Browsing by Author "Chen, Lixian"
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Item The Apelin–Apelin Receptor Axis Triggers Cholangiocyte Proliferation and Liver Fibrosis During Mouse Models of Cholestasis(Wiley, 2021-06) Chen, Lixian; Zhou, Tianhao; White, Tori; O'Brien, April; Chakraborty, Sanjukta; Liangpunsakul, Suthat; Yang, Zhihong; Kennedy, Lindsey; Saxena, Romil; Wu, Chaodong; Meng, Fanyin; Huang, Qiaobing; Francis, Heather; Alpini, Gianfranco; Glaser, Shannon; Medicine, School of MedicineBackground and Aims Apelin (APLN) is the endogenous ligand of its G protein–coupled receptor, apelin receptor (APJ). APLN serum levels are increased in human liver diseases. We evaluated whether the APLN–APJ axis regulates ductular reaction and liver fibrosis during cholestasis. Approach and Results We measured the expression of APLN and APJ and serum APLN levels in human primary sclerosing cholangitis (PSC) samples. Following bile duct ligation (BDL) or sham surgery, male wild-type (WT) mice were treated with ML221 (APJ antagonist) or saline for 1 week. WT and APLN−/− mice underwent BDL or sham surgery for 1 week. Multidrug resistance gene 2 knockout (Mdr2−/−) mice were treated with ML221 for 1 week. APLN levels were measured in serum and cholangiocyte supernatants, and cholangiocyte proliferation/senescence and liver inflammation, fibrosis, and angiogenesis were measured in liver tissues. The regulatory mechanisms of APLN–APJ in (1) biliary damage and liver fibrosis were examined in human intrahepatic biliary epithelial cells (HIBEpiCs) treated with APLN and (2) hepatic stellate cell (HSC) activation in APLN-treated human HSC lines (HHSteCs). APLN serum levels and biliary expression of APLN and APJ increased in PSC samples. APLN levels were higher in serum and cholangiocyte supernatants from BDL and Mdr2−/− mice. ML221 treatment or APLN−/− reduced BDL-induced and Mdr2−/−-induced cholangiocyte proliferation/senescence, liver inflammation, fibrosis, and angiogenesis. In vitro, APLN induced HIBEpiC proliferation, increased nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, reactive oxygen species (ROS) generation, and extracellular signal–regulated kinase (ERK) phosphorylation. Pretreatment of HIBEpiCs with ML221, diphenyleneiodonium chloride (Nox4 inhibitor), N-acetyl-cysteine (NAC, ROS inhibitor), or PD98059 (ERK inhibitor) reduced APLN-induced cholangiocyte proliferation. Activation of HHSteCs was induced by APLN but reduced by NAC. Conclusions The APLN–APJ axis induces cholangiocyte proliferation through Nox4/ROS/ERK-dependent signaling and HSC activation through intracellular ROS. Modulation of the APLN–APJ axis may be important for managing cholangiopathies.Item Downregulation of p16 Decreases Biliary Damage and Liver Fibrosis in the Mdr2 / Mouse Model of Primary Sclerosing Cholangitis(Cognizant Communication Corporation, 2020-11) Kyritsi, Konstantina; Francis, Heather; Zhou, Tianhao; Ceci, Ludovica; Wu, Nan; Yang, Zhihong; Meng, Fanyin; Chen, Lixian; Baiocchi, Leonardo; Kundu, Debjyoti; Kennedy, Lindsey; Liangpunsakul, Suthat; Wu, Chaodong; Glaser, Shannon; Alpini, Gianfranco; Medicine, School of MedicineBiliary senescence and hepatic fibrosis are hallmarks of cholangiopathies including primary sclerosing cholangitis (PSC). Senescent cholangiocytes display senescence-associated secretory phenotypes [SASPs, e.g., transforming growth factor-1 (TGF-1)] that further increase biliary senescence (by an autocrine loop) and trigger liver fibrosis by paracrine mechanisms. The aim of this study was to determine the effect of p16 inhibition and role of the TGF-1/microRNA (miR)-34a/sirtuin 1 (SIRT1) axis in biliary damage and liver fibrosis in the Mdr2/ mouse model of PSC. We treated (i) in vivo male wild-type (WT) and Mdr2/ mice with p16 Vivo-Morpholino or controls before measuring biliary mass [intrahepatic bile duct mass (IBDM)] and senescence, biliary SASP levels, and liver fibrosis, and (ii) in vitro intrahepatic murine cholangiocyte lines (IMCLs) with small interfering RNA against p16 before measuring the mRNA expression of proliferation, senescence, and fibrosis markers. p16 and miR-34a increased but SIRT1 decreased in Mdr2/ mice and PSC human liver samples compared to controls. p16 immunoreactivity and biliary senescence and SASP levels increased in Mdr2/ mice but decreased in Mdr2/ mice treated with p16 Vivo-Morpholino. The increase in IBDM and hepatic fibrosis (observed in Mdr2/ mice) returned to normal values in Mdr2/ mice treated with p16 Vivo-Morpholino. TGF-1 immunoreactivity and biliary SASPs levels were higher in Mdr2/ compared to those of WT mice but returned to normal values in Mdr2/ mice treated with p16 Vivo-Morpholino. The expression of fibrosis/senescence markers decreased in cholangiocytes from Mdr2/ mice treated with p16 Vivo-Morpholino (compared to Mdr2/ mice) and in IMCLs (after p16 silencing) compared to controls. Modulation of the TGF-1/miR-34a/SIRT1 axis may be important in the management of PSC phenotypes.Item Functional Role of the Secretin/Secretin Receptor Signaling During Cholestatic Liver Injury(AASLD, 2020-12) Wu, Nan; Baiocchi, Leonardo; Zhou, Tianhao; Kennedy, Lindsey; Ceci, Ludovica; Meng, Fanyin; Sato, Keisaku; Wu, Chaodong; Ekser, Burcin; Kyritsi, Konstantina; Kundu, Debjyoti; Chen, Lixian; Meadows, Vik; Franchitto, Antonio; Alvaro, Domenico; Onori, Paolo; Gaudio, Eugenio; Lenci, Ilaria; Francis, Heather; Glaser, Shannon; Alpini, Gianfranco; Medicine, School of MedicineLiver diseases are a major health concern and affect a large proportion of people worldwide. There are over 100 types of liver disorders, including cirrhosis, cholangiocarcinoma (CCA), hepatocellular carcinoma, and hepatitis. Despite the relevant number of people who are affected by liver diseases, and the increased awareness with regard to these disorders, the number of deaths corresponding to liver injury is expected to increase in the foreseeable future. One of the possible reasons for this is that a complete comprehension of the mechanisms of hepatic damage involving specific liver anatomical districts is lacking, and, as a consequence, current treatments available are suboptimal. A major burden in the clinical setting are chronic cholestatic liver diseases (e.g., primary biliary cholangitis [PBC], primary sclerosing cholangitis [PSC], biliary atresia), which target the biliary epithelium and are characterized by cholestasis.(1, 2) Because the secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes in the liver)(3, 4) is the major regulator of ductal bile secretion,(5, 6) it is intuitive that this axis plays a key role in the maintenance of biliary homeostasis during the progression of cholangiopathies. For instance, PBC is characterized by reduced bicarbonate secretion, a phenomenon possibly impeding the formation of an HCO3 canalicular film (“bicarbonate umbrella”) on bile ducts, which has protective properties against highly concentrated bile acids (BAs).(1, 7, 8) In this review, we examined the molecular mechanisms by which the Sct/SR axis regulates biliary function and the homeostasis of the biliary epithelium in normal and pathophysiological conditions.Item Inhibition of Secretin/Secretin Receptor Axis Ameliorates NAFLD Phenotypes(Wiley, 2021-10) Chen, Lixian; Wu, Nan; Kennedy, Lindsey; Francis, Heather; Ceci, Ludovica; Zhou, Tianhao; Samala, Niharika; Kyritsi, Konstantina; Wu, Chaodong; Sybenga, Amelia; Ekser, Burcin; Dar, Wasim; Atkins, Constance; Meadows, Vik; Glaser, Shannon; Alpini, Gianfranco; Surgery, School of MedicineBackground & Aims Human non-alcoholic fatty liver disease (NAFLD) is characterized at early stages by hepatic steatosis, which may progress to nonalcoholic steatohepatitis (NASH) when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through downregulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence and liver fibrosis in NAFLD/NASH. Approach & Results In vivo, 4 wk male WT, Sct-/- and Sctr-/- mice were fed a control diet (CD) or high-fat diet (HFD) for 16 wks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry (IHC) and qPCR. Biliary/hepatocyte senescence, ductular reaction and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between WT mice and Sct-/-/Sctr-/- mice. Conclusion The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by upregulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.Item The interplay between mast cells, pineal gland, and circadian rhythm: Links between histamine, melatonin, and inflammatory mediators(Wiley, 2021-03) Pham, Linh; Baiocchi, Leonardo; Kennedy, Lindsey; Sato, Keisaku; Meadows, Vik; Meng, Fanyin; Huang, Chiung-Kuei; Kundu, Debjyoti; Zhou, Tianhao; Chen, Lixian; Alpini, Gianfranco; Francis, Heather; Medicine, School of MedicineOur daily rhythmicity is controlled by a circadian clock with a specific set of genes located in the suprachiasmatic nucleus in the hypothalamus. Mast cells (MCs) are major effector cells that play a protective role against pathogens and inflammation. MC distribution and activation are associated with the circadian rhythm via two major pathways, IgE/FcεRI- and IL-33/ST2-mediated signaling. Furthermore, there is a robust oscillation between clock genes and MC-specific genes. Melatonin is a hormone derived from the amino acid tryptophan and is produced primarily in the pineal gland near the center of the brain, and histamine is a biologically active amine synthesized from the decarboxylation of the amino acid histidine by the L-histidine decarboxylase enzyme. Melatonin and histamine are previously reported to modulate circadian rhythms by pathways incorporating various modulators in which the nuclear factor–binding near the κ light-chain gene in B cells, NF-κB, is the common key factor. NF-κB interacts with the core clock genes and disrupts the production of pro-inflammatory cytokine mediators such as IL-6, IL-13, and TNF-α. Currently, there has been no study evaluating the interdependence between melatonin and histamine with respect to circadian oscillations in MCs. Accumulating evidence suggests that restoring circadian rhythms in MCs by targeting melatonin and histamine via NF-κB may be promising therapeutic strategy for MC-mediated inflammatory diseases. This review summarizes recent findings for circadian-mediated MC functional roles and activation paradigms, as well as the therapeutic potentials of targeting circadian-mediated melatonin and histamine signaling in MC-dependent inflammatory diseases.Item Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2-/- mouse model of primary sclerosing cholangitis (PSC)(Elsevier, 2019-10) Zhou, Tianhao; Kyritsi, Konstantina; Wu, Nan; Francis, Heather; Yang, Zhihong; Chen, Lixian; O'Brien, April; Kennedy, Lindsey; Ceci, Ludovica; Meadows, Vik; Kusumanchi, Praveen; Wu, Chaodong; Baiocchi, Leonardo; Skill, Nicholas J.; Saxena, Romil; Sybenga, Amelia; Xie, Linglin; Liangpunsakul, Suthat; Meng, Fanyin; Alpini, Gianfranco; Glaser, Shannon; Medicine, School of MedicineBACKGROUND: Cholangiocytes are the target cells of cholangiopathies including primary sclerosing cholangitis (PSC). Vimentin is an intermediate filament protein that has been found in various types of mesenchymal cells. The aim of this study is to evaluate the role of vimentin in the progression of biliary damage/liver fibrosis and whether there is a mesenchymal phenotype of cholangiocytes in the Mdr2-/- model of PSC. METHODS: In vivo studies were performed in 12 wk. Mdr2-/- male mice with or without vimentin Vivo-Morpholino treatment and their corresponding control groups. Liver specimens from human PSC patients, human intrahepatic biliary epithelial cells (HIBEpiC) and human hepatic stellate cell lines (HHSteCs) were used to measure changes in epithelial-to-mesenchymal transition (EMT). FINDINGS: There was increased mesenchymal phenotype of cholangiocytes in Mdr2-/- mice, which was reduced by treatment of vimentin Vivo-Morpholino. Concomitant with reduced vimentin expression, there was decreased liver damage, ductular reaction, biliary senescence, liver fibrosis and TGF-β1 secretion in Mdr2-/- mice treated with vimentin Vivo-Morpholino. Human PSC patients and derived cell lines had increased expression of vimentin and other mesenchymal markers compared to healthy controls and HIBEpiC, respectively. In vitro silencing of vimentin in HIBEpiC suppressed TGF-β1-induced EMT and fibrotic reaction. HHSteCs had decreased fibrotic reaction and increased cellular senescence after stimulation with cholangiocyte supernatant with reduced vimentin levels. INTERPRETATION: Our study demonstrated that knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes, which leads to decreased biliary senescence and liver fibrosis. Inhibition of vimentin may be a key therapeutic target in the treatment of cholangiopathies including PSC. FUND: National Institutes of Health (NIH) awards, VA Merit awards.Item Mast Cells Regulate Ductular Reaction and Intestinal Inflammation in Cholestasis Through Farnesoid X Receptor Signaling(Wiley, 2021-11) Meadows, Vik; Kennedy, Lindsey; Ekser, Burcin; Kyritsi, Konstantina; Kundu, Debjyoti; Zhou, Tianhao; Chen, Lixian; Pham, Linh; Wu, Nan; Demieville, Jennifer; Hargrove, Laura; Glaser, Shannon; Alpini, Gianfranco; Francis, Heather; Medicine, School of MedicineBackground & Aim Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF15). Primary sclerosing cholangitis (PSC) patients typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile duct ligated (BDL) MC-deficient mice (KitW-sh), ductular reaction (DR) and liver fibrosis decrease compared to BDL WT; and MC injection exacerbates liver damage in normal mice. Approach & Results In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared to control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in PSC and PSC-IBD patients compared to healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence associated secretory phenotype (SASP), fibrosis and histamine in KitW-sh mice. Inhibition of MC-FXR prior to injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15 and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and Mdr2-/- mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. Conclusion Our studies demonstrate the novel findings that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.Item Melatonin and circadian rhythms in liver diseases: Functional roles and potential therapies(Wiley, 2020-04) Sato, Keisaku; Meng, Fanyin; Francis, Heather; Wu, Nan; Chen, Lixian; Kennedy, Lindsey; Zhou, Tianhao; Franchitto, Antonio; Onori, Paolo; Gaudio, Eugenio; Glaser, Shannon; Alpini, Gianfranco; Medicine, School of MedicineCircadian rhythms and clock gene expressions are regulated by the suprachiasmatic nucleus in the hypothalamus, and melatonin is produced in the pineal gland. Although the brain detects the light through retinas and regulates rhythms and melatonin secretion throughout the body, the liver has independent circadian rhythms and expressions as well as melatonin production. Previous studies indicate the association between circadian rhythms with various liver diseases, and disruption of rhythms or clock gene expression may promote liver steatosis, inflammation, or cancer development. It is well known that melatonin has strong antioxidant effects. Alcohol drinking or excess fatty acid accumulation produces reactive oxygen species and oxidative stress in the liver leading to liver injuries. Melatonin administration protects these oxidative stress-induced liver damage and improves liver conditions. Recent studies have demonstrated that melatonin administration is not limited to antioxidant effects and it has various other effects contributing to the management of liver conditions. Accumulating evidence suggests that restoring circadian rhythms or expressions as well as melatonin supplementation may be promising therapeutic strategies for liver diseases. This review summarizes recent findings for the functional roles and therapeutic potentials of circadian rhythms and melatonin in liver diseases.Item Melatonin receptor 1A, but not 1B, knockout decreases biliary damage and liver fibrosis during cholestatic liver injury(Wiley, 2021) Wu, Nan; Carpino, Guido; Ceci, Ludovica; Baiocchi, Leonardo; Francis, Heather; Kennedy, Lindsey; Zhou, Tianhao; Chen, Lixian; Sato, Keisaku; Kyritsi, Konstantina; Meadows, Vik; Ekser, Burcin; Franchitto, Antonio; Mancinelli, Romina; Onori, Onori; Gaudio, Eugenio; Glaser, Shannon; Alpini, Gianfranco; Medicine, School of MedicineBackground and Aims Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein–coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFβ receptor type I (TGFβRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. Approach and Results Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2−/−) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2−/− mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin’s interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFβR1 signaling, which was reduced by loss of MT1. Conclusions Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFβR1 activation. Blocking GPR50/TGFβR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.Item Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis(Wiley, 2020-03) Kyritsi, Konstantina; Chen, Lixian; O’Brien, April; Francis, Heather; Hein, Travis W.; Venter, Julie; Wu, Nan; Ceci, Ludovica; Zhou, Tianhao; Zawieja, David; Gashev, Anatoliy A.; Meng, Fanyin; Invernizzi, Pietro; Fabris, Luca; Wu, Chaodong; Skill, Nicholas J.; Saxena, Romil; Liangpunsakul, Suthat; Alpini, Gianfranco; Glaser, Shannon S.; Medicine, School of MedicineBackground and aims: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. Approach and results: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. Conclusions: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.