Cholangiopathies are characterized by dysregulation of the balance between biliary growth and loss. We have shown that histamine (HA) stimulates biliary growth via autocrine mechanisms. To evaluate the paracrine effects of mast cell (MC) stabilization on biliary proliferation, sham or BDL rats were treated by IP-implanted osmotic pumps filled with saline or cromolyn sodium (24 mg/kg BW/day (inhibits MC histamine release)) for 1 week. Serum, liver blocks and cholangiocytes were collected. Histidine decarboxylase (HDC) expression was measured using real-time PCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19. MC number was determined using toluidine blue staining and correlated to IBDM. Proliferation was evaluated by PCNA expression in liver sections and purified cholangiocytes. We assessed apoptosis using real-time PCR and IHC for BAX. Expression of MC stem factor receptor, c-kit, and the proteases chymase and tryptase were measured by real-time PCR. HA levels were measured in serum by EIA. In vitro, MCs and cholangiocytes were treated with 0.1% BSA (basal) or cromolyn (25 mM) for up to 48 h prior to assessing HDC expression, HA levels and chymase and tryptase expression. Supernatants from MCs treated with or without cromolyn were added to cholangiocytes before measuring (i) proliferation by MTT assays, (ii) HDC gene expression by real-time PCR and (iii) HA release by EIA. In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM, MC number, and biliary proliferation; (ii) HDC and MC marker expression; and (iii) HA levels. Cromolyn treatment increased cholangiocyte apoptosis. In vitro, cromolyn decreased HA release and chymase and tryptase expression in MCs but not in cholangiocytes. Cromolyn-treated MC supernatants decreased biliary proliferation and HA release. These studies provide evidence that MC histamine is key to biliary proliferation and may be a therapeutic target for the treatment of cholangiopathies.
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 mice. Methods: 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 1X PBS before collecting serum, liver blocks and cholangiocytes. Liver damage was assessed by H&E and ALT levels. IBDM was detected by CK-19 expression and proliferation by Ki-67 immunohistochemistry. Fibrosis was detected by immunohistochemistry, hydroxyproline content and by qPCR for fibrotic markers. Hepatic stellate cell (HSC) activation and TGF-β1 expression/secretion were evaluated. HDC and histamine receptor (HR) expression were detected by qPCR and HA secretion by EIA. To evaluate vascular cells, Von Willebrand (vWF) and VEGF-C expression were measured. In vitro, cultured HSCs were stimulated with cholangiocyte supernatants and α-SMA measured. Results: BDL-induced liver damage was reduced in the 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-β1 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. Conclusion: 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.
Background In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1–H4 histamine receptors (HRs). Objective To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth. Methods In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment. Results Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1–H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (~80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups. Conclusion The novel concept that an autocrine loop (consisting of enhanced histamine synthesis by HDC) sustains cholangiocarcinoma growth is proposed. Drug targeting of HDC may be important for treatment of patients with cholangiocarcinoma.
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