Vasodilator Stimulated Phosphoprotein

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Ulrich Walter - One of the best experts on this subject based on the ideXlab platform.

  • phosphorylation of Vasodilator Stimulated Phosphoprotein prevents platelet neutrophil complex formation and dampens myocardial ischemia reperfusion injury
    Circulation, 2011
    Co-Authors: David Köhler, Ulrich Walter, Rainer Lehmann, Andreas Straub, Thomas Weissmuller, Marion Faigle, Sarah Bender, H P Wendel, Julia Kurz, Kai Zacharowski
    Abstract:

    Background—Recent work has suggested that the formation of platelet-neutrophil complexes (PNCs) aggravates the severity of inflammatory tissue injury. Given the importance of Vasodilator-Stimulated Phosphoprotein (VASP) for platelet function, we pursued the role of VASP on the formation of PNCs and its impact on the extent of myocardial ischemia-reperfusion (IR) injury. Methods and Results—In initial in vitro studies we found that neutrophils facilitated the movement of platelets across endothelial monolayers. Phosphorylation of VASP reduced the formation of PNCs and transendothelial movement of PNCs. During myocardial IR injury, VASP−/− animals demonstrated reduced intravascular formation of PNCs and reduced presence of PNCs within the ischemic myocardial tissue. This was associated with reduced IR injury. Studies using platelet transfer and bone marrow chimeric animals showed that hematopoietic VASP expression was crucial for the intravascular formation of PNCs the presence of PNCs within ischemic myoca...

  • Vasodilator Stimulated Phosphoprotein deficiency potentiates par 1 induced increase in endothelial permeability in mouse lungs
    Journal of Cellular Physiology, 2011
    Co-Authors: J. Profirovic, Ulrich Walter, Alexandra V Andreeva, Radu Neamu, Sasha Pavlovic, Stephen M Vogel, Tatyana A Voynoyasenetskaya
    Abstract:

    Vasodilator-Stimulated Phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. The function of VASP in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP−/− mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP−/− mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may be correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function. J. Cell. Physiol. 226: 1255–1264, 2011. © 2010 Wiley-Liss, Inc.

  • deficiency of Vasodilator Stimulated Phosphoprotein vasp increases blood brain barrier damage and edema formation after ischemic stroke in mice
    PLOS ONE, 2010
    Co-Authors: Peter Kraft, Ulrich Walter, Peter M. Benz, Madeleine Austinat, Marc Brede, Kai Schuh, Guido Stoll, Christoph Kleinschnitz
    Abstract:

    Background Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-Stimulated Phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification.

  • Vasodilator Stimulated Phosphoprotein regulates inside out signaling of β2 integrins in neutrophils
    Journal of Immunology, 2010
    Co-Authors: Ravi K Deevi, Kai Schuh, Madhuri Koneydash, Adrien Kissenpfennig, James A Johnston, Ulrich Walter
    Abstract:

    The monomeric GTPase Rap1 controls functional activation of β2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 and inside-out signaling of β2 integrins. We found that fMLP-induced activation of Rap1 in human polymorphonuclear leukocytes or neutrophils and differentiated PLB-985 cells was blocked by inhibitors of the NO/guanosine-3′,5′-cyclic monophosphate–dependent protein kinase (cGKI) pathway [N-(3-(aminomethyl)benzyl)acetamidine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, DT-3 peptide, 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphothioate, Rp-isomer triethylammonium salt–guanosine-3′,5′-cyclic monophosphate], indicating that the downstream signaling events in Rap1 activation involve the production of NO and guanosine-3′,5′-cyclic monophosphate, as well as the activation of cGKI. Silencing the expression of Vasodilator-Stimulated Phosphoprotein (VASP), a substrate of cGKI, in resting PLB-985 cells or mice neutrophils led to constitutive activation of Rap1. In parallel, silencing VASP in differentiated PLB-985 cells led to recruitment of C3G, a guanine nucleotide exchange factor for Rap1, to the plasma membrane. Expression of murine GFP-tagged phosphodeficient VASP Ser235Ala mutant (murine serine 235 of VASP corresponds to human serine 239) in PLB-985 cells blunted fMLP-induced translocation of C3G to the membrane and activation of Rap1. Thus, bacterial fMLP triggers cGKI-dependent phosphorylation of human VASP on serine 239 and, thereby, controls membrane recruitment of C3G, which is required for activation of Rap1 and β2 integrin-dependent antibacterial functions of neutrophils.

  • inflammation associated repression of Vasodilator Stimulated Phosphoprotein vasp reduces alveolar capillary barrier function during acute lung injury
    The FASEB Journal, 2009
    Co-Authors: Janek Henes, Ulrich Walter, David Köhler, Therese Eldh, Julio C Morotegarcia, Marthe A Schmit, Valbona Mirakaj, Louise Glover, Jorn Karhausen
    Abstract:

    Acute lung injury (ALI) is an inflammatory disorder associated with reduced alveolar-capillary barrier function, increased pulmonary vascular permeability, and infiltration of leukocytes into the alveolar space. Pulmonary function might be compromised, its most severe form being the acute respiratory distress syndrome. A protein central to physiological barrier properties is Vasodilator-Stimulated Phosphoprotein (VASP). Given the fact that VASP expression is reduced during periods of cellular hypoxia, we investigated the role of VASP during ALI. Initial studies revealed reduced VASP expressional levels through cytokines in vitro. Studies in the putative human VASP promoter identified NF-κB as a key regulator of VASP transcription. This VASP repression results in increased paracellular permeability and migration of neutrophils in vitro. In a model of LPS-induced ALI, VASP−/− mice demonstrated increased pulmonary damage compared with wild-type animals. These findings were confirmed in a second model of vent...

Giovanni Pitari - One of the best experts on this subject based on the ideXlab platform.

  • Vasodilator Stimulated Phosphoprotein biomarkers are associated with invasion and metastasis in colorectal cancer
    Biomarkers in cancer, 2018
    Co-Authors: Giovanni Pitari, Paolo Cotzia, Ruth Birbe, Wendy Rizzo, Alessandro Bombonati, Juan P Palazzo, Charalambos C Solomides, Anthony P Shuber, Frank A Sinicrope, David S. Zuzga
    Abstract:

    Background and Aims: The benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) patients remains unclear, emphasizing the need for improved prognostic biomarkers to identify patients at risk of metastatic recurrence. To address this unmet clinical need, we examined the expression and phosphorylation status of the Vasodilator-Stimulated Phosphoprotein (VASP) in CRC tumor progression. VASP, a processive actin polymerase, promotes the formation of invasive membrane structures leading to extracellular matrix remodeling and tumor invasion. Phosphorylation of VASP serine (Ser) residues 157 and 239 regulate VASP function, directing subcellular localization and inhibiting actin polymerization, respectively. Methods: The expression levels of VASP protein, pSer157-VASP, and pSer239-VASP were determined by immunohistochemistry in tumors and matched normal adjacent tissue from 141 CRC patients, divided into 2 cohorts, and the association of VASP biomarker expression with clinicopathologic features and disease recurrence was examined. Results: We report that changes in VASP expression and phosphorylation were significantly associated with tumor invasion and disease recurrence. Furthermore, we disclose a novel 2-tiered methodology to maximize VASP positive and negative predictive value performance for prognostication. Conclusion: VASP biomarkers may serve as prognostic biomarkers in CRC and should be evaluated in a larger clinical study.

  • serine phosphorylation of Vasodilator Stimulated Phosphoprotein vasp regulates colon cancer cell survival and apoptosis
    Life Sciences, 2015
    Co-Authors: Lynette K Rogers, Giovanni Pitari
    Abstract:

    Abstract Aims In colon cancer, disease recurrence and death are associated with abnormal tumor cell survival. Vasodilator-Stimulated Phosphoprotein (VASP) is an actin binding protein regulating cell shape and polarity through the F-actin cytoskeleton, whose activity is controlled by cAMP-dependent phosphorylation at Ser157 and cGMP-dependent phosphorylation at Ser239. This study examined the role of differential VASP Ser phosphorylation in regulating cell survival and apoptosis in human colon carcinoma cells. Main methods Selective inhibition of VASP Ser157 or Ser239 phosphorylation in colon cancer cells was performed with specific phosphomutant constructs. F-actin organization was examined by confocal microscopy, and the balance of cell survival and death assessed by measuring acridine orange and ethidium bromide staining, caspase-3 and BAD-pS112 expression and DNA fragmentation. Key findings In human colon carcinoma cells suppression of VASP Ser157 phosphorylation reduced F-actin content and survival and increased apoptosis, while inhibition of VASP Ser239 phosphorylation increased F-actin content and survival and reduced cell death. Also, while 8Br-cAMP induced VASP Ser157 phosphorylation and reduced cell death, treatments with 8CPT-cGMP elevated VASP Ser239 phosphorylation and promoted apoptosis. Significance These findings suggest that differential VASP Ser phosphorylation represents a unique therapeutic target to control cell survival and death behavior in colon cancer. In particular, pharmacological manipulation of VASP Ser phosphorylation could be exploited to affect the malignant actin cytoskeleton and induce apoptosis in colorectal cancer cells.

  • abstract 3944 serine phosphorylation of Vasodilator Stimulated Phosphoprotein regulates the tumorigenic potential of colon cancer cells
    Cancer Research, 2013
    Co-Authors: Inna Chervoneva, Giovanni Pitari
    Abstract:

    Colorectal cancer is one of the most common and deadly cancers in developed nations. Molecular mechanisms precisely defining colorectal tumorigenesis and cancer progression remain unclear, an obstacle for innovative research applications. The Vasodilator-Stimulated Phosphoprotein (VASP) is an actin-binding protein which critically regulates the cytoskeleton and its dependent functions, including cell shape, adhesion and migration. Of relevance, VASP Ser phosphorylation becomes deregulated in colorectal transformation and promotes invasive protrusion dynamics. Here, VASP Ser phosphorylation was selectively inhibited in colon carcinoma cells employing dominant negative mutants. Effects on cell tumorigenic potential were examined with two mouse models of colorectal cancer. Compared to control conditions, suppression of VASP Ser239 phosphorylation resulted in an enhanced cancer cell capability to establish tumor colonies in both the subcutaneous and peritoneal xenograft model. Increased tumorigenicity reflected, in part, hyper proliferative kinetics of cancer cells bearing VASP Ser239 mutants, which exhibited accelerated growth and DNA synthesis rates. In contrast, selective inhibition of VASP Ser157 slowed cell proliferation and reduced the in vivo tumorigenic potential of colon carcinoma cells, compared to respective controls. These observations support the suggestion that VASP Ser phosphorylation represents a fine signaling mechanism to control intestinal tumorigenesis and invasion, a novel paradigm for targeted anticancer strategies in patients with colorectal cancer. Citation Format: Mehboob Ali, Inna Chervoneva, Giovanni M. Pitari. Serine phosphorylation of Vasodilator-Stimulated Phosphoprotein regulates the tumorigenic potential of colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2013-3944

  • phosphorylation of Vasodilator Stimulated Phosphoprotein ser239 suppresses filopodia and invadopodia in colon cancer
    International Journal of Cancer, 2012
    Co-Authors: David S. Zuzga, Alessandro Bombonati, Joshua Peltaheller, Peng Li, Scott A Waldman, Giovanni Pitari
    Abstract:

    In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through Vasodilator-Stimulated Phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.

  • abstract 1653 Vasodilator Stimulated Phosphoprotein ser157 and ser239 are antimetastatic targets in colon cancer
    Cancer Research, 2011
    Co-Authors: Giovanni Pitari
    Abstract:

    Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Colorectal cancer is the third deadly cancer worldwide. Patient mortality is largely the result of metastatic disease progression, in which tumor cells undergo dramatic morphological changes characterized by the formation of membrane protrusive structures mediating invasion (lamellipodia, filopodia, invadopodia). These events are driven by the activity of actin-regulatory proteins, including the Vasodilator-Stimulated Phosphoprotein (VASP), which reorganize the actin cytoskeleton to promote cell migration and invasion. In colon cancer, cAMP- and cGMP-dependent VASP phosphorylation at Ser157 (pVASP-Ser157) and Ser239 (pVASP-Ser239) represent opposing molecular switches, which regulate the invasive cell shape. Here, pVASP-Ser157 and pVASP-Ser239 differentially affected the metastatic potential of colon cancer cells, including tumor proliferation, colony formation, migration, adhesion and metastasis in vivo. Investigations employed intestinal cancer cells, human tumor cells expressing serine phosphoresistant VASP mutants, cGMP or cAMP analogs, agonists selectively elevating intracellular cGMP or cAMP levels, and orthotopic mouse models of colorectal cancer. Collectively, results demonstrated pVASP-Ser239 suppresses, while pVASP-Ser157 promoted, the metastatic phenotype of colon cancer cells. Thus, differential VASP serine phosphorylation is a novel targeted strategy to prevent colon cancer metastasis. Clinical translation of these findings may provide original therapeutic interventions to reduce mortality in patients with colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1653. doi:10.1158/1538-7445.AM2011-1653

Alexander W Clowes - One of the best experts on this subject based on the ideXlab platform.

  • Vasodilator Stimulated Phosphoprotein regulates proliferation and growth inhibition by nitric oxide in vascular smooth muscle cells
    Arteriosclerosis Thrombosis and Vascular Biology, 2004
    Co-Authors: Lihua Chen, Kanchan Chitaley, Ulrich Walter, Gunter Daum, Martin Eigenthaler, Scott A Coats, Daniel F Bowenpope, Naresh R Thumati, Alexander W Clowes
    Abstract:

    Objective— Vasodilator-Stimulated Phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition. Methods and Results— Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed. Conclusions— Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

  • Vasodilator Stimulated Phosphoprotein is a substrate for protein kinase c
    FEBS Letters, 2004
    Co-Authors: Kanchan Chitaley, Lihua Chen, A Galler, Ulrich Walter, Gunter Daum, Alexander W Clowes
    Abstract:

    Abstract Vasodilator-Stimulated Phosphoprotein (VASP), an actin binding protein localized to areas of focal contacts, is a substrate for the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP)-dependent protein kinases (PKA, PKG). In this study, we show that serum stimulation of vascular smooth muscle cells (SMCs) induces VASP phosphorylation on Ser157, in a mechanism not dependent on PKA or PKG. We tested the possibility that protein kinase C (PKC), a regulator of cytoskeletal function, is involved. PKC inhibition or down-regulation prevented serum-induced phosphorylation of VASP at Ser157 in rat vascular SMCs. Additionally, recombinant PKCα directly phosphorylated Ser157 on VASP. In summary, our data support the hypothesis that PKC phosphorylates VASP and mediates serum-induced VASP regulation.

Susanne Illenberger - One of the best experts on this subject based on the ideXlab platform.

  • the Vasodilator Stimulated Phosphoprotein promotes actin polymerisation through direct binding to monomeric actin
    FEBS Letters, 2002
    Co-Authors: Birgit Waldersharbeck, Brigitte M Jockusch, Sofia Khaitlina, Horst Hinssen, Susanne Illenberger
    Abstract:

    The Vasodilator-Stimulated Phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain. Point mutations within this motif drastically weaken VASP/G-actin interactions, thereby abolishing any actin-nucleating activity of VASP. Additionally, actin nucleation was found to depend on VASP oligomerisation since VASP monomers fail to induce the formation of actin filaments. Phosphorylation negatively affects VASP/G-actin interactions preventing VASP-induced actin filament formation.

  • phosphorylation of the Vasodilator Stimulated Phosphoprotein regulates its interaction with actin
    Journal of Biological Chemistry, 2000
    Co-Authors: Birgit Harbeck, Stefan Huttelmaier, Brigitte M Jockusch, Kathrin Schluter, Susanne Illenberger
    Abstract:

    Abstract The Vasodilator-Stimulated Phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.

  • characterization of the actin binding properties of the Vasodilator Stimulated Phosphoprotein vasp
    FEBS Letters, 1999
    Co-Authors: Stefan Huttelmaier, Birgit Harbeck, Nils Ole Steffens, Tania Meserschmidt, Susanne Illenberger, Brigitte M Jockusch
    Abstract:

    The Vasodilator-Stimulated Phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.

Martin Eigenthaler - One of the best experts on this subject based on the ideXlab platform.

  • Vasodilator Stimulated Phosphoprotein regulates proliferation and growth inhibition by nitric oxide in vascular smooth muscle cells
    Arteriosclerosis Thrombosis and Vascular Biology, 2004
    Co-Authors: Lihua Chen, Kanchan Chitaley, Ulrich Walter, Gunter Daum, Martin Eigenthaler, Scott A Coats, Daniel F Bowenpope, Naresh R Thumati, Alexander W Clowes
    Abstract:

    Objective— Vasodilator-Stimulated Phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition. Methods and Results— Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed. Conclusions— Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

  • enhanced in vivo platelet adhesion in Vasodilator Stimulated Phosphoprotein vasp deficient mice
    Blood, 2004
    Co-Authors: Steffen Massberg, Sabine Gruner, Ildiko Konrad, Maisa Garcia I Arguinzonis, Martin Eigenthaler, Kathrin Hemler, Julia Kersting, Christian Schulz, Iris Muller, Felicitas Besta
    Abstract:

    Platelet adhesion and activation at the vascular wall are the initial steps leading to arterial thrombosis and vascular occlusion. Prostacyclin and nitric oxide inhibit platelet adhesion, acting via cyclic adenosine monophosphate (cAMP)– and cyclic guanosine monophosphate (cGMP)–dependent protein kinases. A major downstream target for both cAMP- and cGMP-dependent protein kinases is the Vasodilator-Stimulated Phosphoprotein ( VASP ). To test the significance of VASP for the regulation of platelet adhesion in vivo , we studied platelet–vessel wall interactions using VASP -deficient ( VASP – / –) mice. Under physiologic conditions, platelet adhesion to endothelial cells was significantly enhanced in VASP null mutants when compared with wild-type mice ( P < .05). Platelet recruitment in VASP null mice involved P-selectin and the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa). Under pathophysiologic conditions, the loss of VASP increased platelet adhesion to the postischemic intestinal microvasculature, to the atherosclerotic endothelium of ApoE -deficient mice, and to the subendothelial matrix following endothelial denudation ( P < .05 vs wild type). Importantly, platelet adhesion in VASP null mutants was unresponsive to nitric oxide. These data show for the first time in vivo that VASP is involved in down-regulation of platelet adhesion to the vascular wall under both physiologic and pathophysiologic conditions.

  • resistance to thienopyridines clinical detection of coronary stent thrombosis by monitoring of Vasodilator Stimulated Phosphoprotein phosphorylation
    Catheterization and Cardiovascular Interventions, 2003
    Co-Authors: Paul Barragan, Ulrich Walter, J L Bouvier, Pierreolivier Roquebert, G Macaluso, Philippe Commeau, Bertrand Comet, Antoine Lafont, Laurence Camoin, Martin Eigenthaler
    Abstract:

    We carried out a prospective evaluation of a new Vasodilator-Stimulated Phosphoprotein (VASP) phosphorylation assay in order to detect patients with high-risk coronary subacute stent thrombosis (SAT) despite thienopyridine regimen. Twenty healthy donors (group 1) without any medication were compared to 16 stented patients (group 2) treated by ticlopidin or clopidogrel initiated 2 days before stenting and aspirin (250 mg/day). No difference in platelet reactivity was noted between group 1 and group 2 treated only with aspirin (72.00% ± 4.17% vs. 69.73% ± 5.62%, respectively; P = NS). Significant differences were found between patients of group 2 treated with aspirin alone (69.73% ± 5.62%), after 2.0 days (60.14% ± 9.60%; P < 0.05), and after 4.8 ± 1.3 days (48.37% ± 11.19%; P < 0.05) with thienopyridine-aspirin. Among 1,684 consecutive stented patients, 16 patients who presented an SAT (group 3) were compared with 30 other stented patients free of SAT (group 4). We found a significant difference between group 3 (63.28% ± 9.56%) and group 4 (39.80% ± 10.9%; P < 0.0001). VASP phosphorylation analysis may be useful for the detection of coronary SAT. Cathet Cardiovasc Intervent 2003;59:295–302. © 2003 Wiley-Liss, Inc.

  • megakaryocyte hyperplasia and enhanced agonist induced platelet activation in Vasodilator Stimulated Phosphoprotein knockout mice
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Wolfgang Hauser, Martin Eigenthaler, Klauspeter Knobeloch, Stepan Gambaryan, Veit Krenn, Jorg Geiger, Margarita Glazova, Elvira Rohde, Ivan D Horak, Ulrich Walter
    Abstract:

    Abstract Vasodilator-Stimulated Phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell–cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin αIIbβ3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP−/− mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin αIIbβ3 activation. Although the limited phenotypic differences between wild-type and VASP−/− mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.

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