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

Abstract

Rentsendorj O, Mirzapoiazova T, Adyshev D, Servinsky LE, Renné T, Verin AD, Pearse DB. Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function. Am J Physiol Lung Cell Mol Physiol 294: L686-L697, 2008. First published February 15, 2008 doi:10.1152/ajplung.00417.2007.-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 PKGI barrier protection because VASP is a cytoskeletal phosphorylation target of PKGI expressed 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 Ser 157 and Ser 239 . 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 PKGI expression introduced by adenoviral infection (Ad.PKG). In the absence of PKGI expression, H2O2 (100 -250 M) 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 H2O2. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H2O2-induced barrier dysfunction but had no effect on cGMPmediated 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/PKGI to attenuate H2O2-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKGI on H2O2-induced endothelial barrier dysfunction in HPAEC.protein kinase G; cAMP; protein kinase A; small interfering RNA; H2O2

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

“…Immunoblots for PDE2A, GFP, and iNOS were performed as previously described (34,39). Briefly, 293A cells were lysed in a Laemmli sample buffer (Bio-Rad) containing 4 mM Na 3VO4, 40 mM NaF, 1% mercaptoethanol, and 1:200 dilution of protease inhibitor cocktail (Sigma-Aldrich).…”

The role of cytoskeleton in the regulation of vascular endothelial barrier function

“…VASP was long known as target for PKA and PKG phoshorylation; however, its involvement in PKA/PKG-mediated barrier protection remains a subject for further investigation. Although the level of phospho-VASP in endothelial junctional complexes seems to correlate with barrier enhancement in adenosinetreated endothelial cells [Comerford et al, 2002], other studies fail to show that elevated VASP phosphorylation is critical for PKA/PKG-dependent barrier enhancement [Schlegel et al, 2007;Rentsendorj et al, 2008].…”

cGMP-Dependent Protein Kinase in the Regulation of Cardiovascular Functions

“…Studies also support a barrier protection effect in the vascular endothelium (Moldobaeva et al, 2006;Rentsendorj et al, 2008). Increasing evidence also suggests that PKG exerts negative inotropic and antihypertrophic actions in the heart (Takimoto et al 2005;Yang et al, 2007;Zhang et al, 2010) as well as a cardioprotective action against ischemia-reperfusion injury (Das et al, 2008;Juhaszova et al, 2009;Xi et al, 2010).…”

“…In human pulmonary artery endothelial cells infected with adenovirus encoding PKG I the cGMP analog prevents the increase in endothelial permeability caused by H 2 O 2. The barrier protection effect was not affected by inhibition of the expression of VASP, a PKG substrate (Moldobaeva et al, 2006;Rentsendorj et al, 2008).…”

“…There are only limited studies on the role of PKG in the regulation of endothelial function, which is related to eNOS activity and endothelial permeability (Butt et al, 2000;Draijer et al, 1995;Moldobaeva et al, 2006;Rentsendorj et al, 2008). Studies using recombinant human eNOS suggest that the enzyme can be phosphorylated at Ser 1177 , Ser 633 , Thr 495 and activated by PKG II in a manner independent of Ca 2+ and calmodulin (Butt et al, 2000).…”