title: Group V phospholipase A2 mediates barrier disruption of human pulmonary endothelial cells caused by LPS in vitro.
abstract: We examined the functional role of 14-kD secretory group V phospholipase A(2) (gVPLA(2)) on the barrier function of pulmonary endothelial cells (ECs) after LPS activation in vitro. Expression of gVPLA(2) was elicited by 20 ng/ml LPS as demonstrated by increased (1) mRNA, (2) protein content, and (3) cell surface expression of gVPLA(2) within 4 hours. The effect of LPS on EC barrier function was measured by transendothelial monolayer electrical resistance (TER). LPS increased permeability across EC monolayers at 2-3 hours, and was sustained for 10 hours or more. Blockade of gVPLA(2) with mouse monoclonal 3G1 (MCL-3G1) monoclonal antibody directed against gVPLA(2) inhibited EC barrier dysfunction elicited by LPS in a time- and concentration-dependent manner; control IgG had no effect on TER. Like LPS, exogenous gVPLA(2) caused increased EC permeability in a time- and concentration-dependent manner; neither gIIaPLA(2), a close homolog of gVPLA(2), nor W31A, an inactive mutant of gVPLA(2), caused a decrease in EC TER. Immunofluorescence analysis revealed comparable F-actin stress fiber and intercellular gap formation for ECs treated with either gVPLA(2) or LPS. Treatment with gVPLA(2) disrupted vascular endothelial-cadherin junctional complexes on ECs. Coincubation of ECs with MCL-3G1 substantially attenuated the structural changes caused by gVPLA(2) or LPS. We demonstrate that (1) gVPLA(2) is constitutively expressed in ECs and is up-regulated after LPS activation, (2) endogenously secreted gVPLA(2) from ECs after LPS increases EC permeability through F-actin and junctional complex rearrangement, and (3) inhibition of endogenous gVPLA(2) from ECs is sufficient to block disruption of the EC barrier function after LPS in vitro.
authors.affiliation: Section of Pulmonary & Critical Care Medicine, Department of Medicine, University of Chicago, IL 60637, USA. sdudek@uic.edu
authors.forename: Steven M
authors.initials: SM
authors.lastname: Dudek
authors.forename: Nilda M
authors.initials: NM
authors.lastname: Muñoz
authors.forename: Anjali
authors.initials: A
authors.lastname: Desai
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Osan
authors.forename: Angelo Y
authors.initials: AY
authors.lastname: Meliton
authors.forename: Alan R
authors.initials: AR
authors.lastname: Leff
mesh_terms: D000199:Actins / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002478:Cells, Cultured; D003911:Dextrans / Q000737:chemistry; D042783:Endothelial Cells / Q000166:cytology*; D054505:Group V Phospholipases A2 / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000378:metabolism*; D008168:Lung / Q000201:enzymology*; D008833:Microcirculation; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D003911:Dextrans; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054505:Group V Phospholipases A2
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