title: Nox2-dependent glutathionylation of endothelial NOS leads to uncoupled superoxide production and endothelial barrier dysfunction in acute lung injury.
abstract: Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.
authors.affiliation: University of Pittsburgh School of Medicine Department of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, Pittsburgh, Pennsylvania;
authors.forename: Feng
authors.initials: F
authors.lastname: Wu
authors.affiliation: University of Pittsburgh School of Medicine Department of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, Pittsburgh, Pennsylvania;
authors.forename: William S
authors.initials: WS
authors.lastname: Szczepaniak
authors.affiliation: University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania; University of Pittsburgh School of Medicine Department of Pharmacology, Pittsburgh, Pennsylvania;
authors.forename: Sruti
authors.initials: S
authors.lastname: Shiva
authors.affiliation: University of Pittsburgh School of Medicine Department of Surgery, Pittsburgh, Pennsylvania;
authors.forename: Huanbo
authors.initials: H
authors.lastname: Liu
authors.affiliation: University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania;
authors.forename: Yinna
authors.initials: Y
authors.lastname: Wang
authors.affiliation: University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania;
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.affiliation: University of Pittsburgh School of Medicine Department of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, Pittsburgh, Pennsylvania;
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania; University of Pittsburgh School of Medicine Department of Anesthesiology, Pittsburgh, Pennsylvania.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Kelley
authors.affiliation: University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania; University of Pittsburgh School of Medicine Department of Surgery, Pittsburgh, Pennsylvania;
authors.forename: Alex F
authors.initials: AF
authors.lastname: Chen
authors.affiliation: University of Pittsburgh School of Medicine Department of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, Pittsburgh, Pennsylvania; University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania;
authors.forename: Mark T
authors.initials: MT
authors.lastname: Gladwin
authors.affiliation: University of Pittsburgh School of Medicine Department of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, Pittsburgh, Pennsylvania; University of Pittsburgh Vascular Medicine Institute, Pittsburgh, Pennsylvania; mcverrybj@upmc.edu.
authors.forename: Bryan J
authors.initials: BJ
authors.lastname: McVerry
mesh_terms: D055371:Acute Lung Injury / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology* / Q000473:pathology; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity*; D008168:Lung / Q000098:blood supply / Q000201:enzymology / Q000473:pathology; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013481:Superoxides / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
chemical_list: D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; C440499:lipopolysaccharide, Escherichia coli O111 B4; D013481:Superoxides; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C056558:CYBB protein, human; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C419706:NOSIP protein, human; D044767:Ubiquitin-Protein Ligases; D005978:Glutathione
keywords: NADPH oxidase; acute respiratory distress syndrome; endothelial barrier function; endothelial nitric oxide synthase uncoupling; nitric oxide