title: Exposure of human keratinocytes to ischemia, hyperglycemia and their combination induces oxidative stress via the enzymes inducible nitric oxide synthase and xanthine oxidase.
abstract: BACKGROUND
  Diabetes mellitus is characterized by a chronic hyperglycemia and might cause skin pathologies resulting from an ischemic insult. A variety of mechanisms have been suggested for the damage provided by ischemia-reperfusion injury (IRI) or for hyperglycemic conditions. Yet, the association between IRI and hyperglycemia together in skin has been poorly investigated even thought they are both present in diabetic patients.
  
  
  OBJECTIVE
  To examine the effect of a dual stress combining IRI and hyperglycemia on human keratinocytes-its ability to cause oxidative damage and inflammatory response via the enzymes xanthine oxidase (XO) and inducible nitric oxide synthase (iNOS).
  
  
  METHODS
  HaCaT cells were used as a model to induce IRI and hyperglycemia. In order to assess the oxidative damage, total antioxidant scavenging capacity (TSC) and GSH/GSSG ratio were evaluated. iNOS expression was evaluated and its metabolite nitric oxide was estimated by measuring nitrite levels. XO activity was assessed by uric acid quantification and by superoxide radical formation. Inflammatory response was determined through interleukin-6 secretion.
  
  
  RESULTS
  Our observations demonstrate different responses of the cells exposed to single stress (IRI) compared to dual stress combining also hyperglycemia. However, cells response exhibited similarity during reperfusion, by enhancing iNOS expression as well as superoxide levels. While ischemia led to changes in TSC and redox state, reperfusion restored them to basal levels. IRI also caused the enhancement of secreted IL-6 and uric acid levels.
  
  
  CONCLUSIONS
  iNOS and XO play a major role in IRI and hyperglycemia. Inhibition of one of these enzymes may be beneficial to skin cells under these conditions.
authors.affiliation: Department of Pharmaceutics, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. meitalp@ekmd.huji.ac.il
authors.forename: Meital
authors.initials: M
authors.lastname: Portugal-Cohen
authors.forename: Ron
authors.initials: R
authors.lastname: Kohen
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival; D005947:Glucose / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007511:Ischemia / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D015603:Keratinocytes / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D018384:Oxidative Stress; D015427:Reperfusion Injury / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D013481:Superoxides / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism*
chemical_list: D015415:Biomarkers; C508600:IL6 protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; D009573:Nitrites; D013481:Superoxides; D014527:Uric Acid; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D014969:Xanthine Oxidase; D005978:Glutathione; D005947:Glucose; D019803:Glutathione Disulfide