title: Involvement of SSAO/VAP-1 in oxygen-glucose deprivation-mediated damage using the endothelial hSSAO/VAP-1-expressing cells as experimental model of cerebral ischemia.
abstract: BACKGROUND
  In the acute phase of ischemic stroke, endothelial cells are activated and induce the expression of adhesion molecules. Vascular adhesion protein 1 (VAP-1) is a proinflammatory protein that mediates leukocyte recruitment through its semicarbazide-sensitive amine oxidase (SSAO) activity (EC 1.4.3.21). Plasmatic SSAO activity predicts the appearance of parenchymal hemorrhages after tissue plasminogen activator treatment in ischemic stroke patients, and it is increased as well in hemorrhagic stroke patients. The aim of this study has been to elucidate the role of SSAO/VAP-1 present in endothelial cells during ischemic stroke conditions.
  
  
  METHODS
  Based on the use of endothelial cells expressing, or not expressing, the human SSAO/VAP-1 protein, we have set up an easy ischemic model using oxygen-glucose deprivation (OGD) as an experimental approach to the stroke process. Different OGD and reoxygenation conditions have been analyzed. Western blotting has been used to analyze the activated apoptotic pathways. Several metalloproteinase inhibitors were also used to determine their role in the SSAO/VAP-1 release from the membrane of endothelial cells to the culture media, as a soluble form. Adhesion assays were also performed in order to assess the SSAO/VAP-1-dependent leukocyte adhesion to the endothelia under different OGD and reoxygenation conditions.
  
  
  RESULTS
  Our results show that SSAO/VAP-1 expression increases the susceptibility of endothelial cells to OGD, and that its enzymatic activity, through specific substrate oxidation, increases vascular cell damage under these experimental conditions. Caspase-3 and caspase-8 are activated during the death process. In addition, OGD constitutes a stimulus for soluble SSAO/VAP-1 release, partly mediated by metalloproteinase-2-dependent shedding. Short-time OGD induces SSAO/VAP-1-dependent leukocyte binding on endothelial cells, which is partly dependent on its enzymatic activity.
  
  
  CONCLUSIONS
  Our results show that SSAO/VAP-1 could participate in some of the processes occurring during stroke. Its expression in endothelial cells increases the OGD-associated cell damage. SSAO/VAP-1 mediates also part of the tissue damage during the reoxygenation process by oxidizing its known enzymatic substrate, methylamine. Also, OGD constitutes a stimulus for its soluble-form release, found elevated in many pathological conditions including stroke. OGD induces SSAO-dependent leukocyte-binding activity, which may have consequences in disease progression, since leukocyte infiltration has shown a determinant role in cerebral ischemia. For all the stroke-related processes in which SSAO/VAP-1 participates, it would be an interesting therapeutic target. Therefore, this model will be a very useful tool for the screening of new molecules as therapeutic agents for cerebral ischemia.
authors: Institut de Neurociències i Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
authors: Ping
authors: P
authors: Sun
authors: Montse
authors: M
authors: Solé
authors: Mercedes
authors: M
authors: Unzeta
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000502:physiology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002545:Brain Ischemia / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000502:physiology*; D015687:Cell Hypoxia; D002470:Cell Survival; D042783:Endothelial Cells / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D036904:Leukocyte Rolling / Q000502:physiology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism / Q000633:toxicity; D010100:Oxygen / Q000494:pharmacology; D010624:Phenelzine / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012663:Semicarbazides / Q000494:pharmacology; D014162:Transfection
chemical_list: D015815:Cell Adhesion Molecules; D008744:Methylamines; D011993:Recombinant Fusion Proteins; D012663:Semicarbazides; C010059:carbamylhydrazine; C027451:methylamine; C076668:AOC3 protein, human; D006631:Amine Oxidase (Copper-Containing); C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D005947:Glucose; D010624:Phenelzine; D010100:Oxygen