Vasodilator Phosphostimulated Protein (VASP) Protects Endothelial Barrier Function During Hypoxia

The endothelial barrier controls the passage of solutes from the vascular space. This is achieved through active reorganization of the actin cytoskeleton. A central cytoskeletal protein involved into this is vasodilator-stimulated phosphoprotein (VASP). However, the functional role of endothelial VASP during hypoxia has not been thoroughly elucidated. We determined endothelial VASP expression through real-time PCR (Rt-PCR), immunhistochemistry, and Western blot analysis during hypoxia. VASP promoter studies were performed using a PGL3 firefly luciferase containing plasmid. Following approval by the local authorities, VASP−/− mice and littermate controls were subjected to normobaric hypoxia (8% O2, 92% N2) after intravenous injection of Evans blue dye. In in vitro studies, we found significant VASP repression in human microvascular and human umbilical vein endothelial cells through Rt-PCR, immunhistochemistry, and Western blot analysis. The VASP promoter construct demonstrated significant repression in response to hypoxia, which was abolished when the binding of hypoxia-inducible factor 1 alpha was excluded. Exposure of wild-type (WT) and VASP−/− animals to normobaric hypoxia for 4 h resulted in an increase in Evans blue tissue extravasation that was significantly increased in VASP−/− animals compared to WT controls. In summary, we demonstrate here that endothelial VASP holds significant importance for endothelial barrier properties during hypoxia

Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor

Protease-activated receptor 1 (PAR-1) mediates thrombin signaling in human endothelial cells. As a G-protein-coupled receptor, PAR-1 transmits thrombin signal through activation of the heterotrimeric G proteins, Gi, Gq, and G12/13. In this study, we demonstrated that zyxin, a LIM-domain-containing protein, is involved in thrombin-mediated actin cytoskeleton remodeling and serum response element (SRE)-dependent gene transcription. We determined that zyxin binds to the C-terminal domain of PAR-1, providing a possible mechanism of involvement of zyxin as a signal transducer in PAR-1 signaling. Data showing that disruption of PAR-1-zyxin interaction inhibited thrombin-induced stress fiber formation and SRE activation supports this hypothesis. Similarly, depletion of zyxin using siRNA inhibited thrombin-induced actin stress fiber formation and SRE-dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. Notably, down-regulation of zyxin did not affect thrombin-induced activation of RhoA or Gi, Gq, and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR-1 that is not mediated by G-proteins. The observation that zyxin targets VASP, a partner of zyxin in regulation of actin assembly and dynamics, to focal adhesions and along stress fibers on thrombin stimulation suggests that zyxin may participate in thrombin-induced cytoskeletal remodeling through recruitment of VASP. In summary, this study establishes a crucial role of zyxin in thrombin signaling in endothelial cells and provides evidence for a novel PAR-1 signaling pathway mediated by zyxin.—Han, J., Liu, G., Profirovic, J., Niu, J., Voyno-Yasenetskaya, T. Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor

Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways

Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y6 receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091–1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G12/13 signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia