Role of vasodilator‐stimulated phosphoprotein in protein kinase A‐induced changes in endothelial junctional permeability

Abstract

At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation. E ndothelial cells that line blood vessels are the primary determinants of vascular permeability [1], and structural and functional integrity of the endothelium is crucial in determining overall vascular permeability. Endothelial injury, such as occurs during leukocyte-endothelial interactions, may result in increased paracellular permeability, decreased barrier function, and subsequent intravascular loss of fluid and local tissue edema [2,3]. During transendothelial migration (TEM), polymorphonuclear leukocyte (PMN)-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent sealing of endothelial monolayers [4,5]. Elevated levels of cAMP could enhance barrier function by activating protein kinase A (PKA) and subsequent phosphorylation of key complex-associated proteins [6,7]. At present, the intracellular signals that link cAMP elevation and vascular barrier function have not been identified.Regulation of junctional permeability is coupled to actin-based systems, a...

Prostaglandins PGE2 and PGI2 promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

“…Despite the extensive investigation of receptor splice variants, tissue specificity and differential activation, physiological significance of these differences remains to be explored Our results clearly demonstrate barrier-enhancing effects of PG in the pulmonary EC. Although involvement of PKA-dependent mechanisms in the PG protective effects on vascular endothelium has been recognized [19,51,52], the role of recently discovered Epac-Rap pathway in PG-mediated barrier regulation is not well understood.…”

“…In addition PG-induced activation of PKA may cause actin cytoskeletal remodeling by Rac-independent mechanisms, for example, via phosphorylation of myosin light chain kinase and reduction of its enzymatic activity [38,65], or via phosphorylation of Rho regulatory protein RhoGDI leading to Rho inhibition [21,38], or via PKA-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) leading to the structural relaxation of the actin cytoskeleton linked to tight junctions via VASP -ZO-1 complex, which result in enhanced endothelial barrier function [52]. Importantly, our studies revealed strong protective effects of PG in the pulmonary EC model of thrombininduced barrier hyperpermeability and in the murine model of ventilator-induced lung injury ( Figure 8).…”

Activation of tumor suppressor protein PTEN and induction of apoptosis are involved in cAMP-mediated inhibition of cell number in B92 glial cells

“…Vasodilator-stimulated phosphoprotein (VASP) is a critical factor in regulating actin dynamics, and an increase in phospho-VASP is related to the increased levels of intracellular cAMP [4,10]. Treatment of B92 cells with IBMX and ISO increased phospho-VASP protein levels significantly in 10 min and slightly in 72 h compared with controls (Fig.…”

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

“…VASP can associate with tight junctions via ZO-1 [Comerford et al, 2002], and bind with adherence junction via α-catenin [Reinhard et al, 2001]. These facts along with VASP ability to promote actin polymerization and assembly are thought to be a basis for VASPdependent regulation of cytoskeleton organization.…”

“…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].…”