype, induction of senescence in nearby cells, and the depletion of stem cell subpopulations. Current evidence for the molecular mechanisms of senescence in the previously mentioned diseases is discussed in detail, with attention to recent advances on the role of pathways associated with senescence-associated phenotype, stress-induced senescence, telomere dysfunction, and autophagy.

Regulation of the extrinsic apoptotic pathway by microRNA-21 in alcoholic liver injury.

IL-6/Stat3 is associated with the regulation of transcription of key cellular regulatory genes (microRNAs) during different types of liver injury. This study evaluated the role of IL-6/Stat3 in regulating miRNA and miR-21 in alcoholic liver disease. By microarray, we identified that ethanol feeding significantly up-regulated 0.8% of known microRNAs in mouse liver compared with controls, including miR-21. Similarly, the treatment of normal human hepatocytes (N-Heps) and hepatic stellate cells (HSCs) with ethanol and IL-6 significantly increased miR-21 expression. Overexpression of miR-21 decreased ethanol-induced apoptosis in both N-Heps and HSCs. The expression level of miR-21 was significantly increased after Stat3 activation in N-Heps and HSCs, in support of the concept that the 5'-promoter region of miR-21 is regulated by Stat3. Using real time PCR, we confirmed that miR-21 activation is associated with ethanol-linked Stat3 binding of the miR-21 promoter. A combination of bioinformatics, PCR array, dual-luciferase reporter assay, and Western blot analysis revealed that Fas ligand (TNF superfamily, member 6) (FASLG) and death receptor 5 (DR5) are the direct targets of miR-21. Furthermore, inhibition of miR-21 by specific Vivo-Morpholino and knock-out of IL-6 in ethanol-treated mice also increased the expression of DR5 and FASLG in vivo during alcoholic liver injury. The identification of miR-21 as an important regulator of hepatic cell survival, transformation, and remodeling in vitro, as well as its upstream modulators and downstream targets, will provide insight into the involvement of altered miRNA expression in contributing to alcoholic liver disease progression and testing novel therapeutic approaches for human alcoholic liver diseases.

Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth.

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Secretin stimulates biliary cell proliferation by regulating expression of microRNA 125b and microRNA let7a in mice.

BACKGROUND & AIMS: Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. METHODS: Cholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile. RESULTS: Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. CONCLUSIONS: After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

The functional role of microRNAs in alcoholic liver injury.

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver-inflammatory agents such as tumour necrosis factor-alpha (TNF-α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, is responsible for TNF-α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR-155 and miR-21, show a positive correlation in up-regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF-α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR-122 and miR-34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.

Molecular mechanisms of stem cell therapy in alcoholic liver disease.

Alcoholic liver disease affects a great number of people worldwide. With limited therapeutic options, stem cell therapy offers significant potential for these patients. To date, a limited number of clinical trials have produced transient clinical responses to cell therapy in patients with alcoholic liver disease. Stem cell therapy to reorganize the postnatal liver is an important theme and mission for patients with chronic liver disorders including alcoholic liver injury. We therefore should redevelop the evidence of cell-based liver regeneration therapy, focusing on targets (disease, patient's status and hepatic function), materials (cells, cytokines and genes), and methodology (stem cell types and their derived microparticles, transplantation route, implantation technology and tissue engineering). In this review, we summarize the recent findings regarding the experimental and clinical use of mesenchymal and liver stem cells, focusing mainly on the treatment of alcoholic liver disorders and their relevance in the field of regenerative medicine, and advances on the role of microvesicles and exosomes in this process. We discuss new advances in stem cell therapy from liver regeneration to liver re-organization, which is involved in the recent progress of on-going clinical trials, basic research in stem cell therapy and liver regeneration, and updated exosomes/microvesicles recovery/repairing technology.