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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.

ABSTRACT: 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.

SUBMITTER: Meadows V

PROVIDER: S-EPMC6878979 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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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<sup>-/-</sup> mice.

Meadows Vik V Kennedy Lindsey L Hargrove Laura L Demieville Jennifer J Meng Fanyin F Virani Shohaib S Reinhart Evan E Kyritsi Konstantina K Invernizzi Pietro P Yang Zhihong Z Wu Nan N Liangpunsakul Suthat S Alpini Gianfranco G Francis Heather H

Biochimica et biophysica acta. Molecular basis of disease 20190913 12

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.<h4>Methods</h4>FVB/NJ and Mdr2<sup>-/-</sup> mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage ...[more]

PMID: 31521820

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Project description:Hepatic fibrosis is marked by activation of hepatic stellate cells (HSCs). Cholestatic injury precedes liver fibrosis, and cholangiocytes interact with HSCs promoting fibrosis. Mast cells (MCs) infiltrate following liver injury and release histamine, increasing biliary proliferation. We evaluated if inhibition of MC-derived histamine decreases biliary proliferation and fibrosis. Wild-type and multidrug resistance 2 knockout mice (9-11 weeks) were treated with cromolyn sodium for 1 week to block MC-derived histamine. Biliary mass and proliferation were evaluated by immunohistochemistry for cytokeratin 19 and Ki-67. Bile flow, bicarbonate excretion, and total bile acids were measured in all mice. Fibrosis was evaluated by sirius red/fast green staining and by quantitative polymerase chain reaction for alpha-smooth muscle actin, fibronectin, collagen type 1a, and transforming growth factor-beta 1. HSC activation was evaluated by quantitative polymerase chain reaction in total liver and immunofluorescent staining in tissues for synaptophysin 9. Histamine serum secretion was measured by enzymatic immunoassay. Mouse liver and human liver samples from control or primary sclerosing cholangitis patients were evaluated for MC markers by quantitative polymerase chain reaction and immunohistochemistry. In vitro, cultured MCs were transfected with histidine decarboxylase short hairpin RNA to decrease histamine secretion and subsequently cocultured with cholangiocytes or HSCs prior to measuring fibrosis markers, proliferation, and transforming growth factor-beta 1 secretion. Treatment with cromolyn sodium decreased biliary proliferation, fibrosis, histamine secretion, and bile flow in multidrug resistance 2 knockout mice. Primary sclerosing cholangitis mice and patients have increased MCs. Knockdown of MC histidine decarboxylase decreased cholangiocyte and HSC proliferation/activation.MCs are recruited to proliferating cholangiocytes and promote fibrosis. Inhibition of MC-derived histamine decreases fibrosis, and regulation of MC mediators may be therapeutic for primary sclerosing cholangitis. (Hepatology 2016;64:1202-1216).

Project description:Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. MCs infiltrate Mdr2-/- mice liver (model of primary sclerosing cholangitis (PSC)). MC-derived histamine increases inflammation, hepatic stellate cell (HSC) activation and fibrosis. The objective was to determine the effects of UDCA treatment on MC infiltration, biliary damage, inflammation and fibrosis in Mdr2-/- mice and human PSC. Wild-type and Mdr2-/- mice were fed bile acid control diet or UDCA (0.5% wt/wt). Human samples were collected from control and PSC patients treated with placebo or UDCA (15?mg/kg/BW). MC infiltration was measured by immunhistochemistry and quantitative polymerase chain reaction (qPCR) for c-Kit, chymase, and tryptase. The HDC/histamine/histamine receptor (HR)-axis was evaluated by EIA and qPCR. Intrahepatic bile duct mass (IBDM) and biliary proliferation was evaluated by CK-19 and Ki-67 staining. Fibrosis was detected by immunostaining and qPCR for fibrotic markers. Inflammatory components were measured by qPCR. HSC activation was measured by SYP-9 staining. Inflammation was detected by qPCR for CD68. In vitro, MCs were treated with UDCA (40??M) prior to HA secretion evaluation and coculturing with cholangiocytes or HSCs. BrDU incorporation and fibrosis by qPCR was performed. UDCA reduced MC number, the HDC/histamine/HR-axis, IBDM, HSC activation, inflammation, and fibrosis in Mdr2-/- mice and PSC patients. In vitro, UDCA decreases MC-histamine release, which was restored by blocking ASBT and FXR?. Proliferation and fibrosis decreased after treatment with UDCA-treated MCs. We conclude that UDCA acts on MCs reducing histamine levels and decreases the inflammatory/hyperplastic/fibrotic reaction seen in PSC. Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. Following liver injury like primary sclerosing cholangitis in mice and humans, MCs infiltrate. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.

Project description:Secretin receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2-/- mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR-/-, Mdr2-/-, and SR-/-/Mdr2-/- mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and ?-SMA; (v) senescence by SA-?-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-?1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2-/- mice was reduced in SR-/-/Mdr2-/- mice. Enhanced senescence levels in cholangiocytes from Mdr2-/- mice were reversed to normal in SR-/-/Mdr2-/- mice. However, senescence was decreased in HSCs from Mdr2-/- mice but returned to normal values in SR-/-/Mdr2-/- mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-?1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-?1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.

Project description:Background & aimsCholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2 -/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2 -/- mice.MethodsWe used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2 -/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2 -/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1β, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining.ResultsAP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1β, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16 -/- xMdr2 -/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1β (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2 -/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1β (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05).ConclusionsOur data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach.Lay summaryPrimary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease.

Project description:Activated mast cells (MCs) release histamine (HA) and MCs infiltrate the liver following bile duct ligation (BDL), increasing intrahepatic bile duct mass (IBDM) and fibrosis. We evaluated the effects of BDL in MC-deficient (KitW-sh ) mice. Wild-type (WT) and KitW-sh mice were subjected to sham or BDL for up to 7 days and KitW-sh mice were injected with cultured mast cells or 1× phosphate-buffered saline (PBS) before collecting serum, liver, and cholangiocytes. Liver damage was assessed by hematoxylin and eosin and alanine aminotransferase levels. IBDM was detected by cytokeratin-19 expression and proliferation by Ki-67 immunohistochemistry (IHC). Fibrosis was detected by IHC, hydroxyproline content, and by qPCR for fibrotic markers. Hepatic stellate cell (HSC) activation and transforming growth factor-beta 1 (TGF-?1) expression/secretion were evaluated. Histidine decarboxylase (HDC) and histamine receptor (HR) expression were detected by qPCR and HA secretion by enzymatic immunoassay. To evaluate vascular cells, von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-C expression were measured. In vitro, cultured HSCs were stimulated with cholangiocyte supernatants and alpha-smooth muscle actin levels were measured. BDL-induced liver damage was reduced in BDL KitW-sh mice, whereas injection of MCs did not mimic BDL-induced damage. In BDL KitW-sh mice, IBDM, proliferation, HSC activation/fibrosis, and TGF-?1 expression/secretion were decreased. The HDC/HA/HR axis was ablated in sham and BDL KitW-sh mice. vWF and VEGF-C expression decreased in BDL KitW-sh mice. In KitW-sh mice injected with MCs, IBDM, proliferation, fibrosis, and vascular cell activation increased. Stimulation with cholangiocyte supernatants from BDL WT or KitW-sh mice injected with MCs increased HSC activation, which decreased with supernatants from BDL KitW-sh mice.MCs promote hyperplasia, fibrosis, and vascular cell activation. Knockout of MCs decreases BDL-induced damage. Modulation of MCs may be important in developing therapeutics for cholangiopathies. (Hepatology 2017;65:1991-2004).

Dataset Information

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.

Publications

Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2<sup>-/-</sup> mice.

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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.