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Blocking H1/H2 histamine receptors inhibits damage/fibrosis in Mdr2-/- mice and human cholangiocarcinoma tumorigenesis.

ABSTRACT: Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild-type and multidrug-resistant knockout (Mdr2-/- ) mice were treated by osmotic minipumps with saline, mepyramine, or ranitidine (10 mg/kg body weight/day) or a combination of mepyramine/ranitidine for 4 weeks. Liver damage was assessed by hematoxylin and eosin. We evaluated (1) H1/H2HR expression, (2) MC presence, (3) L-histidine decarboxylase/histamine axis, (4) cholangiocyte proliferation/bile duct mass, and (5) fibrosis/hepatic stellate cell activation. Nu/nu mice were implanted with Mz-ChA-1 cells into the hind flanks and treated with saline, mepyramine, or ranitidine. Tumor growth was measured, and (1) H1/H2HR expression, (2) proliferation, (3) MC activation, (4) angiogenesis, and (5) epithelial-mesenchymal transition (EMT) were evaluated. In vitro, human hepatic stellate cells were evaluated for H1HR and H2HR expression. Cultured cholangiocytes and CCA lines were treated with saline, mepyramine, or ranitidine (25 ?M) before evaluating proliferation, angiogenesis, EMT, and potential signaling mechanisms. H1/H2HR and MC presence increased in human PSC and CCA. In H1/H2HR antagonist (alone or in combination)-treated Mdr2-/- mice, liver and biliary damage and fibrosis decreased compared to saline treatment. H1/H2HR antagonists decreased tumor growth, serum histamine, angiogenesis, and EMT. In vitro, H1/H2HR blockers reduced biliary proliferation, and CCA cells had decreased proliferation, angiogenesis, EMT, and migration. Conclusion: Inhibition of H1/H2HR reverses PSC-associated damage and decreases CCA growth, angiogenesis, and EMT; because PSC patients are at risk of developing CCA, using HR blockers may be therapeutic for these diseases. (Hepatology 2018).

SUBMITTER: Kennedy L

PROVIDER: S-EPMC6165706 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Blocking H1/H2 histamine receptors inhibits damage/fibrosis in Mdr2<sup>-/-</sup> mice and human cholangiocarcinoma tumorigenesis.

Kennedy Lindsey L Hargrove Laura L Demieville Jennifer J Karstens Walker W Jones Hannah H DeMorrow Sharon S Meng Fanyin F Invernizzi Pietro P Bernuzzi Francesca F Alpini Gianfranco G Smith Steven S Akers Austin A Meadows Vik V Francis Heather H

Hepatology (Baltimore, Md.) 20180601 3

Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild-type and multidrug-resistant knockout (Mdr2<sup>-/-</sup> ) mice were treated by osmotic minipumps with saline, mepyramine ...[more]

PMID: 29601088

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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 α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 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-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 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:UnlabelledHepatic 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.ConclusionMCs 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).

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Blocking H1/H2 histamine receptors inhibits damage/fibrosis in Mdr2-/- mice and human cholangiocarcinoma tumorigenesis.

Publications

Blocking H1/H2 histamine receptors inhibits damage/fibrosis in Mdr2<sup>-/-</sup> mice and human cholangiocarcinoma tumorigenesis.

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