title: Angiopoietin-1 requires oxidant signaling through p47phox to promote endothelial barrier defense.
abstract: BACKGROUND
  Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.
  
  
  RESULTS
  Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47-/-) littermates.
  
  
  CONCLUSIONS
  These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.
authors.affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.
authors.forename: Chandra C
authors.initials: CC
authors.lastname: Ghosh
authors.affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.
authors.forename: Aditi
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: Department of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: David
authors.affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.
authors.forename: Katelyn E
authors.initials: KE
authors.lastname: Milam
authors.affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.
authors.forename: Jon T
authors.initials: JT
authors.lastname: Hunter
authors.affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.
authors.forename: Samir M
authors.initials: SM
authors.lastname: Parikh
mesh_terms: D042683:Angiopoietin-1 / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007365:Intercellular Junctions; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors; D051379:Mice; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; C467628:Angpt1 protein, mouse; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013917:Thrombin