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Originally published In Press as doi:10.1074/jbc.M606938200 on April 23, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19676-19684, July 6, 2007
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Osteopontin Overexpression Inhibits in Vitro Re-endothelialization via Integrin Engagement*Formula

Daria Leali{ddagger}, Emanuela Moroni{ddagger}, Federico Bussolino§, and Marco Presta{ddagger}1

From the {ddagger}Unit of General Pathology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 25123 Brescia, Italy and the §Institute for Cancer Research and Treatment, Department of Oncological Sciences, University of Torino, 10100 Turin, Italy

Received for publication, July 21, 2006 , and in revised form, March 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellular matrix protein osteopontin (OPN) plays a nonredundant role in atherosclerosis and restenosis. Here we investigated the impact of OPN up-regulation in an in vitro model of re-endothelialization after mechanical injury of the endothelial cell monolayer. Murine aortic endothelial (MAE) cells interact via {alpha}v integrins with the integrin-binding Arg-Gly-Asp OPN sequence and adhere to immobilized OPN. On this basis, MAE cells were stably transfected with a wild-type OPN cDNA (OPN-MAE cells), with an OPN mutant lacking the Arg-Gly-Asp sequence ({Delta}RGD-OPN-MAE cells), or with vector alone (mock-MAE cells). When compared with mock-MAE and {Delta}RGD-OPN-MAE cells, OPN-MAE cells showed a reduced sprouting activity in fibrin gel, a reduced motility in a Boyden chamber assay, and a reduced capacity to repair the wounded monolayer. Accordingly, OPN-MAE cells at the edge of the wound were unable to form membrane ruffles, to reorganize their cytoskeleton, and to activate the focal adhesion kinase and the small GTPase Rac1, key regulators of the cell entry into the first phase of the cell migration cycle. Accordingly, wounded OPN-MAE cells failed to activate the intracellular signals RhoA and ERK1/2, involved in the later phases of the cell migration cycle. Also, parental MAE cells showed reduced re-endothelialization after wounding when seeded on immobilized OPN and exhibited increased adhesiveness to OPN-enriched extracellular matrix. In conclusion, OPN up-regulation impairs re-endothelialization by inhibiting the first phase of the cell migration cycle via {alpha}v integrin engagement by the extracellular matrix-immobilized protein. This may contribute to the adverse effects exerted by OPN in restenosis and atherosclerosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several vascular pathologies, including atherosclerosis and restenosis, are related to injury of the endothelial monolayer lining the inner surface of the vessel wall. Also, the loss of endothelium that occurs after vascular trauma, microvascular transplantation, and reconstructive surgery may lead to stenosis and vessel occlusion. Furthermore, in vitro and in vivo endothelialization of prosthetic grafts is of pivotal importance in reducing/preventing their thrombogenic properties. Thus, the process of endothelialization after loss of the endothelial monolayer integrity plays an important role in different pathological and clinical settings (1–4).

Osteopontin (OPN)2 is a phosphorylated acidic RGD-containing glycoprotein that binds certain CD44 variants and integrin receptors (5, 6). OPN exists both as an immobilized extracellular matrix (ECM) component and as a soluble molecule implicated in inflammation, cell-mediated immunity, tissue remodeling, and tumor metastases (6–8).

OPN has recently emerged as a key factor in vascular remodeling and in the development of atherosclerosis (9–11). Indeed, OPN overexpression in mice results in increased medial thickening with age and a larger neointima formation after mechanical injury (12). Conversely, OPN blockade by neutralizing antibodies and OPN deficiency in OPN null mice prevent neointimal thickening and attenuate atherosclerosis (11, 13). Several stimuli can induce OPN up-regulation in endothelium, including interleukin-1, interferon-{gamma}, glucocorticoids, and the angiogenic growth factors vascular endothelial growth factor and (VEGF) fibroblast growth factor-2 (FGF2) (6, 14–16). Also, OPN expression is up-regulated in regenerating endothelium after balloon catheter denudation and in restenotic human coronary atherosclerotic plaques (17, 18). Indeed, OPN immunoreactivity has been observed in acellular ECM areas of human coronary atherosclerotic specimens (17), indicating that OPN is associated with ECM of injured blood vessels in vivo. However, the impact of endogenous OPN on damaged endothelium during the process of re-endothelialization after injury remains unknown.

In the present study, we utilized murine aortic endothelial (MAE) cells transfected with a murine OPN cDNA or with an OPN mutant lacking the integrin-binding Arg-Gly-Asp amino acid sequence ({Delta}RGD-OPN) to investigate the impact of OPN up-regulation in an in vitro model of endothelialization after mechanical injury of the endothelial cell monolayer (see Ref. 19 and references therein). The results demonstrate that OPN overexpression dramatically impairs re-endothelialization by inhibiting endothelial cell motility. The inhibition requires {alpha}v integrin engagement by ECM-deposited, immobilized OPN.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Recombinant murine OPN and {Delta}RGD-OPN were expressed in E. coli as glutathione S-transferase (GST) fusion proteins and purified as described (20). Vitronectin was from BD Biosciences, and fibronectin was from Sigma. Synthetic RGD mimetics SCH221153 and SCH216687 were obtained from C. Kumar (Schering-Plough Research Institute, Kenilworth, NJ).

Cell Cultures and Transfection—Immortalized BALB/cMAE cells (21) were grown in DMEM (Invitrogen) added with 10% FCS (Integro, Zaandam, The Netherlands). The retroviral pBABE-OPN, pBABE-{Delta}RGD-OPN, and empty pBABE expression vectors were used to generate stable transfectants (16) that were grown in DMEM, 10% FCS plus 4.0 µg/ml puromycin (Sigma). Puromycin-resistant clones were tested for OPN expression by Western blotting of the serum-free supernatants using goat polyclonal anti-OPN antibodies (R&D Systems, Minneapolis, MN). Murine OPN was purified from the conditioned medium of OPN-transfected MAE cells as described (16).

Cell Proliferation Assay—Parental and transfected MAE cells were seeded at 10,000 cells/cm2 in 24-well dishes. After overnight incubation, cells were incubated in fresh medium containing 10% FCS. After 6 days, cells were fixed in 3.7% paraformaldehyde, 0.1 M sucrose in phosphate-buffered saline (PBS) and stained with methylene blue/Azur II (1/1, v/v). After solubilization in 10% acetic acid (300 µl/well), plates were read with a microplate reader at 595 nm.

Cell Adhesion Assay—Aliquots (100 µl) of PBS containing the adhesive molecule under test were added to polystyrene nontissue culture microtiter plates. After 16 h of incubation at 4 °C, wells were washed with cold PBS and overcoated for 1 h at 37 °C with 1.0 mg/ml bovine serum albumin. Next, cells were seeded onto coated wells (25,000 cells/well) for 2 h at 37 °C in DMEM without serum. In some experiments, MAE cells were seeded onto OPN-coated wells in the absence or in the presence of 30 µM SCH 221153 or SCH 216687. Then wells were washed with PBS. Adherent cells were fixed in 3.7% paraformaldehyde, 0.1 M sucrose in PBS and stained with methylene blue/Azur II (1/1, v/v). After solubilization in 10% acetic acid (100 µl/well), plates were read with a microplate reader at 595 nm.

In Situ Gelatin Degradation Assay—DQTM-gelatin (Molecular Probes, Inc., Eugene, OR) is a fluorescence-quenched gelatin substrate that will fluoresce upon proteolytic cleavage. Glass coverslips were coated for 16 h at 4 °C with DQ gelatin (100 µg/ml). Then cells were seeded on the coverslip at 70,000 cells/cm2 and cultured in a humidified chamber for 16 h at 37 °C. Cells were then fixed with 3% paraformaldehyde, and gelatin-degraded areas were visualized under a fluorescence microscope.

Wounding of the Endothelial Monolayer—Cells were seeded at 70,000 cells/cm2 in 10-cm diameter dishes and maintained in complete medium until they reached confluence. Multiple wounds crossing the entire dish (60 wounds/dish) were then created in cell monolayers with a 1.0-mm wide rubber policeman. After two washes in DMEM without serum, cells were incubated in fresh medium added with 10% FCS. In some experiments, mock-MAE cell monolayers were incubated with various protease inhibitors. At different time points, the percentage of cells at the edge of the wound showing cell membrane ruffles were counted at x400 magnification. After 16 h, wounds were photographed, and the percentage of repaired area was quantified by computerized analysis of the digitized images (22). When indicated, cell monolayers on glass coverslips were wounded, fixed 20 min or 3 h after wounding, and analyzed for microtubule organization center (MTOC) repositioning and cytoskeletal organization by immunocytochemistry. In some experiments, cells were seeded at 70,000 cells/cm2 and allowed to adhere on wells coated with increasing concentrations of purified OPN or with 160 nM vitronectin for 2 h at 37 °C. Then monolayers were wounded, and the repair was assessed after 24 h.

Three-dimensional Fibrin Gel Assay—Cell aggregates were prepared on agarose-coated plates and embedded in fibrin gel as described (23). Then complete medium containing 100 ng/ml recombinant FGF2 (R&D Systems) was added on the top of the gel in the presence of 10 µg/ml aprotinin (Sigma). Radially growing cell sprouts were photographed after 24–48 h at x40 magnification, and the area of cell clusters was quantified by computerized analysis of the digitized images (22).

Chemotaxis Assay—Cell migration assay was performed using a 48-well microchemotaxis chamber (Neuroprobe, Pleasanton, CA). Polyvinylpyrrolidone-free polycarbonate filters (Nucleopore; Corning Glass) with a pore size of 8 µm were boiled for 1 h in water containing 5 mg/liter gelatin. Chemotactic stimulus (DMEM added with 10% FCS) was placed in the lower chamber. Cells (55,000 cells/well) were suspended in DMEM without serum and added in the upper chamber. In some experiments, mock-MAE cells were preincubated with protease inhibitors for 2 h at 37 °C and then added in the upper chamber in the presence of the inhibitor. Cells were allowed to migrate for 4 h at 37 °C in a humidified atmosphere with 5% CO2. The filter was then removed, and cells on the upper side were scraped off with a rubber policeman. Migrated cells were fixed in methanol, stained with Giemsa solution (Diff-Quick; Baxter Diagnostics, Rome, Italy), and counted from five random high power fields at x100 magnification in each well.

MTOC Reorientation—Repositioning of the MTOC was evaluated by immunostaining with an anti-pericentrin antibody (Babco, Richmond, CA) as described (22). Briefly, cell monolayers were fixed 3 h after wounding in ice-cold methanol for 10 min at -20 °C. After saturation with goat serum in PBS, cells were incubated with the primary antibody, followed by biotinylated anti-rabbit IgG (Molecular Probes, Eugene, OR) and Texas Red avidin (Vector Laboratories, Burlingame, CA). Finally, cells were stained with 0.5 µg/ml 4',6-diamidino-2-phenylindole to visualize nucleus position. To determine MTOC position, 130–140 cells in the first row of cells adjacent to the wound and in the inner monolayer were then selected per experimental point. Each cell was visually divided by drawing two perpendicular lines across the cell nucleus, thus defining a front quadrant covering 25% of the cell surface and containing the leading edge. The percentage of migrating and quiescent cells with MTOC located inside the front quadrant was then calculated. Statistically, a random distribution of the MTOC around the nucleus, as observed in a quiescent monolayer, will result in 25% of cells with MTOC located in the front quadrant.

Immunocytochemistry—Wounded monolayers were washed, fixed in 3% paraformaldehyde plus 2% sucrose in PBS, and permeabilized with 0.5% Triton X-100. After saturation with goat serum in PBS, cells were incubated with rhodamine-phalloidin (Sigma), a monoclonal anti-paxillin antibody (BD Transduction Laboratories, Lexington, KY), a rabbit polyclonal affinity-purified anti-phospho-FAK(Tyr(P)397) antibody (BIOSOURCE), or a monoclonal anti-{alpha}-tubulin antibody followed by fluorescinated anti-mouse IgG (all from Sigma) or Alexa-Fluor-488-conjugated anti-rabbit IgG (Molecular Probes). In a second set of experiments, cells were seeded on glass coverslips at 25,000 cells/cm2 in DMEM added with 10% FCS. After overnight incubation, subconfluent cells were immunostained as described. Cells were photographed at x63 magnification with an epifluorescence microscope.

RhoA, Rac1, and ERK1/2 Activation Assays—Wounds were created in cell monolayers with a 1.0-mm-wide rubber policeman. Then cells were incubated in fresh medium added with 10% FCS. At different time points, the cells were washed with ice-cold PBS and lysed for 20 min on ice in GST-fish buffer (50 mM Tris, pH 7.4, 10% glycerol, 100 mM NaCl, 1.0% Nonidet P-40, 2.0 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1.0 mM phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 20,800 x g for 5 min at 4 °C. Aliquots (250 µg) of the extracted material were incubated for 1 h at 4 °C with 30 µg of GST-rhotekin binding domain or GST-PAK binding domain (a generous gift from J. Collard, Division of Cell Biology, Netherlands Cancer Institute) coupled to glutathione-Sepharose beads (Amersham Biosciences). The beads were washed three times with cold GST-fish buffer and eluted in sample buffer containing 40 mM dithiothreitol. Bound RhoA-GTP, Rac1-GTP, or Cdc42-GTP eluted from the beads and total RhoA/Rac1/Cdc42 from the original cell lysate (30 µg) were detected by Western blot analysis using a monoclonal anti-RhoA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), a monoclonal anti-Rac1 antibody, or a monoclonal anti-Cdc42 antibody (BD Transduction Laboratories). Aliquots (30 µg) of the original extracted material were also analyzed by Western blotting with the following antibodies: monoclonal anti-phospho-ERK1/2 antibody and monoclonal anti-ERK2 antibody (Santa Cruz Biotechnology).

Preparation of ECM-coated Wells—Cells were seeded at 80,000/cm2 in 96-well plates. After overnight incubation (for the cell spreading assay) or after a further 48-h incubation in serum-free medium (for the OPN detection assay), cells were removed by exposure to 0.5% Triton X-100 plus 20 mM NH4OH for 5 min at room temperature, followed by four washes in PBS (24).

Cell Spreading Assay—ECM-coated wells were incubated for 1 h at 37 °C with DMEM containing 3 µg/ml of a goat neutralizing anti-OPN antibody or goat IgGs (Santa Cruz Biotechnology). After washing, mock-MAE cells were seeded on top of the matrices at 25,000/cm2. Fifteen min after seeding, the percentages of cells showing a spread morphology (25–27) were counted from three random fields in each well under an inverted microscope at x200 magnification.

ECM-associated OPN Detection Assay—ECM-coated wells were blocked with 5.0% bovine serum albumin in PBS plus 0.05% Tween 20 (1 h at 37 °C) and incubated with 0.2 µg/ml of neutralizing goat anti-OPN antibody (1 h at 37 °C) in 5% bovine serum albumin. Then wells were washed and incubated with biotin-labeled rabbit anti-goat IgG (1 h at 37 °C) followed by horseradish peroxidase-labeled streptavidin incubation (1:5000 dilution; 1 h at room temperature; Amersham Biosciences). Finally, 50 µl of chromogen substrate tetramethylbenzidine were added (BD Pharmingen, San Diego, CA), and absorbance values were read at 405 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
OPN Overexpression Inhibits in Vitro Endothelialization—Immortalized BALB/c MAE cells retain several endothelial cell features, including the capacity to respond to angiogenic growth factors (e.g. FGF2) (21) and to interact with OPN (16). Semiquantitative RT-PCR analysis (data not shown) indicates that these cells express the standard isoform of CD44 receptor, unable to bind OPN (29), but do not express its OPN-binding v6 or v7 isoforms (29). However, MAE cells express the integrin subunits {alpha}v, {alpha}5, β1, β3, and β5, all involved in OPN interaction (7, 18, 30). Accordingly, MAE cells adhere to immobilized GST-OPN fusion protein but not to the GST-{Delta}RGD-OPN mutant in which the integrin-binding RGD sequence has been deleted (20) (Fig. 1A). Also, MAE cell adhesion to immobilized OPN was prevented by the selective {alpha}vβ3/{alpha}vβ5 integrin antagonist SCH 221153 (31) (Fig. 1B). Taken together, the data indicate that {alpha}v integrins play a major role in the interaction of OPN with MAE cells.

On this basis, MAE cells were transfected with a retroviral expression vector harboring the murine cDNA encoding for wild-type OPN or the {Delta}RGD-OPN mutant. Various OPN-overexpressing cell clones were obtained that release significant amounts of OPN in their culture medium (Fig. 2A) when compared with vector-alone transfectants (mock-MAE cells). Semiquantitative Western blot analysis indicated that OPN transfectants secrete ~4.0 µg of OPN/24 h/106 cells, similar to that observed for FGF2-stimulated MAE cells (see supplemental Fig. S1 and Ref. 16). Also, in keeping with previous observations on the same cells (16), secreted OPN cross-reacts with anti-phosphoserine Abs (data not shown). No significant modifications in the expression of integrin and CD44 isoforms were observed in OPN transfectants when compared with parental cells (data not shown).

OPN overexpression did not affect the proliferation rate of the different transfectants when grown in the presence of 10% FCS (supplemental Fig. S2) or 0.4% FCS (data not shown). Also, mock-MAE cells and OPN transfectants attach and spread to immobilized ECM proteins fibronectin and vitronectin with similar efficiency (supplemental Fig. S3).

Next, OPN- and {Delta}RGD-OPN-MAE cells were assessed in an in vitro model of endothelialization after mechanical injury of the endothelial cell monolayer (19). As shown in Fig. 2, B and C, OPN transfectants show a reduced capacity to repair the wounded monolayer when compared with parental, mock-MAE, and {Delta}RGD-OPN-MAE cells. Similar results were obtained with several OPN-overexpressing cell clones originated by independent OPN cDNA transfections. Also, when one of the OPN-MAE clones spontaneously lost the expression of the transgene after several months in culture, it reverted its phenotype, thus reacquiring the capacity to repair the wounded monolayer (data not shown).


Figure 1
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  		FIGURE 1.
RGD-dependent adhesion of MAE cells to immobilized OPN. A, MAE cells were seeded onto nontissue culture plastic 96-well plates coated with GST-OPN (•), GST-{Delta}RGD-OPN ({circ}), or GST ({square}) at the indicated doses and allowed to adhere for 2 h at 37 °C. Then adherent cells were fixed and stained, and the plates were read with a microplate reader at 595 nm. B, nontissue culture plastic 96-well plates were incubated with 30 nM purified OPN. Then MAE cells were seeded onto coated plates in the absence or in the presence of the {alpha}vβ3/{alpha}vβ5 integrin antagonist SCH 221153 or of control SCH 216687 (both at 30 µM). Cell adhesion was evaluated as described. *, p < 0.01.

 
Consistent with the hypothesis that OPN overexpression may impair endothelial cell migration, OPN-MAE cells were unable to invade a three-dimensional fibrin gel when stimulated to migrate in response to recombinant FGF2 (Fig. 3, A and B). Instead, no inhibition of endothelial cell sprouting was observed in {Delta}RGD-OPN-MAE cells. Accordingly, when compared with mock- and {Delta}RGD-OPN-transfected cells, OPN-MAE cells show a reduced ability to migrate through a gelatin-coated filter in response to 10% FCS in a Boyden chamber assay (Fig. 3C). It must be pointed out that OPN transfectants adhere to the gelatin-coated filter in a manner undistinguishable from that shown by parental, mock-MAE, and {Delta}RGD-OPN-MAE cells (data not shown).

Taken together, the data indicate that OPN overexpression leads to the inhibition of in vitro endothelialization and that this inhibitory effect is the result of an autocrine interaction between OPN protein and {alpha}v integrin receptors. This interaction is missing in {Delta}RGD-OPN transfectants characterized by a migratory behavior similar to that shown by mock-transfected and parental MAE cells.


Figure 2
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  		FIGURE 2.
OPN overexpression inhibits in vitro re-endothelialization. A, aliquots (10 µg) of serum-free conditioned media of vector alone, OPN, and {Delta}RGD-OPN-transfected MAE cell clones were probed with anti-OPN antibody in Western blot. A broad immunoreactive 60–65 kDa band was observed in all of the transfectants, no OPN proteolytic degradation product(s) being detectable in any cell line (not shown). The same band was detectable in parental and mock-MAE cell conditioned media only after longer exposure of the gel (not shown). B, confluent monolayers were wounded with a 1.0-mm wide rubber policeman (T0, open bars). After 16 h, wounds were photographed, and the percentage of repaired area was quantified by computerized analysis of the digitized images (black bars). OPN transfectants were significantly different (*, p < 0.01) from mock-MAE and {Delta}RGD-OPN-MAE cells. C, representative images of OPN-MAE and {Delta}RGD-OPN-MAE cell monolayers photographed 16 h after wounding. Original magnification was x40.

 
Proteases, including urokinase-type plasminogen activator and metalloproteases (MMPs), play a role in endothelial cell migration (32). Also, OPN/integrin interaction may affect protease production in target cells (reviewed in Ref. 8). In keeping with previous observations on parental MAE cells (21), OPN transfectants express undetectable levels of urokinase-type plasminogen activator. By contrast, gelatin zymography revealed that MAE cells release significant levels of MMP-2 and MMP-9 (data not shown). Although a decrease in MMP-2/9 production was observed in OPN-MAE cells maintained under serum-free conditions when compared with mock and {Delta}RGD-OPN transfectants, these differences were abolished when intact or wounded cell monolayers were maintained in complete medium added with serum (data not shown). Accordingly, all of the clones were able to degrade a fluorogenic gelatin substrate during a 16-h incubation at 37 °C in complete medium (supplemental Fig. S4A). On this basis, we tested the ability of various protease inhibitors to affect MAE cell migration in a chemotaxis assay and after wounding of the confluent monolayer. Preincubation of mock-MAE cells with the broad spectrum protease inhibitor 1,10-phenanthroline, the serine protease inhibitor {epsilon}-aminocaproic acid, or the carboxyprotease inhibitor pepstatin (34) did not affect their ability to migrate through a gelatin-coated filter in a Boyden chamber assay (data not shown). Also, these inhibitors, as well as a series of MMP inhibitors specific for different members of the MMP family, had only a limited effect, if any, on the ability of MAE cells to repair the wounded monolayer (supplemental Fig. S4B). Taken together, these observations indicate that the reduced motility of OPN-overexpressing cells is not related to their proteolytic potential. This prompted us to investigate in more detail the cell migration process after mechanical wounding of the OPN-MAE cell monolayer.


Figure 3
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  		FIGURE 3.
OPN overexpression inhibits MAE cell migration. A, representative images of mock-transfected and OPN-transfected cell aggregates grown for 48 h in three-dimensional fibrin gel in the presence of 100 ng/ml FGF2. Note the reduced sprouting of OPN-MAE versus mock-MAE cells. Original magnification was x40. B, quantification of the sprouting activity of mock-MAE, OPN-MAE, and {Delta}RGD-OPN-MAE cell aggregates grown for 48 h in three-dimensional fibrin gel in the absence (open bars) or in the presence (black bars) of 100 ng/ml FGF2. Data are expressed as relative area of the cell clusters as assessed by computerized analysis of the digitized images. C, the indicated mock-MAE, OPN-MAE, and {Delta}RGD-OPN-MAE cell clones were evaluated in a Boyden chamber assay for their chemotactic response to fresh medium containing 0% FCS (open bars) or 10% FCS (black bars). In B and C, OPN-transfectants were significantly different (*, p < 0.01) from mock-MAE cells and {Delta}RGD-OPN transfectants.

 
OPN Overexpression Inhibits the Cell Entry in the First Phase of the Cell Migration Cycle—Migration of endothelial cells after wounding of the cell monolayer is preceded by the rapid repositioning of the MTOC from a random distribution to a biased localization in front of the nucleus, toward the direction of cell migration (35). Consistently, 3 h after wounding, the MTOC was located at the leading edge in the front of the nucleus in 62 ± 14% and 67 ± 5% of OPN-MAE and mock-MAE cells adjacent to the wound, respectively. Also, similar levels of active GTP-bound Cdc42, a member of the Rho-GTPase family mediating MTOC reorientation (36, 37), were found in the two cell lines at different time points after wounding (ranging from 30 to 180 min) (data not shown). Thus, OPN up-regulation does not affect MTOC repositioning, indicating that both mock-MAE and OPN-MAE cells are able to polarize toward the chemotactic stimulus.

Migration is a cyclic multistep process (38, 39). The first phase of the cell migration cycle requires lamellipodia extension following microtubule polymerization from the cell center toward the leading edge. This causes activation of the small GTPase Rac1, leading to actin polymerization and focal complex formation at the end of the protrusions (39, 40). OPN-MAE cells adjacent to the wound showed a ruffling activity that was significantly reduced when compared with wounded mock-MAE cells (Fig. 4). Also, 3 h after wounding, mock-MAE cells at the edge of the wound, but not OPN-MAE cells, showed well organized microtubules and actin microfilaments together with the formation of paxillin-immunoreactive focal complexes at the front of migrating cells (Fig. 5). FAK plays a prominent role in integrin signaling and is involved in the activation of an adhesion signaling complex that leads to Rac activation (39, 41). Twenty min after wounding, immunostaining using a specific anti-phospho-FAK(Tyr(P)397) antibody revealed the activation of FAK in mock-MAE cells at the edge of the wound that was absent in OPN transfectants (Fig. 6A). Accordingly, wounding of the monolayer caused a significant increase of the levels of active GTP-bound Rac1 in mock-MAE cells. In contrast, no increase in active GTP-bound Rac1 was observed in wounded OPN-MAE cell monolayers (Fig. 6B). Moreover, different from mock-MAE cells, no significant levels of active GTP-bound RhoA and phospho-ERK1/2, intracellular signals involved in the later phases of the cell migration cycle (39, 41), were observed in OPN-MAE cells 3 h after wounding of the monolayer (Fig. 6C). Similar results were obtained for all of the clones tested. Taken together, the data indicate that OPN up-regulation hampers the migration of MAE cells by impairing their ability to enter the first phase of the cell migration cycle.

Immobilized OPN Hampers MAE Cell Migration and Increases Cell Adhesiveness—Increasing adhesive ligand concentrations may reduce cell motility as a consequence of an increased cell-substratum adhesiveness (42). To assess whether this holds true also for immobilized OPN, parental MAE cells were seeded at high cell density on increasing concentrations of immobilized OPN and were allowed to adhere for 2 h at 37 °C. Then, half of the cell cultures were counted, whereas the remaining confluent monolayers were mechanically wounded; repair was assessed 24 h thereafter. As shown in Fig. 7, purified OPN exerts a potent cell-adhesive activity for MAE cells that form a confluent monolayer on wells coated with OPN concentrations of ≥0.3 nM. As anticipated, increasing concentrations of immobilized OPN progressively impair MAE cell motility, 60% inhibition of wound healing being observed for cells seeded on wells coated with 160 nM OPN. Similar results were obtained when MAE cells were allowed to adhere on wells coated with high concentrations (160 nM) of the {alpha}vβ3 prototypic ligand vitronectin (Fig. 7).


Figure 4
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  		FIGURE 4.
Ruffling activity in OPN overexpressing MAE cells. Confluent mock-MAE cells ({circ}) and OPN-MAE cells (•) were wounded with a 1.0-mm-wide rubber policeman and incubated with complete medium. At different time points, the percentage of cells showing membrane ruffles was counted. Top, representative images of mock-MAE and OPN-MAE cell monolayers photographed 3 h after wounding. The arrows point to membrane ruffles. Original magnification was x400.

 
On this basis, parental MAE cells were seeded onto wells coated with ECM deposited by confluent OPN-MAE cell cultures. This ECM contains OPN levels significantly higher than the ECM deposited by mock-MAE cells (A405 nm values in the OPN detection assay (see "Experimental Procedures") being equal to 0.250 ± 0.010 and 0.085 ± 0.015 for OPN-MAE-ECM and mock-MAE-ECM, respectively). MAE cells attach rapidly to both mock-MAE ECM and OPN-MAE ECM. However, 15 min after attachment, only 20–30% of cells adherent to mock-MAE ECM show a spread morphology (25–27) when compared with 60–70% of cells adherent to OPN-MAE ECM (Fig. 8). This difference was abolished by preincubation of ECMs with neutralizing anti-OPN antibodies.


Figure 5
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  		FIGURE 5.
OPN overexpression affects cytoskeleton organization at the edge of the wound. Confluent mock-MAE and OPN-MAE cells were wounded with a 1.0-mm-wide rubber policeman. After 3 h of incubation with complete medium, cells were stained for microtubules with anti-{alpha}-tubulin antibody, for actin microfilaments with rhodamine-phalloidin, and for focal contacts with anti-paxillin antibody. Original magnification was x630.

 


Figure 6
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  		FIGURE 6.
Integrin signaling in OPN overexpressing MAE cells. A, confluent mock-MAE and OPN-MAE cells were wounded with a 1.0-mm-wide rubber policeman. After 20 min of incubation with complete medium, cells were immunostained with an anti-phospho-FAK(Tyr(P)397) antibody. Original magnification was x630. In parallel, wounded monolayers were lysed at the indicated time points and analyzed for Rac1 activation (B) and for RhoA activation and ERK1/2 phosphorylation (C) as described under "Experimental Procedures."

 


Figure 7
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  		FIGURE 7.
MAE cell adhesion and migration on immobilized OPN. MAE cells were seeded at 70,000 cells/cm2 onto nontissue culture plastic 6-well plates coated with the indicated doses of purified OPN or with 160 nM vitronectin. Cells were allowed to adhere for 2 h at 37 °C. Then adherent cells were fixed and stained, and the plates were read with a microplate reader at 595 nm ({circ}). In parallel, adhered monolayers were wounded with a 1.0-mm-wide rubber policeman. After overnight incubation in complete medium, wounds were photographed, and the repaired area was quantified by computerized analysis of the digitized images (•). Data are expressed as a percentage of maximal adhesion ({circ}) or wound repair (•).

 
Together, these results are consistent with the hypothesis that ECM-associated OPN may increase MAE cell-substratum adhesiveness, thus reducing cell motility.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results demonstrate that constitutive, long lasting OPN up-regulation dramatically impairs re-endothelialization by inhibiting endothelial cell motility. The inhibition requires {alpha}v integrin engagement by immobilized OPN deposited into the subendothelial ECM. This causes an increase in endothelial cell adhesiveness to the substratum, leading to inhibition of the cell entry into the first phase of the cell migration cycle.

OPN binds different integrins and CD44 splicing isoforms (6). Various experimental evidence implicates {alpha}v integrins as the OPN receptors responsible for the observed effects of OPN up-regulation on cell movement: (i) parental and OPN-transfected MAE cells express OPN-binding integrins but do not express any CD44 variant known to interact with OPN; (ii) the selective {alpha}vβ3/{alpha}vβ5 integrin antagonist SCH 221153 (31) fully prevents MAE cell adhesion to immobilized OPN; (iii) the {Delta}RGD-OPN mutant does not mediate MAE cell adhesion; (iv) {Delta}RGD-OPN overexpression does not affect MAE cell motility; (v) high concentrations of immobilized OPN and of the prototypic {alpha}vβ3 ligand vitronectin inhibit the motility of adherent MAE cells.

Soluble OPN exerts a chemotactic effect on endothelial and smooth muscle cells following integrin engagement (20, 43). Accordingly, we have observed that free OPN and GST-OPN, but not GST-{Delta}RGD-OPN, are chemotactic for MAE cells (data not shown). Also, soluble OPN does not hamper re-endothelialization of the wounded monolayer. Indeed, we did not observe any change in the rate of wound repair or in the capacity of parental MAE cells at the edge of the wound to form membrane ruffles when cells were exposed immediately after wounding to increasing concentrations of soluble purified OPN (ranging from 0.3 to 300 nM). Similar results were obtained when cell monolayers were incubated with soluble OPN before wounding, thus confirming the inability of free OPN to inhibit endothelialization.


Figure 8
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  		FIGURE 8.
MAE cell adhesion on OPN-enriched ECM. A, spreading of MAE cells on ECM deposited by mock-MAE and OPN-MAE cells. ECM-coated wells were incubated with vehicle (open bars), 3.0 µg/ml of goat neutralizing anti-OPN antibody (black bars), or irrelevant goat IgG (dashed bars). Then, MAE cells were seeded and allowed to spread on ECM. Fifteen min after seeding, the percentage of cells showing a spread morphology were counted from three random fields in each well under an inverted microscope at x200 magnification. *, significantly different from mock-MAE-ECM controls, p < 0.01. B, parental MAE cells were seeded on the top of mock-MAE-ECM or OPN-MAE-ECM and photographed 15 min thereafter to visualize attached cells with a spread morphology (arrows). Magnification was x200.

 
Integrins play a pivotal role in mediating cell adhesion during migration (44, 45). Migration speed is limited by the rate of lamellipodia protrusion and stabilization of new adhesions at the cell front and by the rate of adhesive release at the cell rear. Cells migrate optimally at an intermediate level of cell-substratum adhesiveness, insufficient or excessive substrate interactions being both detrimental for cell motility and maximum migration occurring at an optimal, intermediate level of integrin receptor occupancy (42, 45). Our data indicate that OPN deposition in the ECM by OPN-MAE cells causes an increase in cell adhesiveness with a consequent impairment of cell motility. Accordingly, subconfluent OPN-MAE cells show a firmly adherent morphology characterized by flatter cobblestone-like appearance when compared with the elongated, fibroblast-like morphology, with frequent lamellipodia and microspikes, observed in parental and mock-MAE cells (see supplemental Fig. S3B).3

FAK plays an important role in promoting integrin-stimulated cell migration (39, 41). In keeping with their reduced motility, wounded OPN-MAE cells fail to phosphorylate FAK-(Tyr(P)397), the major FAK autophosphorylation site that triggers the aggregation of an adhesion signaling complex that leads to Rac1 activation (39, 41). Accordingly, wounded OPN transfectants do not activate Rac1, a key regulator of cell entry into the first phase of the cell migration cycle (39, 40). Consequently, also the activations of RhoA and ERK1/2, both involved in the later phases of the cell migration cycle (39, 41), are hampered in these cells. These data are in keeping with the incapacity of OPN-MAE cells to reorganize their cytoskeleton and to form membrane ruffles at the edge of the wound.

Previous observations had shown that high levels of immobilized fibronectin impairs cell migration (42). Similarly, we have found that high levels of immobilized OPN inhibit the wound healing ability of adherent endothelial cells. Similar results were obtained with vitronectin, thus confirming that a highly adhesive {alpha}v integrin engagement is sufficient to limit cell movement. Accordingly, "freezing" integrins in a high avidity binding state results in the inhibition of cell migration (46). Recent findings (47) have led to the hypothesis that a high density fibronectin substrate may cause focal adhesions that are too stable and unable to signal, thus impairing cell motility (48). Our data confirm and extend these observations and suggest that a long lasting, high avidity engagement by immobilized OPN may down-regulate {alpha}v integrin signaling. Further studies are required to elucidate the molecular basis of this down-regulation.

OPN-MAE cells secrete OPN at levels similar to those observed in parental cells treated with recombinant FGF2, a well known stimulator of endothelial cell motility (33). This apparent contradiction may be explained when considering the long lasting versus the transient OPN up-regulation occurring in the two conditions (i.e. stable cDNA transfection versus growth factor-mediated induction). Also, FGF2 triggers the activation of various intracellular signaling pathways and induces a complex modification of the gene expression profile in endothelial cells, resulting in the up-regulation of transcripts encoding for proteins involved in various cell functions, including cell proliferation, stress response, cell adhesion, and cytoskeleton organization (28). This complex phenotype may modulate the inhibitory effect exerted by ECM-deposited OPN on cell motility.

OPN acts as a key factor in vascular remodeling and atherosclerosis (9–11). Proinflammatory and/or proangiogenic stimuli induce OPN up-regulation in endothelium (14–16). OPN expression is up-regulated in regenerating endothelium after balloon catheter denudation and in restenotic human coronary atherosclerotic plaques (17, 18). Relevant to this point, OPN is associated with the ECM of the injured blood vessel in vivo (17). Thus, the relative concentration of OPN and its possibility to exist both as a free molecule and as an immobilized ECM component may result in the fine tuning of the endothelial cell migration process after blood vessel injury. OPN-transgenic mice undergo increased medial thickening and larger neointima formation after mechanical damage (12). The adverse effects of OPN up-regulation after blood vessel injury have been related to stimulation of smooth muscle cell recruitment. Our data suggest that ECM-bound OPN may contribute to these effects also by delaying re-endothelialization via an autocrine inhibitory action on damaged endothelium.

Loss of the endothelial monolayer integrity may lead to stenosis and vessel occlusion. Also, endothelialization of prosthetic grafts is of pivotal importance in reducing their thrombogenic properties. Our findings suggest that the blockade of endogenous OPN at the site of injury may be viewed as a therapeutic approach to accelerate the endothelialization process and to prevent smooth muscle cell activation and recruitment. This hypothesis is in keeping with the observation that neutralizing anti-OPN antibodies and suppression of OPN expression in OPN null mice affect neointimal thickening and attenuate atherosclerosis (11, 13).


   FOOTNOTES
 
* This study was supported by grants from Ministero dell'Istruzione, Università e Ricerca (Centro di Eccellenza per l'Innovazione Diagnostica e Terapeutica, Fondo per gli Investimenti della Ricerca di Base, and Cofin projects), Fondazione Berlucchi, and Istituto Superiore di Sanità (Progetto Oncotecnologico) (to M. P.); from Associazione Italiana per la Ricerca sul Cancro (to M. P. and F. B.); and from Regione Piemonte (to F. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at www.jbc.org) contains supplemental Figs. S1–S4. Back

1 To whom correspondence should be addressed: General Pathology and Immunology, Dept. of Biomedical Sciences and Biotechnology, Viale Europa 11, 25123 Brescia, Italy. Tel.: 39-0303717311; Fax: 39-0303701157; E-mail: presta{at}med.unibs.it.

2 The abbreviations used are: OPN, osteopontin; ECM, extracellular matrix; MAE, murine aortic endothelial; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; MTOC, microtubule organization center; MMP, matrix metalloprotease; FAK, focal adhesion kinase. Back

3 D. Leali, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. S. Mitola and Dr. M. Rusnati for helpful discussion and criticisms of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tassiopoulos, A. K., and Greisler, H. P. (2000) J. Biomater. Sci. Polym. Ed. 11, 1275-1284[CrossRef][Medline] [Order article via Infotrieve]
  2. Davignon, J., and Ganz, P. (2004) Circulation 109, 27-32[CrossRef]
  3. Fishman, J. A., Ryan, G. B., and Karnovsky, M. J. (1975) Lab. Investig. 32, 339-351[Medline] [Order article via Infotrieve]
  4. Ross, R. (1999) N. Engl. J. Med. 340, 115-126[Free Full Text]
  5. Denhardt, D. T., and Guo, X. (1993) FASEB J. 7, 1475-1482[Abstract]
  6. Denhardt, D. T., Noda, M., O'Regan, A. W., Pavlin, D., and Berman, J. S. (2001) J. Clin. Investig. 107, 1055-1061[Medline] [Order article via Infotrieve]
  7. O'Regan, A. W., Nau, G. J., Chupp, G. L., and Berman, J. S. (2000) Immunol. Today 21, 475-478[CrossRef][Medline] [Order article via Infotrieve]
  8. Furger, K. A., Rajashree, K. M., Tuck, A. B., Bramwell, V. H. C., and Chambers, A. F. (2001) Curr. Mol. Med. 1, 621-632[CrossRef][Medline] [Order article via Infotrieve]
  9. Ikeda, T., Shirasawa, T., Esaki, Y., Yoshiki, S., and Hirokawa, K. (1993) J. Clin. Investig. 92, 2814-2820[Medline] [Order article via Infotrieve]
  10. Giachelli, C. M., Bae, N., Almeida, M., Denhardt, D. T., Alpers, C. E., and Schwartz, S. M. (1993) J. Clin. Investig. 92, 1686-1696[Medline] [Order article via Infotrieve]
  11. Liaw, L., Lombardi, D. M., Almeida, M. M., Schwartz, S. M., deBlois, D., and Giachelli, C. M. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 188-193[Abstract/Free Full Text]
  12. Isoda, K., Nishikawa, K., Kamezawa, Y., Yoshida, M., Kusuhara, M., Moroi, M., Tada, N., and Ohsuzu, F. (2002) Circ. Res. 91, 77-82[Abstract/Free Full Text]
  13. Matsui, Y., Rittling, S. R., Okamoto, H., Inobe, M., Jia, N., Shimizu, T., Akino, M., Sugawara, T., Morimoto, J., Kimura, C., Kon, S., Denhardt, D., Kitabatake, A., and Uede, T. (2003) Arterioscler. Thromb. Vasc. Biol. 23, 1029-1034[Abstract/Free Full Text]
  14. Singh, K., Balligand, J. L., Fischer, T. A., Smith, T. W., and Kelly, R. A. (1995) J. Biol. Chem. 270, 28471-28478[Abstract/Free Full Text]
  15. Senger, D. R., Ledbetter, S. R., Claffey, K. P., Papadopoulos-Sergiou, A., Peruzzi, C. A., and Detmar, M. (1996) Am. J. Pathol. 149, 293-305[Abstract]
  16. Leali, D., Dell'Era, P., Stabile, H., Sennino, B., Chambers, A. F., Naldini, A., Sozzani, S., Nico, B., Ribatti, D., and Presta, M. (2003) J. Immunol. 171, 1085-1093[Abstract/Free Full Text]
  17. O'Brien, E. R., Garvin, M. R., Stewart, D. K., Hinohara, T., Simpson, J. B., Schwartz, S. M., and Giachelli, C. M. (1994) Arterioscler. Thromb. 14, 1648-1656[Abstract/Free Full Text]
  18. Liaw, L., Lindner, V., Schwartz, S. M., Chambers, A. F., and Giachelli, C. M. (1995) Circ. Res. 77, 665-672[Abstract/Free Full Text]
  19. Lee, T. Y., and Gotlieb, A. I. (1999) Cell Tissue Res. 297, 435-450[CrossRef][Medline] [Order article via Infotrieve]
  20. Xuan, J. W., Hota, C., Shigeyama, Y., D'Errico, J. A., Somerman, M. J., and Chambers, A. F. (1995) J. Cell Biochem. 57, 680-690[CrossRef][Medline] [Order article via Infotrieve]
  21. Bastaki, M., Nelli, E. E., Dell'Era, P., Rusnati, M., Molinari-Tosatti, M. P., Parolini, S., Auerbach, R., Ruco, L. P., Possati, L., and Presta, M. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 454-464[Abstract/Free Full Text]
  22. Belleri, M., Ribatti, D., Nicoli, S., Cotelli, F., Forti, L., Vannini, V., Stivala, L. A., and Presta, M. (2005) Mol. Pharmacol. 67, 1451-1459[Abstract/Free Full Text]
  23. Gualandris, A., Rusnati, M., Belleri, M., Nelli, E. E., Bastaki, M., Molinari-Tosatti, M. P., Bonardi, F., Parolini, S., Albini, A., Morbidelli, L., Ziche, M., Corallini, A., Possati, L., Vacca, A., Ribatti, D., and Presta, M. (1996) Cell Growth Differ. 7, 147-160[Abstract]
  24. Falcone, D. J., McCaffrey, T. A., Haimovitz-Friedman, A., Vergilio, J. A., and Nicholson, A. C. (1993) J. Biol. Chem. 268, 11951-11958[Abstract/Free Full Text]
  25. Runyan, R. B., Versalovic, J., and Shur, B. D. (1988) J. Cell Biol. 107, 1863-1871[Abstract/Free Full Text]
  26. Yoshimura, M., Nishikawa, A., Nishiura, T., Ihara, Y., Kanayama, Y., Matsuzawa, Y., and Taniguchi, N. (1995) J. Biol. Chem. 270, 2298-2304[Abstract/Free Full Text]
  27. Yu, D. H., Qu, C. K., Henegariu, O., Lu, X., and Feng, G. S. (1998) J. Biol. Chem. 273, 21125-21131[Abstract/Free Full Text]
  28. Dell'Era, P., Coco, L., Ronca, R., Sennino, B., and Presta, M. (2002) Oncogene 21, 2433-2440[CrossRef][Medline] [Order article via Infotrieve]
  29. Katagiri, Y. U., Sleeman, J., Fujii, H., Herrlich, P., Hotta, H., Tanaka, K., Chikuma, S., Yagita, H., Okumura, K., Murakami, M., Saiki, I., Chambers, A. F., and Uede, T. (1999) Cancer Res. 59, 219-226[Abstract/Free Full Text]
  30. Denhardt, D. T., Giachelli, C. M., and Rittling, S. R. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 723-749[CrossRef][Medline] [Order article via Infotrieve]
  31. Kumar, C. C., Malkowski, M., Yin, Z., Tanghetti, E., Yaremko, B., Nechuta, T., Varner, J., Liu, M., Smith, E. M., Neustadt, B., Presta, M., and Armstrong, L. (2001) Cancer Res. 61, 2232-2238[Abstract/Free Full Text]
  32. van Hinsbergh, V. W., Engelse, M. A., and Quax, P. H. (2006) Arterioscler. Thromb. Vasc. Biol. 26, 716-728[Abstract/Free Full Text]
  33. Presta, M., Dell'Era, P., Mitola, S., Moroni, E., Ronca, R., and Rusnati, M. (2005) Cytokine Growth Factor Rev. 16, 159-178[CrossRef][Medline] [Order article via Infotrieve]
  34. Montesano, R., and Orci, L. (1985) Cell 42, 469-477[CrossRef][Medline] [Order article via Infotrieve]
  35. Ueda, M., Graf, R., MacWilliams, H. K., Schliwa, M., and Euteneuer, U. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9674-9678[Abstract/Free Full Text]
  36. Etienne-Manneville, S., and Hall, A. (2001) Cell 106, 489-498[CrossRef][Medline] [Order article via Infotrieve]
  37. Etienne-Manneville, S., and Hall, A. (2002) Nature 420, 629-635[CrossRef][Medline] [Order article via Infotrieve]
  38. Raftopoulou, M., and Hall, A. (2004) Dev. Biol. 265, 23-32[CrossRef][Medline] [Order article via Infotrieve]
  39. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T., and Horwitz, A. R. (2003) Science 302, 1704-1709[Abstract/Free Full Text]
  40. Waterman-Storer, C. M., Worthylake, R. A., Liu, B. P., Burridge, K., and Salmon, E. D. (1999) Nat. Cell Biol. 1, 45-50[CrossRef][Medline] [Order article via Infotrieve]
  41. Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J. T., and Horwitz, A. F. (2004) Nat. Cell Biol. 6, 154-161[CrossRef][Medline] [Order article via Infotrieve]
  42. Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A., and Horwitz, A. F. (1997) Nature 385, 537-540[CrossRef][Medline] [Order article via Infotrieve]
  43. Liaw, L., Almeida, M., Hart, C. E., Schwartz, S. M., and Giachelli, C. M. (1994) Circ. Res. 74, 214-224[Abstract/Free Full Text]
  44. Cox, E. A., Sastry, S. K., and Huttenlocher, A. (2001) Mol. Biol. Cell 12, 265-277[Abstract/Free Full Text]
  45. Cox, E. A., and Huttenlocher, A. (1998) Microsc. Res. Tech. 43, 412-419[CrossRef][Medline] [Order article via Infotrieve]
  46. Kuijpers, T. W., Mul, E. P., Blom, M., Kovach, N. L., Gaeta, F. C., Tollefson, V., Elices, M. J., and Harlan, J. M. (1993) J. Exp. Med. 178, 279-284[Abstract/Free Full Text]
  47. Gupton, S. L., and Waterman-Storer, C. M. (2006) Cell 125, 1361-1374[CrossRef][Medline] [Order article via Infotrieve]
  48. Schwartz, M. A., and Horwitz, A. R. (2006) Cell 125, 1223-1225[CrossRef][Medline] [Order article via Infotrieve]

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