Vasodilator-stimulated phosphoprotein: crucial for activation of Rac1 in endothelial barrier maintenance

The endothelium provides a selective barrier separating blood vessels and the interstitial tissue.1 The intercellular cleft is sealed by tight junctions and mechanical cohesion is provided by adherens junctions. Additionally, integrin-mediated anchoring of endothelial cells to the extracellular matrix contributes to endothelial barrier functions.1 All of these junctions are composed of transmembrane proteins that are linked directly or indirectly to the actin cytoskeleton via adaptor proteins. Therefore, it is conceivable that actin dynamics modulate junctional integrity and endothelial barrier functions in vivo.2 Because GTPases of the Rho family regulate cell adhesion and cytoskeletal reorganization, they are important regulators of the endothelial barrier.3 In particular, Rac1 is well known to be required for maintenance of endothelial barrier functions. Recently, the actin-binding vasodilator-stimulated phosphoprotein (VASP) has been recognized to act in concert with Rac1 in endothelial barrier regulation.4,5 Here, we will discuss the current knowledge on the mechanisms underlying VASP-dependent endothelial barrier regulation. The 46 kDa protein VASP belongs to the ENA/VASP family proteins which are known to be involved in the regulation of actin dynamics, cell adhesion, and cell migration.6 The ENA/VASP family members share a tripartite structure, consisting of (i) an N-terminal segment (EVH1), necessary for interaction with various junction-associated proteins, (ii) a central core containing a polyproline-rich region, and (iii) a C-terminal segment (EVH2) for tetramerization and binding to filamentous actin.6,7 In endothelial cells, VASP is phosphorylated by serine/threonine kinases, such as cAMP- and cGMP-dependent protein kinases (PKA and PKG), as well as by AMP-activated protein kinase (AMPK),8,9 which target residues of endothelial VASP. PKA and PKG phosphorylate serine 157 (S157) and serine 239 (S239), respectively, whereas AMPK selectively phosphorylates threonine 278 (T278).8,10 Phosphorylation of VASP at S157 induces VASP localization to the cell periphery but has a minor impact on F-actin assembly, whereas VASP phosphorylation at S239 or T278 was shown to be associated with impaired actin filament formation.10 Meanwhile, it is well established that VASP is critical for maintenance of endothelial barrier functions in vivo and in vitro.4,5,11–17 Evidence came from investigations on VASP-deficient endothelial cells in vitro when permeability coefficients calculated from FITC-dextran fluxes across endothelial monolayers were significantly increased in VASP-deficient cells compared with wild-type (WT) endothelium.4 Interestingly, when studied by immunostaining, neither tight junction nor adherens junction integrity appeared to be affected by VASP or ENA/VASP deficiency.4,13 However, laser tweezer experiments revealed that VASP-deficient endothelium displayed reduced integrin-mediated binding which was paralleled by a loss of focal adhesions at the cell periphery.12 Moreover, cytoskeletal anchorage of VE-cadherin and β1-integrin was reduced in VASP-deficient endothelium as revealed by Triton extraction. Furman et al.13 generated ENA/VASP triple null mice and found that ENA/VASP-deficient mice exhibited severe embryonic oedema which was the first in vivo study demonstrating that ENA/VASP family proteins are crucial to maintain endothelial barrier properties. Additionally, transfection of endothelial cells with a construct that inactivates ENA/VASP proteins resulted in increased endothelial permeability.13 In another study, siRNA-induced repression of VASP in endothelial cells increased paracellular permeability, whereas overexpression of VASP protected endothelial barrier functions.15 Recent studies revealed that VASP expression is altered under pathophysiological conditions and that this mechanism contributes to endothelial barrier breakdown. Rosenberger et al.15 demonstrated hypoxia-dependent binding of HIF-1 to the human VASP promoter and identified HIF-1-dependent repression of VASP underlying hypoxia-regulated barrier dysfunction. Moreover, tumour necrosis factor-α, interleukin 1β, and interleukin 6 in endothelial cells led to binding of NF-κB to the VASP promoter and down-regulation of VASP. Conversely, overexpression of VASP completely blocked increased endothelial permeability in response to these cytokines.14 Previously, VASP-dependent stabilization of endothelial barrier functions has primarily been attributed to mechanisms directly affecting actin dynamics. VASP deficiency or protein inactivation in endothelial cells led to reduced F-actin content.13 In addition, ENA/VASP-deficient endothelium failed to reorganize stress fibres in response to physiological shear stress and G-actin incorporation at barbed ends of stress fibres was also dramatically reduced.13 Another study used immunoprecipitation to show that VASP interacts with the SH3 domain of α-II-spectrin in endothelial cells,11 suggesting that α-II-spectrin–VASP complexes regulate the cortical actin cytoskeleton and thereby contribute to endothelial barrier stabilization. Since in this study, phosphorylation of VASP at S157 by PKA was found to inhibit the formation of α-II-spectrin–VASP complexes, this scenario would result in increased endothelial permeability. However, because cAMP and PKA usually are associated with stabilization of the endothelial barrier,3 the relevance of this mechanism for endothelial barrier regulation remains to be defined. We observed that VASP was recruited to cell borders in microvascular endothelium under conditions of cytotoxic necrotizing factor-1 (CNF-1)-mediated Rac1 activation.4,18 Similarly, in endothelium transfected with a constitutively active pEGFP-Rac1 mutant, VASP and Rac1 colocalized at cell borders.4,5 Therefore, we initially hypothesized that VASP may act as a downstream effector of Rac1 to mediate barrier-protective effects via reorganization of the cortical actin cytoskeleton. However, Rac1 activation by CNF-1 was equally effective to stabilize endothelial barrier functions, to induce actin reorganization, and to cause recruitment of cortactin—a downstream effector of Rac1—to cell borders in both VASP-deficient and WT endothelium.4 Because VASP-deficient endothelial cells displayed a reduced baseline activity of Rac1 when compared with WT endothelium, this observation led to the new hypothesis that VASP was required for Rac1 activation. This was substantiated by experiments in which reconstitution of VASP in VASP-deficient endothelium significantly augmented Rac1 activity and increased barrier properties.5 In addition, cAMP-mediated Rac1 activation in VASP-deficient endothelial cells was impaired. The idea that VASP participates in Rac1 activation was also supported by experiments where VASP and an S239D phosphomimetic VASP mutant were overexpressed in WT endothelium which resulted in increased Rac1 activity.17 Moreover, it was shown in vivo and in vitro that the barrier-stabilizing effects of cGMP and PKG are mediated by Rac1 activation, a process which required VASP phosphorylation at S239.17 Thus, these data provide evidence for a completely new mechanism by which VASP stabilizes the endothelial barrier, i.e. by facilitating activation of Rac1 downstream of PKA and PKG. At present, it remains largely unclear how exactly phosphorylated VASP contributes to Rac1 activation. The observation that VASP is involved in cAMP-mediated endothelial barrier stabilization by facilitating Rac1 activation supports the idea that VASP upon S157 phosphorylation by PKA translocates to endothelial tight junctions to mediate the barrier-stabilizing effects of cAMP16 (Figure 1). Because both PKA-anchoring proteins (AKAPs) and VASP were required for cAMP-induced Rac1 activation,5 VASP may act as part of a scaffolding protein complex controlling GEFs or GAPs to activate Rac1. Moreover, in VASP-deficient cells, recruitment of integrins to the cell periphery was also compromised.12 Because this process is thought to contribute to Rac1 activation and strengthening of endothelial adherens junctions via interaction of paxillin with β-catenin,19 it is possible that integrin recruitment to the cell periphery and proper adhesion of integrins to the extracellular matrix are prerequisites to enable cAMP-mediated Rac1 activation and stabilization of adherens junctions. Mechanisms of VASP-dependent endothelial barrier regulation. VASP stabilizes endothelial barrier functions by cAMP- and cGMP-mediated signalling pathways. VASP via zyxin and vinculin is associated with actin filaments, whereas paxillin is linked to endothelial adherens junctions via β-catenin. S157 phosphorylation of VASP via PKA induces translocation of VASP to cell junctions and binding of VASP to ZO-1. cAMP- and cGMP-mediated signalling may facilitate cortical Rac1 activation in a VASP-dependent manner which appears to be crucial for endothelial barrier stabilization. FAK, focal adhesion kinase; PKA, cAMP-dependent protein kinase A; PKG, cGMP-dependent protein kinase G; EPAC, exchange protein directly activated by cAMP; ZO-1/2/3, zonula occludens protein1/2/3; α, β = α- and β-catenin; ECM, extracellular matrix. In conclusion, VASP is critically involved in endothelial barrier stabilization by regulation of Rac1 activity downstream of both cAMP- and cGMP-mediated signalling. Further studies are required to elucidate the molecular mechanisms underlying this process. We are grateful to Michael Christof for figure illustration. The studies from our laboratory were supported by grants from the Deutsche Forschungsgemeinschaft (SFB 688, TP A4). Conflict of interest: none declared.