Browsing by Author "Hargrove, Laura"

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    Amelioration of Large Bile Duct Damage by Histamine-2 Receptor Vivo-Morpholino Treatment
    (Elsevier, 2020-05) Kennedy, Lindsey; Meadows, Vik; Kyritsi, Konstantina; Pham, Linh; Kundu, Debjyoti; Kulkarni, Rewa; Cerritos, Karla; Demieville, Jennifer; Hargrove, Laura; Glaser, Shannon; Zhou, Tianhao; Jaeger, Victoria; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2-/- (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2-/- mice using vivo-morpholino. Wild-type and Mdr2-/- mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated β-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-β1, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-β1 and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-/--mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC.
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    Biliary damage and liver fibrosis are ameliorated in a novel mouse model lacking l-histidine decarboxylase/histamine signaling
    (Nature Publishing Group, 2020-02-13) Kennedy, Lindsey; Meadows, Vik; Demieville, Jennifer; Hargrove, Laura; Virani, Shohaib; Glaser, Shannon; Zhou, Tianhao; Rinehart, Evan; Jaeger, Victoria; Kyritsi, Konstantina; Pham, Linh; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Primary sclerosing cholangitis (PSC) is characterized by biliary damage and fibrosis. Multidrug resistance-2 gene knockout (Mdr2−/−) mice and PSC patients have increased histamine (HA) levels (synthesized by l-histidine decarboxylase, HDC) and HA receptor (HR) expression. Cholestatic HDC−/− mice display ameliorated biliary damage and hepatic fibrosis. The current study evaluated the effects of knockout of HDC−/− in Mdr2−/− mice (DKO) on biliary damage and hepatic fibrosis. WT, Mdr2−/− mice and homozygous DKO mice were used. Selected DKO mice were treated with HA. We evaluated liver damage along with HDC expression and HA serum levels. Changes in ductular reaction were evaluated along with liver fibrosis, inflammation and bile acid signaling pathways. The expression of H1HR/PKC-α/TGF-β1 and H2HR/pERK/VEGF-C was determined. In vitro, cholangiocyte lines were treated with HA with/without H1/H2 inhibitors before measuring: H1/H2HR, TGF-β1 and VEGF-C expression. Knockout of HDC ameliorates hepatic damage, ductular reaction, fibrosis, inflammation, bile acid signaling and H1HR/PKC-α/TGF-β1 and H2HR/pERK/VEGF-C signaling. Reactivation of the HDC/HA axis increased these parameters. In vitro, stimulation with HA increased HR expression and PKC-α, TGF-β1 and VEGF-C expression, which was reduced with HR inhibitors. Our data demonstrate the key role for the HDC/HA axis in the management of PSC progression.
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    Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice
    (Elsevier, 2019-12-01) Meadows, Vik; Kennedy, Lindsey; Hargrove, Laura; Demieville, Jennifer; Meng, Fanyin; Virani, Shohaib; Reinhart, Evan; Kyritsi, Konstantina; Invernizzi, Pietro; Yang, Zhihong; Wu, Nan; Liangpunsakul, Suthat; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis. Methods: FVB/NJ and Mdr2-/- mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber. Results: Biliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2-/- mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2-/- mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation. Conclusion: Cholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.
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    Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice
    (Elsevier, 2019) Meadows, Vik; Kennedy, Lindsey; Hargrove, Laura; Demieville, Jennifer; Meng, Fanyin; Virani, Shohaib; Reinhart, Evan; Kyritsi, Konstantina; Invernizzi, Pietro; Yang, Zhihong; Wu, Nan; Liangpunsakul, Suthat; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Introduction Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis. Methods FVB/NJ and Mdr2−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber. Results Biliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation. Conclusion Cholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.
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    Mast cells (MCs) induce ductular reaction mimicking liver injury in mice via MC-derived TGF-β1 signaling
    (Wolters Kluwer, 2021) Kyritsi, Konstantina; Kennedy, Lindsey; Meadows, Vik; Hargrove, Laura; Demieville, Jennifer; Pham, Linh; Sybenga, Amelia; Kundu, Debjyoti; Cerritos, Karla; Meng, Fanyin; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Background and aims: Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF-β1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF-β1. Approach and results: Wild-type, KitW-sh (MC-deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l-histidine decarboxylase were injected with vehicle or PKH26-tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF-β1 receptor inhibitor (TGF-βRi), LY2109761 (10 μM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK-19) immunohistochemistry and F4/80 staining coupled with real-time quantitative PCR (qPCR) for interleukin (IL)-1β, IL-33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP-9), desmin, and alpha smooth muscle actin (α-SMA). TGF-β1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC-TGF-β1 expression/secretion were measured after TGF-βRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC-conditioned medium, and biliary proliferation/senescence was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and qPCR; HSC activation evaluated for α-SMA, SYP-9, and collagen type-1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF-β1 secretion, and angiogenesis. Injection of MC-TGF-βRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF-β1. Conclusions: Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC-derived TGF-β1 may be a target in chronic cholestatic liver disease.
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    Mast Cells Promote Nonalcoholic Fatty Liver Disease Phenotypes and Microvesicular Steatosis in Mice Fed a Western Diet
    (Wolters Kluwer, 2021) Kennedy, Lindsey; Meadows, Vik; Sybenga, Amelia; Demieville, Jennifer; Chen, Lixian; Hargrove, Laura; Ekser, Burcin; Dar, Wasim; Ceci, Ludovica; Kundu, Debjyoti; Kyritsi, Konstantina; Pham, Linh; Zhou, Tianhao; Glaser, Shannon; Meng, Fanyin; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Background and aims: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. Approach and results: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. Conclusions: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.
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    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 Medicine
    Background & 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.