title: Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension.
abstract: OBJECTIVE
  Hepatic stellate cells (HSCs) are vital to hepatocellular function and the liver response to injury. They share a phenotypic homology with astrocytes that are central in the pathogenesis of hepatic encephalopathy, a condition in which hyperammonemia plays a pathogenic role. This study tested the hypothesis that ammonia modulates human HSC activation in vitro and in vivo, and evaluated whether ammonia lowering, by using l-ornithine phenylacetate (OP), modifies HSC activation in vivo and reduces portal pressure in a bile duct ligation (BDL) model.
  
  
  METHODS
  Primary human HSCs were isolated and cultured. Proliferation (BrdU), metabolic activity (MTS), morphology (transmission electron, light and immunofluorescence microscopy), HSC activation markers, ability to contract, changes in oxidative status (ROS) and endoplasmic reticulum (ER) were evaluated to identify effects of ammonia challenge (50 μM, 100 μM, 300 μM) over 24-72 h. Changes in plasma ammonia levels, markers of HSC activation, portal pressure and hepatic eNOS activity were quantified in hyperammonemic BDL animals, and after OP treatment.
  
  
  RESULTS
  Pathophysiological ammonia concentrations caused significant and reversible changes in cell proliferation, metabolic activity and activation markers of hHSC in vitro. Ammonia also induced significant alterations in cellular morphology, characterised by cytoplasmic vacuolisation, ER enlargement, ROS production, hHSC contraction and changes in pro-inflammatory gene expression together with HSC-related activation markers such as α-SMA, myosin IIa, IIb, and PDGF-Rβ. Treatment with OP significantly reduced plasma ammonia (BDL 199.1 μmol/L±43.65 vs. BDL+OP 149.27 μmol/L±51.1, p<0.05) and portal pressure (BDL 14±0.6 vs. BDL+OP 11±0.3 mmHg, p<0.01), which was associated with increased eNOS activity and abrogation of HSC activation markers.
  
  
  CONCLUSIONS
  The results show for the first time that ammonia produces deleterious morphological and functional effects on HSCs in vitro. Targeting ammonia with the ammonia lowering drug OP reduces portal pressure and deactivates hHSC in vivo, highlighting the opportunity for evaluating ammonia lowering as a potential therapy in cirrhotic patients with portal hypertension.
authors.affiliation: Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Jalan
authors.affiliation: Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: De Chiara
authors.affiliation: Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK.
authors.forename: Vairappan
authors.initials: V
authors.lastname: Balasubramaniyan
authors.affiliation: Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK.
authors.forename: Fausto
authors.initials: F
authors.lastname: Andreola
authors.affiliation: Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK.
authors.forename: Varun
authors.initials: V
authors.lastname: Khetan
authors.affiliation: Division of Surgery, University College London, Royal Free, London, UK.
authors.forename: Massimo
authors.initials: M
authors.lastname: Malago
authors.affiliation: Regenerative Medicine & Fibrosis Group, Institute for Liver & Digestive Health, University College London, Royal Free, London, UK.
authors.forename: Massimo
authors.initials: M
authors.lastname: Pinzani
authors.affiliation: Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK. Electronic address: r.mookerjee@ucl.ac.uk.
authors.forename: Rajeshwar P
authors.initials: RP
authors.lastname: Mookerjee
authors.affiliation: Regenerative Medicine & Fibrosis Group, Institute for Liver & Digestive Health, University College London, Royal Free, London, UK. Electronic address: k.rombouts@ucl.ac.uk.
authors.forename: Krista
authors.initials: K
authors.lastname: Rombouts
mesh_terms: D000641:Ammonia / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000473:pathology; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D006975:Hypertension, Portal / Q000188:drug therapy*; D008297:Male; D009952:Ornithine / Q000031:analogs & derivatives / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D000641:Ammonia; C572232:ornithine phenylacetate; D009952:Ornithine
keywords: (BDL); (ER); (GS); (HE); (OP); (hHSC); Ammonia; Bile duct ligation; Endoplasmic reticulum stress; Glutamine synthetase; Hepatic encephalopathy; Human hepatic stellate cells; Ornithine phenylacetate; Oxidative stress