title: Early lipopolysaccharide-induced reactive oxygen species production evokes necrotic cell death in human umbilical vein endothelial cells.
abstract: BACKGROUND
  Endothelial dysfunction is a crucial step in the pathogenesis of cardiovascular diseases. Reactive oxygen species (ROS) generated in response to lipopolysaccharide (LPS) during sepsis promotes progressive endothelial failure. Typically, LPS-stimulated leukocytes produce pro-inflammatory cytokines, which trigger endothelial ROS production through NAD(P)H oxidase (Nox) activation, in a process that takes hours. Noteworthy, endothelial cells exposed to LPS may also generate ROS in just a few minutes. However, the mechanisms underlying this early event and its deleterious effect in endothelial function are unknown. Here, we investigated the mechanisms of early LPS-induced ROS generation and its effect in endothelial cell viability.
  
  
  METHODS
  Human umbilical vein endothelial cells were exposed to LPS for 1-40 min to study ROS generation, cytokines expression, and signaling transduction by confocal microscopy, real-time PCR (RT-PCR), western blot, and immunoprecipation. Fourty-eight hour treatments were used to determine cell death by MTT assay, cell counting, and flow cytometry. Contribution of specific Nox isoform was evaluated using a siRNAs approach.
  
  
  RESULTS
  LPS rapidly evoked a cytokine-independent ROS production, eliciting a rapid increase in p47phox phosphorylation by a phospholipase C/conventional protein kinase C and PI3-K signaling. It is noteworthy that the early LPS-induced ROS production triggered significant endothelial necrosis, which was prevented by a previous, but not a posterior, antioxidant treatment. The early LPS-induced ROS production as well as endothelial necrosis was totally dependent of Nox2 and Nox4 activity.
  
  
  CONCLUSIONS
  Endothelial cells exposure to LPS triggers an early ROS production. Remarkably, this single early ROS production is enough to generate extensive endothelial cell death by necrosis dependent on the activity of Nox2 and Nox4. Because, in sepsis, ROS production can cause endothelial dysfunction, results here provided may be relevant when considering the development of strategies for sepsis therapy.
authors.affiliation: Departamento de Ciencias Biologicas, Facultad de Ciencias de la Salud, Universidad Andres Bello, Santiago, Chile. fsimon@unab.cl
authors.forename: Felipe
authors.initials: F
authors.lastname: Simon
authors.forename: Ricardo
authors.initials: R
authors.lastname: Fernández
mesh_terms: D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009336:Necrosis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D016207:Cytokines; D017931:DNA Primers; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases