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 Ser(157) and Ser(239). To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H(2)O(2) 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, H(2)O(2) (100-250 microM) caused a transient increased permeability and pSer(157)-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser(157) phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H(2)O(2). With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H(2)O(2)-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 H(2)O(2)-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG(I) on H(2)O(2)-induced endothelial barrier dysfunction in HPAEC.