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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2051.)
© 2000 American Heart Association, Inc.

Vascular Biology

Vasodilator-Stimulated Phosphoprotein Is Involved in Stress-Fiber and Membrane Ruffle Formation in Endothelial Cells

Caroline J. Price; Nicholas P. J. Brindle

From the Cardiovascular Research Institute and Department of Surgery, University of Leicester, Leicester, UK.

Correspondence to Dr Nicholas P.J. Brindle, Cardiovascular Research Institute and Department of Surgery, University of Leicester, RKCSB, PO Box 65, Leicester LE2 7LX UK. E-mail npjb1{at}leicester.ac.uk

	Abstract

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Abstract—Vasodilator-stimulated phosphoprotein (VASP) is highly expressed in vascular endothelial cells, where it has been implicated in cellular reorganization during angiogenesis, as well as in endothelial retraction and changes in vessel permeability. However, the cellular functions of VASP are not known. In this study, we have expressed wild-type and mutant forms of VASP in endothelial cells to determine in what aspects of cytoskeletal behavior this protein participates. Expression of wild-type VASP induces marked membrane ruffling and formation of prominent stress fibers in bovine aortic endothelial cells. Deletion of the proline-rich domain of VASP abolishes its ability to bind profilin but does not affect ruffling or stress fiber formation. Further deletions reveal a sequence within the carboxy-terminal domain that is responsible for in vivo bundle formation. Ruffling occurs only on the expression of forms of VASP that possess bundling activity and the capacity to bind zyxin/vinculin-derived peptide. The ability of distinct subdomains within VASP to bind adhesion proteins and induce F-actin bundling in vivo suggests that this protein could function in the aggregation and tethering of actin filaments during the formation of endothelial cell–substrate and cell-cell contacts. These data provide a mechanism whereby VASP can influence endothelial migration and organization during capillary formation and modulate vascular permeability via effects on endothelial cell contractility.

Key Words: endothelium • vasodilator-stimulated phosphoprotein • actin • angiogenesis • vasodilators

	Introduction

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Vasodilator-stimulated phosphoprotein (VASP) is a substrate for cAMP- and cGMP-dependent protein kinases and was first isolated as a major phosphorylated protein in endothelial cells and platelets after stimulation with vasodilators such as prostaglandins and NO donors.¹ In humans, VASP is highly expressed in vascular endothelial and smooth muscle cells and platelets.^{2 3} Its expression is elevated during the endothelial reorganization phase of capillary morphogenesis in vitro.⁴ Human VASP is a 380–amino acid protein with a predicted molecular mass of 39.8 kDa. There are 3 principal phosphorylation sites in VASP, Ser157, Ser239, and Thr278.^{3 5} Phosphorylation of VASP in endothelial cells occurs in response to agents that increase cyclic nucleotides and is associated with the inhibition of thrombin-induced cell retraction.⁶ In brain capillary endothelial cells, VASP phosphorylation stimulated by NO has been suggested to be involved in changes in endothelial permeability.⁷ VASP is localized to focal adhesions, along stress fibers, and in areas of highly dynamic membrane activity, such as extending lamellipodia and filopodia.^{3 8} Despite this evidence implicating VASP in aspects of endothelial cell behavior involving the cytoskeleton, its roles in these cells are not known.

VASP is the archetypal member of the Ena/VASP family of proteins. This protein family includes the Drosophila-enabled protein (Ena) and its relative, mammalian Ena (Mena), as well as the Ena/VASP-like (Evl) protein. Ena/VASP proteins are characterized by having 3 domains, an amino-terminal Ena/VASP homology domain denoted EVH-1, a proline-rich domain, and a carboxy-terminal–charged Ena/VASP homology domain designated EVH-2.⁹ VASP binds profilin, zyxin, vinculin, and the bacterial coat protein ActA.^{10 11 12 13} Binding of the G-actin binding protein profilin is likely to be mediated by the tandem repeat of 3 GP₅ motifs within VASP.^{11 14 15 16} The amino-terminal EVH-1 domain of VASP is responsible for binding ActA as well as the adhesion proteins zyxin and vinculin.¹⁷ In these proteins, VASP binds to a short sequence, designated the FP₄ motif or ABM-1 motif, which is composed of phenylalanine followed by 4 or 5 prolines and flanked by acidic residues.^{12 17 18} Zyxin and ActA have multiple FP₄/ABM-1 motifs, whereas vinculin has one.

The ability of VASP to bind cytoskeletal proteins and the intracellular localization of VASP suggest that VASP contributes to some aspect of cytoskeletal dynamics in mammalian cells. Transgenic approaches do support an important role for VASP in platelet activation but, as yet, have provided no clues as to its function in vascular cells.^{19 20} The most likely reason for this, and for the mild phenotypes of the VASP-deficient animals, is functional compensation by other members of the Ena/VASP family. Indeed, VASP has been shown to be able to substitute for Ena in Ena-null mutants of Drosophila.²¹ Therefore, as an alternative approach to gain insight into the functions of VASP in vascular endothelial cells, we have constructed and expressed a series of deleted forms of the protein. The effects of these constructs on the endothelial cytoskeleton were examined. We show that VASP induces membrane ruffling and stress-fiber formation in endothelial cells. Use of the deletion mutants allowed definition of specific regions within VASP participating in these activities. Our data provide the first indication of functions for VASP in vascular endothelial cells and are consistent with the involvement of this protein in endothelial movement and retraction.

	Methods

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Materials
Generation of full-length human VASP has been described previously.¹² Deleted forms of VASP were generated by using existing restriction sites or introducing appropriate new restriction sites by site-directed mutagenesis with use of the Altered Sites Mutagenesis System (Promega Ltd), as per the manufacturer’s directions. For expression, VASP constructs were ligated into the pFLAG epitope-tag vector (Sigma-Aldrich). Profilin II cDNA was a kind gift from Dr Henrik Leffers, Aarhus University, Aarhus, Denmark.²² The sequences of all clones were confirmed by using a 373A ABI automated DNA sequencer. A monoclonal antibody recognizing VASP was obtained from Transduction Laboratories. All other reagents were from sources described previously.¹²

Binding Studies
Radiolabeled VASP and deleted forms of VASP were tested for their ability to bind immobilized FP₄ peptide or control peptide coupled to Actigel (Sterogene), glutathione S-transferase (GST), GST-profilin, or GST full-length VASP. The FP₄ peptide (839-EPDFPPPPPDLE-850) encompassing the VASP binding motif from vinculin and the control peptide that was derived from an adjacent proline-rich region of vinculin (859-APPKPPLPEGEVPPPRPPPPE-879) have been described previously.¹² Radiolabeled VASP and deleted forms of VASP were produced by using T7 RNA polymerase in a rabbit reticulocyte lysate in vitro transcription/translation system (Promega Ltd) in the presence of [³⁵S]methionine and [³⁵S]cysteine with a specific activity of 37 TBq/mmol (Trans³⁵S-Label, ICN Biomedicals), as per the manufacturer’s instructions. Binding studies were performed as described previously.¹² Bound proteins were eluted into Laemmli sample buffer, containing 100 mmol/L dithiothreitol, and separated by SDS-PAGE.²³ Fluorography was as described previously.¹²

Cell Culture and Transfection
Bovine aortic endothelial cells were obtained as described previously and cultured in DMEM containing 10% FCS, 100 µg/mL streptomycin, and 100 U/mL penicillin under 5% CO₂/95% air in a humidified incubator at 37°C.²⁴ Cells were used between passages 3 and 7. For immunofluorescence studies, cells were plated onto sterile glass coverslips. For transfection, cells were plated at 40% to 50% confluence 24 hours before transfection. Cells were then washed with PBS (140 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na₂HPO₄, and 1.8 mmol/L KH₂PO₄, pH 7.3) and transfected with 2.5 µg DNA and 10 µL Superfect (Quiagen Ltd) in 35-mm dishes, as detailed in the manufacturer’s instructions. After transfection, cells were washed in PBS and maintained for 24 hours in complete growth medium. In some experiments, cells were serum-starved for 24 hours before use.

Immunoprecipitation and Western Blotting
Cells were rinsed in PBS and lysed by the addition of ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L phenylmethylsulfonyl fluoride,1 µg/mL leupeptin, 1 µg/mL aprotinin, and 1 µg/mL pepstatin). Lysates were cleared by centrifugation at 12 000g for 10 minutes and then incubated for 2 hours or overnight at 4°C with anti-FLAG monoclonal antibody and protein G–agarose beads. Immune complexes were washed extensively with lysis buffer, and proteins were eluted by boiling in Laemmli sample buffer in the presence of 100 mmol/L dithiothreitol. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Epitope-tagged VASP was detected by probing the membranes with monoclonal antibody recognizing the FLAG epitope tag. Immunoreactive bands were detected with a peroxidase-conjugated secondary antibody and the ECL chemiluminescent detection system (Amersham International Plc).

Immunofluorescence Microscopy
Immunofluorescence was essentially as described by Symons and Mitchison.²⁵ Cells were washed in PBS and fixed by incubation for 15 minutes at room temperature in 4% formaldehyde in cytoskeletal buffer (10 mmol/L MES, pH 6.1, 138 mmol/L KCl, 3 mmol/L MgCl₂, 2 mmol/L EGTA, and 0.32 mol/L sucrose). Permeabilization was accomplished by incubation for 10 minutes at room temperature in cytoskeletal buffer containing 0.5% Triton X-100. Cells were washed with cytoskeletal buffer containing 0.1% Triton X-100 and blocked for 10 minutes in antibody buffer (137 mmol/L NaCl, 25 mmol/L Tris, pH 7.4, 2.7 mmol/L KCl, 0.1% Triton X-100, and 2% BSA). Coverslips were incubated at 37°C with antibody buffer and primary antibody. After 60 minutes, coverslips were washed extensively with antibody buffer before incubation with fluorescently labeled secondary antibodies at 37°C. After 45 minutes, coverslips were washed extensively. After FLAG immunodetection, filamentous actin was stained by probing with fluorescein-conjugated phalloidin at 2 µg/mL in cytoskeletal buffer for 30 minutes before extensive washing in antibody buffer and mounting in 220 mmol/L diazobicyclo-octane in 90% (vol/vol) glycerol and 10% (vol/vol) PBS, pH 8.6.²⁶ Cells were viewed by use of a Bio-Rad MRC 600 Laser Scanning Confocal Microscope.

	Results

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Expression of VASP Induces Ruffling and F-Actin Bundle Formation in Endothelial Cells
Indirect evidence suggests that VASP is involved in some aspect of cytoskeletal dynamics in vascular endothelial cells. To gain insight into the functions of VASP in the endothelium, bovine aortic endothelial cells were transfected with constructs encoding wild-type and mutant forms of the protein, and F-actin organization was examined. The distribution of expressed wild-type VASP, examined in cells by immunofluorescence microscopy of epitope-tagged protein, reveals a distribution similar to that of endogenous VASP, being observed in focal adhesions, along stress fibers, and in lamellipodia (Figure 1A). In all figures depicting transfected cells expressing VASP constructs, the expressing cells are revealed by anti-FLAG immunofluorescence and shown in the right panels to allow location of the relevant cells in the F-actin–stained field. Examination of the F-actin organization in endothelial cells reveals the nontransfected cells to have fine bundles of actin filaments and minimal membrane ruffling, as shown in Figure 1B. In contrast, transfected cells expressing full-length VASP show conspicuous membrane ruffles and prominent thick stress fibers (Figure 1B). These data indicate that increased expression of full-length VASP leads to the induction of marked ruffling and filament bundling in endothelial cells. On the basis of fluorescence intensity of transfected and nontransfected cells after staining with antibodies against VASP, the expression level of VASP in transfected cells was determined to be, on average, between 4- and 6-fold that of the endogenous protein. In the experiments described in the present study, subconfluent cells were used partly to allow examination of peripheral ruffling. However, VASP-overexpressing cells can achieve confluence and continue to demonstrate prominent actin stress fibers (data not shown). As detailed earlier, VASP is known to bind profilin, FP₄-containing cytoskeletal proteins, and full-length VASP. Therefore, deletion mutants of VASP were used to test which, if any, of these interactions were required for the ruffling and stress fiber–inducing effects of the full-length protein.

View larger version (72K): [in this window] [in a new window]   Figure 1. Actin filament reorganization is induced by expression of VASP in endothelial cells. A, Distribution of endogenous VASP and expressed epitope-tagged VASP in endothelial cells. Endogenous VASP was detected by using a monoclonal anti-VASP antibody (VASP). For expressed VASP, endothelial cells were transfected with plasmids encoding full-length (FL) VASP, and the distribution of expressed protein was determined by detection with an antibody recognizing the FLAG epitope tag (FLAG). The optical section reveals distribution to focal adhesions, stress fibers, and cell perimeter. B, Endothelial cells transfected with plasmids expressing FL VASP, as indicated. F-actin organization was determined by staining with FITC-phalloidin (actin), and cells expressing the VASP constructs were identified by anti-FLAG immunofluorescence (FLAG). Examples of the fine-filament bundles in cells not expressing transfected VASP are indicated with arrows, and basal ruffling is indicated by an arrowhead. In the expressing cell, an example of a prominent stress fiber is indicated by a double-headed arrow, and the marked membrane ruffling is indicated by a double arrowhead. Bar=25 µm.

Binding Interactions of Deleted Forms of VASP
The structure of VASP and deletion mutants used in the present study are shown schematically in Figure 2. Before examination of their effects on the cytoskeleton, the ability of wild-type and deleted forms of VASP to bind to FP₄ peptide, profilin, and full-length VASP was analyzed (Figure 3). Full-length VASP, the EVH-1 domain, and GP5 VASP were produced by in vitro transcription/translation and tested for their capacity to bind immobilized FP₄ peptide and control peptide. As shown in Figure 3A, full-length, EVH-1, and GP₅ VASP all bind to FP₄ peptide but not appreciably to control peptide. For examination of binding to profilin and VASP, full-length, GP₅, and C VASP were produced by in vitro transcription/translation, and binding to immobilized GST-profilin or GST-VASP was assessed; nonspecific binding was assessed by using immobilized GST (Figure 3B). Full-length VASP binds profilin II; however, GP₅ and C VASP do not (Figure 3B). Thus, deletion of residues 175 to 196, which disrupts (GP₅)₃ by removing 2 of the tandem repeats but not that between residues 169 and 174 or the single GP₅ motif between residues 117 and 122, abolishes the binding of profilin. Full-length and GP₅ VASP are both able to bind to full-length VASP, but C VASP is not (Figure 3B).

View larger version (25K): [in this window] [in a new window]   Figure 2. Domain structure of VASP and deletion mutants. Schematic representation of the domain structure of VASP shows EVH-1, EVH-2, and GP5 domains. Deletion mutants of VASP and their designation in the present study are shown.

View larger version (37K): [in this window] [in a new window]   Figure 3. Binding of FL and deleted forms of VASP to FP4 peptide, profilin, and FL VASP. A, Binding of FL, VASP EVH-1 domain (EVH-1), and C VASP (C) to FP4 peptide (FP4) and control peptide (C). Radiolabeled FL and deleted forms of VASP produced by in vitro transcription/translation (IVT/T) were incubated with agarose-coupled peptides, as indicated for each lane, for 2 hours at 4°C. B, Binding of FL, GP5 VASP (GP5), and C VASP (C) to GST, GST-profilin, and GST-VASP. Radiolabeled FL and deleted forms of VASP produced by IVT/T were incubated with glutathione-agarose bound GST, GST-profilin, or GST-VASP, as indicated for each panel, for 2 hours at 4°C. In the experiments depicted in panels A and B, bound protein was collected by centrifugation, washed extensively, eluted by boiling in electrophoresis sample buffer, and analyzed by SDS-PAGE. Bound radiolabeled VASP and deletion mutants were detected after fluorography, as described in Experimental Procedures. IVT/T lanes show the sizes of the relevant IVT/T products used in the binding assays.

Effects of VASP Deletion Mutants on Endothelial Cell Ruffling and Stress Fiber Formation
Human VASP has a calculated M_r of 39.8 kDa and has an apparent molecular mass of 46 kDa under the conditions of SDS-PAGE. Phosphorylation of VASP on Ser157 causes a shift in mobility to an apparent M_r of 50 kDa.⁵ Western blots of cells expressing epitope-tagged VASP mutants are shown in Figure 4; full-length, GP₅, and C VASP mutants, but not EVH-1 (which lacks Ser157), are present as doublets, similar to the doublet previously observed for the endogenous wild-type protein,⁵ indicating that they exist in phosphorylated and dephosphorylated forms in the endothelial cells. The predominant form of the expressed full-length, GP₅ and C deletion mutants is the higher mobility form, suggesting that expressed VASP is largely unphosphorylated on Ser157.

View larger version (57K): [in this window] [in a new window]   Figure 4. Expression and mobility of VASP mutants. Endothelial cells were transfected with plasmids expressing FL and deleted forms of VASP, as indicated. Twenty-four hours after transfection, cells were lysed, and expressed proteins were immunoprecipitated and resolved by SDS-PAGE. Expressed VASP was detected by Western blotting with an antibody recognizing the FLAG epitope tag. Mobility of molecular mass markers (in kilodaltons) is shown to the left of each panel.

Membrane ruffling usually reflects actin filament assembly.²⁷ Binding of profilin to VASP could provide a means for the local accumulation of polymerization-competent G-actin, which, in the presence of nucleating activity, can lead to filament assembly and membrane ruffling.²⁸ Therefore, we examined whether profilin binding is required for the VASP-induced ruffling observed in the present study. Expression of GP₅ VASP, which is able to bind both FP₄ and VASP but not profilin, surprisingly still results in the marked membrane ruffling and stress fiber formation similar to those induced by wild-type VASP (Figure 5). This suggests an alternative mechanism whereby VASP induces endothelial ruffling. The C form of VASP is unable to bind to any known partners and was therefore tested for its effects on the endothelial cytoskeleton (Figure 5). Prominent bundles of F-actin are observed in C VASP–expressing endothelial cells. However, the induction of marked ruffling seen with full-length or GP₅ VASP is not observed with C VASP, indicating that the ability to induce stress fibers is independent of the effects of VASP on ruffling. Expression of the EVH-1 domain of VASP, VASP 1-245, and VASP 225-340 does not induce the marked membrane ruffles seen with full-length VASP (Figures 1 and 6). Ruffling is observed only with forms of VASP that can bind FP₄ and localize to the cell periphery (Figures 1, 3, and 5).

View larger version (95K): [in this window] [in a new window]   Figure 5. Effects of GP5 and C VASP on actin organization in endothelial cells. Endothelial cells were transfected with plasmids expressing GP5-disrupted and C-terminal–deleted VASP, as indicated. F-actin organization was determined by staining with FITC-phalloidin (actin), and cells expressing the VASP constructs were identified by anti-FLAG immunofluorescence (FLAG). Bar=25 µm.

View larger version (55K): [in this window] [in a new window]   Figure 6. A subdomain within EVH-2 induces actin filament bundles in vivo. Endothelial cells were transfected with plasmids expressing deleted forms of VASP, as indicated. F-actin distribution was determined by staining with FITC-phalloidin (actin), and cells expressing deleted forms of VASP were identified by anti-FLAG immunofluorescence (FLAG). Bar=25 µm.

A Subdomain Within the EVH-2 Domain Induces F-Actin Bundles in Endothelial Cells
The finding that VASP induces F-actin bundle formation in endothelial cells was unexpected and may be highly significant for endothelial organization and retraction. Therefore, it was of interest to identify the domain responsible for this activity. The EVH-1 domain of VASP is able to bind FP₄ peptide but not profilin or VASP (Figure 3). EVH-1 expression fails to induce bundle formation in vivo (Figure 6). Similarly, VASP 1-245 has no effects on cytoskeletal organization (Figure 6). Because VASP 1-325 induces bundle formation but VASP 1-245 does not, we asked whether the sequence between these residues is sufficient for in vivo bundling activity. Expression of VASP sequence 225-340 results in prominent bundle formation in vivo (Figure 6).

	Discussion

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The data reported in the present study implicate VASP in membrane ruffling and stress fiber formation in vascular endothelial cells. VASP has been hypothesized to have a role in membrane ruffling²⁸ by virtue of its ability to recruit profilin, a 14-kDa polypeptide that binds G-actin and stimulates the exchange of ADP for ATP on the monomer.²⁹ The data in the present study are the first to demonstrate the direct actions of VASP on membrane ruffling. Rather surprisingly, however, experiments with the GP₅ mutant indicate this did not require profilin binding to VASP. This would suggest a previously unsuspected mechanism whereby the phosphoprotein can contribute to membrane ruffling. It is possible that although profilin recruitment is not be essential for VASP induced ruffling, it could be required for optimum activity. The mechanisms whereby VASP causes ruffling will require further investigation.

VASP has not previously been suspected of involvement in stress fiber formation, and it is of significance that we find the induction of prominent stress fibers in endothelial cells in response to VASP expression. This suggests that the high levels of VASP found in endothelial cells in vivo² likely contribute to the conspicuous stress fibers observed in these cells in blood vessels.^{30 31} In addition to providing an important structural component in endothelium, stress fibers are involved in endothelial retraction^{32 33 34} and in the detection and signal transduction of shear stress.^{35 36 37} Our findings provide a possible mechanistic link between the known effects of cyclic nucleotides on VASP phosphorylation and endothelial retraction.^{6 7} It is possible that phosphorylation of VASP on specific sites could influence its activity in stress fiber formation and, hence, endothelial contractility.

A possible mechanism for the effects of VASP on stress fiber formation is provided by identification of the subdomain responsible for this activity. Our findings that removal of residues 340-380 prevents VASP-VASP binding but not bundle formation in endothelial cells indicates that multimerization of VASP is not required for the formation of F-actin bundles in vivo. Alignment of sequences of Ena/VASP family members corresponding to the region of VASP shown in the present study to induce F-actin bundles reveals 2 highly conserved motifs corresponding to VASP 225-245 and VASP 262-277 (data not shown). The second motif includes a positive charge cluster. Such a region could act to induce filament bundling via its interaction and to charge neutralizing effects on acidic actin molecules within filaments.³⁸ During preparation of the present article, it was reported that VASP 259-380 binds F-actin and forms bundles in vitro.^{39 40} This is consistent with our findings on the effects VASP 225-340 in vivo and the scheme outlined in the present study.

In conclusion, the present study demonstrates that VASP is intimately involved in membrane ruffling and stress fiber formation in vascular endothelial cells. This provides a rationale for the increased expression of VASP seen in endothelial organization in angiogenesis.⁴ This phase of angiogenesis requires endothelial cells to migrate, involving peripheral actin assembly, as well as to undergo shape change, a process in which actin bundles would be expected to contribute to individual cell shape changes involved in lumen formation. It has already been shown that VASP phosphorylation status is altered under conditions leading to endothelial retraction.^{6 7} Given the involvement of actin bundles in cell retraction,^{32 33 34} it is likely that VASP phosphorylation modulates its ability to participate in actin bundling. This possibility is under investigation. Finally, the prominence of stress fibers in endothelial cells within blood vessels and their importance in vascular function are consistent with the finding of high expression levels of VASP in endothelial cells in vivo.

	Acknowledgments

 
We are very grateful to Chris R. d’Lacey for help with confocal microscopy, and we thank the University of Leicester for financial support.

Received January 28, 2000; accepted May 2, 2000.

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