title: Reactive oxygen species mediate endotoxin-induced human dermal endothelial NF-kappaB activation.
abstract: BACKGROUND
  Microvascular endothelial cell "activation" by endotoxin is an early and critical phenomenon underlying organ dysfunction related to sepsis. Dermal endothelial cells contribute to many of the manifestations of septic shock, such as cutaneous interstitial edema and loss of peripheral vasomotor regulation. Human dermal endothelial cell activation by endotoxin (lipopolysaccharide [LPS]) is characterized by the generation of reactive oxygen species (ROS) that enhance nuclear translocation of the transcription factor kappa-B (NF-kappaB).
  
  
  METHODS
  We tested our hypothesis by stimulating human dermal microvascular endothelial cells (HMEC.1) with endotoxin and assaying for endothelial generation of ROS and nuclear translocation of NF-kappaB subunits. HMEC.1 cultures were treated individually with LPS, hydrogen peroxide, or xanthine, xanthine oxidase, and ferrous sulfate (xanthine/XO/Fe(2+)). Nuclear proteins were isolated and consensus sequence binding was assessed by electrophoretic mobility shift assay (EMSA). 2',7'-Dichlorofluorescin diacetate and confocal microscopy were used to examine ROS production in LPS-stimulated HMEC.1.
  
  
  RESULTS
  Nuclear translocation of the p65/p50 NF-kappaB heterodimer was detectable 30 min after stimulation with LPS alone or the xanthine/XO/Fe(2+) combination, but not with hydrogen peroxide. Antioxidant N-acetylcysteine (NAC) inhibited LPS-stimulated ROS production in HMEC.1. Antioxidant prior to or simultaneously with LPS exposure, but not following LPS, also prevented NF-kappaB activation. NAC was ineffective at inhibiting NF-kappaB translocation at increased LPS concentrations.
  
  
  CONCLUSIONS
  Dermal endothelial cells, a microvascular cell type that may contribute to the systemic response to blood-borne endotoxemia, generate ROS in the absence of other inflammatory cells. These LPS-activated endothelial cells, in turn, rapidly translocate transcription factor NF-kappaB to cell nuclei, a process regulated in part by intracellular ROS.
authors.affiliation: Department of Surgery, Division of Burns, Trauma, Critical Care, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
authors.forename: Emily L
authors.initials: EL
authors.lastname: Chan
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Murphy
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D005296:Ferrous Compounds / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012867:Skin / Q000098:blood supply*; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
chemical_list: D005296:Ferrous Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D019820:Xanthine; C029569:diacetyldichlorofluorescein; C020748:ferrous sulfate; D006861:Hydrogen Peroxide; D014969:Xanthine Oxidase; D000111:Acetylcysteine