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This Article Full Text Full Text (PDF) All Versions of this Article: 294/4/L686    most recent 00417.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rentsendorj, O. Articles by Pearse, D. B. Search for Related Content PubMed PubMed Citation Articles by Rentsendorj, O. Articles by Pearse, D. B.

Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function

¹Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University, Baltimore, Maryland; ²Department of Medicine, Section of Pulmonary and Critical Care, The University of Chicago Center for Integrative Science, Chicago, Illinois; ³Institute for Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-University Würzburg, Würzburg, Germany; and ⁴Vascular Biology Center, Medical College of Georgia, Augusta, Georgia

Submitted 9 October 2007 ; accepted in final form 13 February 2008

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG_I). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG_I barrier protection because VASP is a cytoskeletal phosphorylation target of PKG_I expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG_I-mediated phosphorylation of Ser¹⁵⁷ and Ser²³⁹. To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H₂O₂ in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKG_I expression introduced by adenoviral infection (Ad.PKG). In the absence of PKG_I expression, H₂O₂ (100–250 µM) caused a transient increased permeability and pSer¹⁵⁷-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser¹⁵⁷ phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H₂O₂. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H₂O₂-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKG_I. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG_I to attenuate H₂O₂-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG_I on H₂O₂-induced endothelial barrier dysfunction in HPAEC.

protein kinase G; cAMP; protein kinase A; small interfering RNA; H₂O₂

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