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Cardiovascular Research 2002 53(4):952-962; doi:10.1016/S0008-6363(01)00547-8
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Vitronectin is up-regulated after vascular injury and vitronectin blockade prevents neointima formation

Pascale Dufourcq*, Thierry Couffinhal, Philipe Alzieu, Danièle Daret, Catherine Moreau, Cécile Duplàa and Jacques Bonnet

Institut National de la Santé et de la Recherche Médicale, INSERM Unité 441, Athéroclérose, Avenue du Haut-Lévêque, 33600 Pessac, France

* Corresponding author. Tel.: +33-5-5789-1975; fax: +33-5-5636-8979 pascale.dufourcq{at}biophar.u-bordeaux2.fr

Received 8 August 2001; accepted 20 November 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Smooth muscle cell (SMC) migration involves interactions with extracellular matrix (ECM) and is an important process in response to arterial wall injury. We investigated the expression and the functional role of vitronectin (VN) in the response after vascular injury. Methods: VN and {alpha}vβ3/β5 integrin expressions were investigated after balloon carotid injury of Sprague–Dawley rats. Adventitial delivery of blocking antibodies to VN, {alpha}vβ5 and β3 integrins were performed to assess their roles in neointima formation. In vitro, migration assays were carried out on human SMC. Results: Immunohistochemistry and in situ hybridization for VN showed an upregulation of VN during the early time points of intima formation. {alpha}vβ3/β5 integrins expression correlated with VN expression. After 7 days, blocking antibodies to VN, {alpha}vβ5 and β3 induced a significant decrease on intimal area associated with a decrease in intimal cell counts. A slight decrease in intimal cell proliferation without any effect on apoptosis was observed after VN blockade. In vitro, migrating SMC strongly expressed VN after injury and neutralizing anti-VN antibody inhibited SMC migration. Blocking experiment with anti-{alpha}vβ5 and -{alpha}vβ3 integrin antibodies showed that not only VN–{alpha}vβ3 but also VN–{alpha}vβ5 interactions are required for SMC migration. Conclusion: This study characterizes the VN–ECM interaction in SMC and supports the role of VN in mediating SMC migration and neointimal formation in response to injury.

KEYWORDS ECM, extracellular matrix; MMP, matrix metalloproteinase; PAI-1, plasminogen activator inhibitor-1; SMC, smooth muscle cell; tPA, tissue-plasminogen activator; uPA, urokinase; VN, vitronectin

This article is referred to in the Editorial by A.C. Newby (pages 779–781) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Atherosclerotic stenosis and its ischemic complications lead to the need for arterial reconstruction. Coronary angioplasty and other transcatheter procedures used to treat stenosis induce an acute form of vascular injury which can result in restenosis thus limiting the long-term benefit of revascularisation. Restenosis after angioplasty results from both intimal hyperplasia and arterial remodeling and it is the balance between the two processes which determines the final luminal size [1]. Intimal formation is a consequence of smooth muscle cell (SMC) accumulation in the neointima due to the combined processes of proliferation and migration from the media [2]. Moreover, recent reports support the hypothesis that adventitial cells can translocate from the adventitia to the intima and participate in the formation of the neointima [3].

Growth factors as well as extracellular matrix proteins (ECM) can stimulate vascular cell migration [2]. Among the latter, expressions of fibronectin, osteopontin, thrombospondin and collagen increase after vascular injury and are involved in SMC migration through interaction with cell surface receptors, which transduce extracellular information to the intracellular machinery [12–14]. In addition to promoting migration, ECM can influence the state of differentiation of SMC as well as the proliferative response [15,16].

Vitronectin (VN) is a glycoprotein present in plasma and serum at about 200–300 µg/ml and also localized into the extracellular matrices of various tissues [4]. The liver emerges as a major source, although other normal organs and pathological tissues synthesize VN [5,6]. Vitronectin is a multifunctional protein involved in adhesion and migration of neural crest cells and keratinocytes [6,7], in the induction of neurite outgrowth and differentiation [8] and myocyte differentiation in Drosophila embryos [9]. Vitronectin interacts with several proteins critical for coagulation and fibrinolysis such as PAI-1, u-PA and its receptor [10]. Vitronectin also contains an RGD sequence which binds the {alpha}v-integrin family of receptors ({alpha}vβ3, {alpha}vβ5, {alpha}vβ1 and {alpha}vβ8) and the platelet receptor {alpha}IIbβ3 [4,11].

VN accumulates in atherosclerotic plaques [17] and we recently demonstrated that SMC express VN mRNA and synthesize the protein [18]. In culture, SMC express both {alpha}vβ3 and {alpha}vβ5 integrins which interact with VN and co-localize with VN in atherosclerotic plaques [18,19]. VN induces migration of SMC in a Boyden chamber assay by a mechanism which appears to require the {alpha}vβ3 integrin [20]. In addition, RGD peptides that compete for integrin binding or anti-{alpha}vβ3 blocking antibody reduce neointima development in animal models [21–23]. These data suggest that VN through its interactions with {alpha}v integrins (VN receptors) may play an important physiopathologic role in vascular diseases.

The purpose of this study was to investigate the expression of VN after vascular injury and VN involvement in the neointima formation after angioplasty through its effects on migration. We aimed to characterize the role of {alpha}vβ5 integrin in this process as {alpha}vβ5 was found to co-localize with VN and as its function is not well defined in vessel wall. We first characterized the expression of VN in a model of rat carotid injury and migrating SMC in vitro. Then, we investigated the effect of selective blockade of VN, {alpha}vβ5 and {alpha}vβ3 integrins on SMC migration in culture and on neointima formation after balloon injury.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cell culture
SMC were isolated from the media of human adult aorta by enzyme digestion, cultured in Ham's F10 medium supplemented with 10% fetal calf serum (FCS), 5 mM HEPES, 50 U/ml penicillin and 50 mg/ml streptomycin as previously described [18].

2.2. Migration assays
2.2.1. Wound assay
Confluent SMC monolayer was wounded with a single-edge razor-blade [23] and allowed to migrate 24 h in serum-free medium with or without blocking antibodies. To inhibit protein synthesis, cycloheximide (12 µg/ml) was added to the cells after the wound.

2.2.2. Migration assays on extracellular matrix
The surface of the two-chamber slides (Labtek, Nunc Inc.) was coated with VN or FN (5 µg/cm2) as described previously [18]. The cells were placed in the center of the ring. At confluence, the ring was removed, SMC were allowed to migrate for 8 h in serum-free medium with or without blocking antibodies.

Cells migrating across the wound line or across the ring were counted using the 10x objective.

Immunofluorescence staining with anti-vitronectin (VIT-2, Sigma) was carried out as described previously [18].

2.3. Arterial injury model
Balloon injury was performed on the right common carotid artery of Sprague–Dawley rats. The animals were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg; Sanofi). A 2F Fogarty balloon catheter (Baxter) was introduced into the common carotid artery through an arteriotomy performed in the external carotid artery [24]. The National Institute for Health and Medical Research (Inserm, France) approved all protocols. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.4. Tissue processing
Immunohistochemistry and in situ hybridization were performed on a total of 54 animals. The analyses were carried out at the following times points after carotid dilatation: 30 min, 2, 4, 8, 12, 24, 48, 72 h, 7, 10, 14, and 30 days. Animals were sacrificed with an overdose of sodium pentobarbital. Whole injured and non-injured carotids were resected along with adjacent tissues, then rinsed in PBS to remove excess blood. Tissues were immediately fixed overnight in methanol or in 4% PFA, respectively, for immunohistochemistry or in situ hybridization, prior to paraffin embedding. All tissues were embedded in a longitudinal way, then carotids were cut until the lumen of the artery was reached and the gross anatomic inspection gave two parallel bands of artery separated by the lumen.

2.5. Immunohistochemistry and in situ hybridization
All methods for VN cDNA cloning and in situ hybridization have been described [18]. In vivo immunostaining for VN (VIT-2; Sigma), {alpha}vβ3/β5 integrins (Gibco), {alpha}-actin (1A4; Sigma), PCNA (Dako) was carried out as previously described [18].

2.6. In vivo blocking experiments: osmotic pump implant
To perform in vivo blocking experiments, a polyethylene microcatheter was placed adjacent to the carotid artery and secured by suturing it to the adjacent cervical musculature after carotid injury. The distal end of the microcatheter was passed through the lateral neck musculature and connected to an Alzet osmotic pump placed under the rat's neck. The pump was filled with a solution of 50 µg/ml of blocking antibody or control antibody in PBS. The osmotic pump containing antibodies was primed ex-vivo, 36 h in PBS at 37 °C before being placed in the rat. Fifty µg/ml of blocking antibody anti-VN (M4; CliniScience), -{alpha}vβ5 integrin (P1F6; Pharmingen), -β3 subunit integrin (Pharmingen) or control antibody were used. Antibodies were delivered continuously over 7 days at 2 µl/h on the adventitial surface of the carotid artery. At 7 days after injury, rats (n=4 for each group) were sequentially perfused at 110 mmHg for 5 min with PBS and 4% PBS–paraformaldehyde via an intra-aortic cannula. Injured and non-injured carotid arteries were dissected and processed for tissue analysis.

2.7. Morphometric and data analysis
Morphometric analysis were performed 7 days after injury and antibodies blockade. All histomorphometric and planimetry measurements were made with Sigma ScanPro software. Results represented the average of intimal or medial areas measured every 400-µm spaced section along the complete carotid artery (a minimum of 20 400-µm spaced sections per animal were analyzed from one end to the other). Total intimal and medial cells, PCNA-positive and apoptotic cells were manually counted in high-magnification video images for each layer. Results represented the average of eight independent high-magnification video images recorded and counted from each animal. Results are expressed in percentage of total intimal cell count.

2.8. Apoptosis analysis
Apoptosis analysis was performed 7 days after injury. To detect apoptosis in situ, fragmented DNA was nick-end labeled with biotinylated dUTP introduced by terminal deoxynucleotidyl transferase and then stained with avidin-conjugated peroxidase [25].

2.9. Antibodies
Anti-vitronectin antibodies: the monoclonal antibodies (mAb) against human vitronectin VIT-2 (Sigma) were used for in vitro and in vivo immunostaining, and the mAb M4 (CliniScience) was used for in vitro and in vivo blocking experiments. Anti-{alpha}vβ3 integrin antibodies: the mAb LM609 (Chemicon) was used for in vitro blocking experiments, the rabbit polyclonal antibody anti-{alpha}vβ3/β5 (Gibco-BRL) previously demonstrated to react with rat tissue [2] was used for rat tissue immunostaining. The mAb anti-β3 integrin subunit (Pharmingen) was used for in vivo blocking experiments. Anti-{alpha}vβ5 integrin antibodies: the mAb P1F6 (Pharmingen) was used for in vitro and in vivo blocking experiments. Anti-β1 integrin (DF5) subunits were purchased from Tebu. Anti-PCNA mAb and anti-{alpha}-actin mAb (1A4) were purchased, respectively, from Dako and Sigma. A non-immune, isotype-matched immunoglobulin (Sigma) was used as a negative control for monoclonal antibodies and preimmune rabbit serum was used as a negative control for polyclonal antibodies.

2.10. Statistics
Data were analyzed by ANOVA using the StatView 4.02 program (Abacus Concepts). A P value of less than 0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Vitronectin expression after balloon injury
The expression of VN in normal and injured rat carotid was investigated by immunohistochemistry and in situ hybridization (Figs. 1 and 2Go). In normal carotid, VN protein and mRNA expression were very low in the media and in the adventitia (Fig. 1A).


Figure 1
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  		Fig. 1 Immunostaining of vitronectin and its receptors after rat carotid injury. Normal artery (A) or injured arteries were stained with mAb anti-VN antibody 8 h (B), 3 days (C), 7 days (E) and 14 days (F) after carotid injury. As control, artery was labeled with non-specific IgG antisera (D). On serial sections, VN receptors staining was realized 3 days (G) and 14 days (H) after carotid injury or on non-injuried carotid with anti-{alpha}vβ3/{alpha}vβ5 integrins antibody (I). Positive staining is in brown and the nuclei were in purple after hematoxylin counterstaining. Arrows indicate internal and external elastic lamina. Magnification x10.

 

Figure 2
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  		Fig. 2 In situ hybridization to detect vitronectin mRNA in carotid after injury. Hybridization with antisense (A,C,E) and sense (B,D,F) of VN riboprobe were performed at 8 h (A,B), 3 days (B,D) and 7 days (E,F) after carotid injury. VN mRNA was revealed in blue and nuclei were counterstained in green with methyl green labeling. Arrows indicate internal and external elastic lamina. M: media. Magnification x10.

 
3.1.1. Adventitia
At 8 h after balloon injury, weakly positive VN protein staining was observed in adventitial cells (Fig. 1B). At 2 and 3 days after injury, adventitial cells closest to the external elastic lamina exhibited more marked VN expression (Fig. 1C). Adventitial VN protein labeling increased until 7 days after the injury (Fig. 1E), then it remained strong and stable until 30 days. VN mRNA labeling was detected in the adventitia after 8 h (Fig. 2A), its expression increased up to 7 days and remained stable until 30 days. No staining was observed with the sense probe (Fig. 2B,D,F)

3.1.2. Media
At 4–8 h, a loss of VN staining was observed (Fig. 1B). Twenty-four h after balloon injury, medial SMC of the external layers strongly expressed VN protein. At 3 days, VN protein labeling was observed in all medial layers with a higher level in the luminal SMC layer (Fig. 1C). After 7 days, VN protein medial expression (Fig. 1F) decreased and returned to the basal level at 30 days. VN mRNA expression was detected in SMC as soon as 24 h post-injury and was strongly expressed at 3 days (Fig. 2C). No mRNA expression was observed in the media after 7 days (Fig. 2E).

3.1.3. Neointima
In the neointima, a strong increase of VN protein staining was observed at 7 and 10 days after the injury and it was higher than in the underlying media (Fig. 1E). At 14 days, although a strong labeling persisted at the luminal border of the neointima, VN protein expression decreased in the deep intima and became diffuse (Fig. 1F). At 30 days, the staining was diffuse and faint. After 7 days, the intimal thickening constituted by several SMC layers showed positive VN mRNA labeling compared to the negative underlying media (Fig. 2E). VN mRNA expression decreased thereafter. The labeling with an antibody against {alpha}-actin showed that in the media and neointima, VN mRNA positive cells were {alpha}-actin positive cells (not shown).

Expression of VN receptors was analyzed by immunohistochemistry on the serial sections used for VN staining with an antibody against {alpha}vβ3 and {alpha}vβ5 integrins. In normal carotid, VN receptor expression was present throughout the media. Three days after carotid injury, the strong expression of VN in the SMC of the medial luminal side was correlated with elevated expression of {alpha}vβ3/β5 receptors in the same area (Fig. 1G). At 7 days, {alpha}vβ3/β5 integrin expression was predominant in the neointima. At 14 days, a stronger {alpha}vβ3/β5 integrin expression was observed in the luminal SMC, similar to VN expression (Fig. 1H), whereas a diffuse staining remained in a deep intima. So, after balloon injury, VN receptor expression appeared to co-localize with VN.

3.2. In vitro migration of SMC
In response to a wound injury of the confluent human cell layer in serum-free medium, SMC were seen migrating during the following 24 h. In this model, the restitution of the wounded area was totally inhibited by cycloheximide (CHX), a protein synthesis inhibitor (10±4 vs. 100%, respectively, for CHX and control; n=3), supporting active synthesis of protein by SMC as being necessary for migration. By immunofluorescent staining (Fig. 3), migrating SMC (arrow) were shown to express more VN as compared to non migrating (arrow ahead) ones after wound assay (Fig. 3A). VN staining in migrating cell has a fibrillar distribution (Fig. 3B). Blocking experiments were performed to investigate whether VN, {alpha}vβ3 and {alpha}vβ5 integrins were required for SMC migration. Anti-VN antibody induced a dose dependent inhibition of SMC migration after a wound injury compared to IgG control. Monoclonal anti-VN antibody at 10 µg/ml induced an inhibition of SMC migration by 52±5% (P<0.0001, as compared to control IgG; n=9; Fig. 4A). The presence of anti-{alpha}vβ3 or -{alpha}vβ5 integrin mAbs (1 µg/ml) induced, respectively, a 16±2 and 28±6% decrease of SMC migration after 24 h (P<0.05, n=5; Fig. 4B). We did observe significant additional inhibition of the migration in the presence of both mAb anti-{alpha}vβ5 and -{alpha}vβ3 (39±3%). In contrast, mAb anti-β1 integrin subunit (DF5) failed to inhibit SMC migration suggesting that there is no apparent role for {alpha}vβ1 integrin in this process.


Figure 3
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  		Fig. 3 Immunofluorescence staining of vitronectin in migratory human SMC. At 24 h after wound injury, SMC were fixed and stained with mAb for VN (A,B) or control IgG (C). The nuclei were counterstained in blue (Hoechst). The wound was represented as a white line, migrating cells with an arrow, and non-migrating cell with an arrow ahead. Magnification x20 on panel A, x40 on panel B,C, and x100 on insert.

 

Figure 4
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  		Fig. 4 Effect of neutralizing antibodies on SMC migration after wound assay and on vitronectin. (A,B) After a wounding assay SMC migrated in serum-free medium (SFM) or in presence of control IgG (IgG, 10 µg/ml), mAb anti-VN (M4, 0.5–10 µg/ml), -{alpha}vβ3 (LM609, 1 µg/ml), -{alpha}vβ5 (P1F6, 1 µg/ml) or -{alpha}vβ3 plus -{alpha}vβ5 (1 µg/ml of each), or mAb anti-β1 (DF5, 1 µg/ml). After 24 h, migrating cells was quantified. (C) Effect of mAb anti-{alpha}vβ3 and {alpha}vβ5 (1 µg/ml) on SMC migration on VN-coated slide. SMC migrated on VN in serum-free medium and were quantified in presence or not of mAb anti-{alpha}vβ3, -{alpha}vβ5 (1 µg/ml) after 8 h. Each point represented the average of five ti nine independent experiments; in each experiment more than ten fields were quantified. Data were expressed as mean±S.E. * P<0.01, ** P<0.001, *** P<0.0001.

 
To confirm the role of VN–{alpha}vβ5 interactions in SMC migration, a second migration assay was developed. In this experiment, SMC were migrating on VN-coated slides without any injury. Addition of anti-{alpha}vβ3 blocking and anti-{alpha}vβ5 integrin mAbs (1 µg/ml) induced a significant decrease in SMC migration on VN compared to control IgG (26±2 and 38±4%, respectively, P<0.0001, n=9) (Fig. 4C).

In conclusion, these data show that migrating SMC expressed VN and required it to migrate after injury. SMC migration involved VN–{alpha}vβ3 but also VN–{alpha}vβ5 interactions.

3.3. In vivo blocking experiments
Continuous local delivery by osmotic pump of the different blocking antibodies to the adventitial layer of balloon-injured carotid arteries was performed during 7 days (n=4 for each group) (Fig. 5). Carotids treated for 7 days with anti-VN, anti-{alpha}vβ5 or anti-β3 antibodies and sacrificed 7 days after injury, displayed a significant reduction of neointima area as compared to control carotids treated with an irrelevant IgG (Fig. 5A and Table 1). Medial areas were the same between the different groups, giving a significantly reduced I/M ratio in the two blocking conditions, VN and {alpha}vβ5, as compared to IgG (Table 1). The I/M ratio of β3 integrin blockade was not significantly different compared to I/M ratio of control IgG (Table 1).


Figure 5
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  		Fig. 5 Anti-vitronectin, -{alpha}vβ5 and β3 antibodies inhibit rat carotid neointima thickening. (A) Representative photomicrographs of Masson trichrome-stained sections of rat carotid arteries from control IgG (a), and anti-VN (b), anti-{alpha}vβ5 (c), anti-β3 (d) mAbs-treated rat at 7 days following carotid injury. Original magnification x10. (B) Arterial areas (mm2) were measured 7 days after balloon injury in carotid intima and media and the ratio values Intimal/Medial area were calculated. Data were expressed as mean±S.E., ** P<0.0001, * P<0.05.

 

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  		Table 1 Effect of blocking antibodies on intima formation and on intimal cell proliferation and apoptosis after 7 days following arterial injury

 
Decrease in intimal area results from a reduction in cell number or a reduction in ECM accumulation. Total intimal cell count was significantly reduced with all blocking antibody conditions compared to control antibody, with a higher cell decrease in VN blocking experiment (Table 1). Cell density was not different in between each group (Table 1), meaning that there is no variation in ECM accumulation. So, the reduction in intimal area was correlated with a reduction in intimal cell contents. Intimal cell reduction could be the result of either a decrease in SMC migration (that could not be monitored per se) and/or a decrease in intimal SMC proliferation and/or an increase in intimal cell apoptosis. A significant reduction in proliferative index was observed in the carotids treated with anti-VN antibody as compared to those treated with control antibody (Fig. 6 and Table 1). Neither β3 nor {alpha}vβ5 integrin blockade had an effect on intimal cell proliferation index as compared to control IgG (Fig. 6 and Table 1). No difference in intimal apoptosis was observed at 7 days in the experiments of VN blockade or {alpha}vβ5 integrin blockade as compared to control blockade (Table 1), whereas β3 integrin blockade induced a significant increase in apoptotic cells in the intima (Table 1).


Figure 6
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  		Fig. 6 Effect of blocking anti-VN, -{alpha}vβ5 and -β3 antibodies on intimal cell proliferation. (A) Representative photomicrograph of PCNA-staining of rat carotid arteries in the area of intimal thickening from control IgG (IgG), anti-VN (VN), anti-{alpha}vβ5 ({alpha}vβ5) and anti-β3 (β3) mAbs-treated rat at 7 days following carotid injury. PCNA positive cells were labeled in brown in the nucleus. The slides were counterstaining with hematoxylin. Original magnification x40.

 
In conclusion, VN blockade has a slight effect on SMC proliferation but not on cell apoptosis. So, the large reduction in I/M ratio induced by VN blockade results from a decrease in SMC migration and proliferation. The {alpha}vβ5 integrin blockade has no effect on proliferation or apoptosis, so I/M ratio reduction was due to a decrease in SMC migration. The modest effect of β3 integrin subunit blockade on I/M reduction could be attributed to an increase of apoptosis in addition to its previously demonstrated role on migration.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we present several lines of evidence that VN is an important mediator in neointima formation after arterial injury. First, VN is upregulated early after vascular injury and it synthesized at the edge of migrating cell. Second, inhibition of VN by blocking antibody inhibits SMC migration in culture and prevents neointima formation following carotid arterial injury. Third, VN receptors blockade demonstrated that {alpha}vβ5 integrin are required for neointima formation and SMC migration.

It has been demonstrated, that in the carotid arteries after balloon injury model, medial SMC started to migrate 3 days after the procedure in the intima then proliferated to constitute the neointima [26]. Our recent data on the rat carotid model showed that important adventitial changes occurred in the first 48 h post-injury with an extensive cell loss by apoptosis and oncosis in the adventitia and in the media, followed by a rapid proliferation [41]. Moreover, adventitial cells could migrate from the adventitia to participate in intimal formation [3,41]. The present study shows that although VN levels are low in normal rat carotids, VN protein expression is upregulated during the early time point of arterial response after injury and follows the edge of cell migration. The upregulation of VN protein correlates with an induction of VN mRNA in the media and in the neointima. This suggests that after injury, vascular cells could synthesize VN protein as in atherosclerotic lesions [18]. The time-course of VN receptor expressions are in agreement with previous studies in other animal models [22,27–29]. This study shows that VN expression is co-localized with its receptors expression, {alpha}vβ3/β5 integrins, on the adventitial cells as well as on SMC and are coordinately upregulated during SMC migration and neointima formation.

As VN is highly expressed early after injury, we investigated whether VN could be involved in the early cellular response after arterial injury. In vivo arterial wall VN-blockade and VN receptor-blockade were performed during 7 days after injury. Adventitial delivery of blocking antibody by an osmotic pump showed that VN blockade reduces the intimal area and total intimal cells. There was no effect on apoptosis whereas a decrease in SMC proliferation in the intima was observed. Although it is difficult to follow cell migration in the carotid model, in vitro data support the fact that VN blockade function could also inhibit cell migration. Indeed, migrating SMC strongly expressed VN and required VN to migrate independently of proliferation. This is in agreement with several studies, which demonstrated that VN is chemotactic and haptotatic for SMC and has a stronger effect compared to the other ECM proteins [20,30]. All together, these data suggest that the benefic effect of VN blockade on neointima formation could be due to an inhibition of cell proliferation and SMC migration.

Recent studies described {alpha}vβ5 integrin in vascular disease and suggested a role in SMC migration [28,29] but a direct evidence for a role of {alpha}vβ5–VN interactions in SMC migration and neointima formation was still missing. This present study demonstrates that specific {alpha}vβ5 integrin blockade by antibody prevents neointima formation decreasing intimal cell number without modifying cell proliferation and apoptosis. In vitro, we demonstrated that {alpha}vβ5 integrin is required, as {alpha}vβ3, for SMC migration after a wound injury or on VN-coated surface. The {alpha}vβ5 integrins were organized in focal adhesion over all the cell surface of migrating cells (data not shown) suggesting that VN–{alpha}vβ5 interaction should be required to activate intracellular signaling pathways after integrin ligation and/or clustering [11]. It has been shown that human keratinocytes and tumor cells express VN receptors and use {alpha}vβ5 integrin for cellular locomotion [36,37]. So, the reduction in intimal thickening after {alpha}vβ5 blockade could be due to a decrease in SMC migration. Our study showed that local adventitial blockade of β3 integrin reduces neointima formation by decreasing intimal cell content, without modification in cell proliferation. However, an increase in intimal apoptosis was observed. This is in agreement with the recent studies, which demonstrated that inhibition of β3 integrin function by neutralizing antibodies or peptides prevent neointima formation [21,40,38] associated with inhibition of SMC migration [23] and/or apoptosis activation [39]. Involvement of VN–{alpha}vβ3 interactions in SMC migration in vitro are in agreement with previous studies showed that soluble VN induced SMC migration involving {alpha}vβ3 integrin [20] and that after wound injury SMC in serum conditions migrated in a {alpha}vβ3 integrin-dependent manner [23]. VN–{alpha}vβ3 interaction was described as the pathway required for SMC migration. Here, we provided data showing that VN–{alpha}vβ5 interactions are also an important pathway involved in SMC migration and in neointima formation.

This study demonstrates for the first time the in vivo role of VN and suggests that blockade of its interaction with cognate integrins could be a target to prevent intimal thickening. The notion that motile cells would provide their own VN substratum for migration is suggested in other systems. Vitronectin is found associated with late stage malignant astrocytoma cells and is preferentially expressed at the invading tumor margin [6]. Vitronectin is also detected in the early embryo closely associated with the surface of neural crest cells in migration [7]. Besides its ability to act as a migratory ligand for the integrin, VN has other properties that makes it an important candidate for the control of the vascular response to injury. Vitronectin is able to induce specific gene expression involved in ECM degradation such as MMP2 [31], tPA and urokinase [32]. Vitronectin protects microvascular and tumor cells from apoptotic death by increasing anti-apoptotic proteins Bcl-2 and Bcl-x [33,34]. Finally, VN regulates smooth muscle contractility via {alpha}v and β1 integrins [35].

In conclusion, VN could be an important mediator of tissue repair after arterial injury, permitting the migration and proliferation of cells into the newly formed intima. Beside {alpha}vβ3 integrin, VN interaction with {alpha}vβ5 integrin is essential for this migration. Local inhibition of VN function prevents neointima formation after arterial injury suggesting that targeting VN–integrin interaction may be efficient way to limit restenosis after angioplasty.

Time for primary review 22 days.


   Acknowledgements
 
Supported in part by a grant from the Fédération Française de Cardiologie, Paris, France and by the Conseil Régional D'Aquitaine


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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