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

Abstract

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKGI). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKGIbarrier protection because VASP is a cytoskeletal phosphorylation target of PKGIexpressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKGI-mediated phosphorylation of Ser157and Ser239. To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H2O2in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKGIexpression introduced by adenoviral infection (Ad.PKG). In the absence of PKGIexpression, 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 Ser157phosphorylation 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 PKGI. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKGIto attenuate H2O2-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKGIon H2O2-induced endothelial barrier dysfunction in HPAEC.