Reactive oxygen species mediate endotoxin-induced human dermal endothelial NF-κB activation

Emily L. Chan, Joseph T. Murphy

Research output: Contribution to journalArticlepeer-review

36 Scopus citations


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-κB). 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-κB subunits. HMEC.1 cultures were treated individually with LPS, hydrogen peroxide, or xanthine, xanthine oxidase, and ferrous sulfate (xanthine/XO/Fe2+). 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-κB heterodimer was detectable 30 min after stimulation with LPS alone or the xanthine/XO/Fe2+ 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-κB activation. NAC was ineffective at inhibiting NF-κB 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-κB to cell nuclei, a process regulated in part by intracellular ROS.

Original languageEnglish (US)
Pages (from-to)120-126
Number of pages7
JournalJournal of Surgical Research
Issue number1
StatePublished - May 1 2003


  • 2′,7′-dichlorofluorescin diacetate
  • Human dermal microvascular endothelial cells
  • Lipopolysaccharide
  • NF-κB
  • Reactive oxygen species

ASJC Scopus subject areas

  • Surgery


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