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Cardiovascular Research 2000 48(3):464-472; doi:10.1016/S0008-6363(00)00201-7
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Inhibition of vascular smooth muscle cell adhesion and migration by c7E3 Fab (abciximab): a possible mechanism for influencing restenosis

Julia H Barona,*, Elena P Moiseevaa, David P de Bonoa, Keith R Abramsb and Anthony H Gershlicka

aDivision of Cardiology, Department of Medicine and Therapeutics, University of Leicester, Leicester, UK
bDepartment of Epidemiology and Public Health, University of Leicester, Leicester, UK

* Corresponding author. Tel.: +44-116-256-3038; fax: +44-116-287-5792

Received 27 January 2000; accepted 7 July 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Brief intravenous administration of chimeric antibody c7E3 Fab during coronary angioplasty has been shown in some studies to provide long term protection against coronary events. Smooth muscle cell (SMC) adhesion and migration are key initial steps in the development of restenosis. The purpose of this study was to investigate the effect of c7E3 Fab on adhesion and migration of SMC to the extracellular matrix (ECM) proteins osteopontin (Opn) and vitronectin (Vn). Methods: Adhesion of human vascular SMCs to ECM proteins was quantified using a CyQUANT assay kit. Migration of SMCs to Vn, Opn and PDGF was studied using a modified Boyden's chamber migration assay. Integrin expression was determined by immunoprecipitation. Results: c7E3 Fab reduced SMC adhesion on Vn and Opn to 69.2±3.3% (P<0.001) and 52.5±4.8% (P<0.001) respectively, compared to adhesion without antibody present. This reduction was the same as that for anti-{alpha}vβ3 integrin antibody LM609 (P = 0.5). The combination of anti-{alpha}vβ5 integrin antibody and c7E3 Fab had a greater effect than either antibody alone (P<0.001). c7E3 Fab reduced SMC migration to Vn and Opn to 51.6±8.9% (P<0.001) and 20.3±6.1% (P<0.001) respectively, compared to migration in the absence of antibodies. Again, similar results were seen with LM609. PDGF-induced SMC migration was also inhibited by c7E3 Fab (P = 0.004) and LM609 (P = 0.001), but to much less an extent. The migration SMCs from a culture found not to express the {alpha}vβ3 integrin was unaffected by these antibodies, strengthening the argument that c7E3 Fab inhibits SMC function via this integrin. Conclusions: c7E3 Fab inhibits the adhesion and migration of SMCs via the {alpha}vβ3 integrin. The inhibition, however, is partial, and varied depending on type of ECM protein and {alpha}vβ3 integrin expression. Some of the clinical benefits of c7E3 Fab may be due to its effect on SMCs.

KEYWORDS Extracellular matrix; Monoclonal antibodies; Receptors; Restenosis; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Administration of c7E3 Fab (abciximab, Reo Pro) to prevent thrombotic complications following percutaneous coronary interventions has been associated with long-term reductions in subsequent coronary events or need for revascularisation in some trials [1]. This observation led to speculation that this antibody may also influence restenosis.

Although the antibody c7E3 Fab was developed to be specific for the glycoprotein IIb/IIIa integrin expressed on the surface of activated platelets [2,3], it has recently been confirmed that it also binds the {alpha}vβ3 integrin [4]. This receptor is found on the surface of several cells, including human vascular smooth muscle cells (SMCs) [5,6]. It is upregulated following vascular injury [7,8], and is involved in SMC adhesion and migration [9–14], both of which are important initial steps in the development of the neointimal tissue that is responsible for vessel restenosis [5]. Animal studies have demonstrated that blockade of the {alpha}vβ3 integrin after angioplasty results in reduction of neointimal formation [14–16].

The parent IgG of c7E3 Fab, m7E3, has been shown to reduce thrombospondin- and thrombin-induced SMC proliferation in vitro, but has no effect on PDGF- or serum-induced proliferation [7]. c7E3 Fab has been shown to reduce adhesion of {alpha}vβ3-expressing M21 melanoma cells in vivo [4]. Its effect on human vascular SMC adhesion and migration, however, has not been reported. In this study, the influence of c7E3 Fab on adhesion and migration to SMC substrates vitronectin (Vn) and osteopontin (Opn) was studied. Both these extracellular matrix proteins are present in increased quantities at the site of vascular injury [16–18], and are implicated in adhesion and migration processes mostly mediated via the {alpha}vβ3 integrin [9–12,16,17]. Platelet derived growth factor (PDGF) was included in the migration studies as it is an important chemotactic agent following vascular injury [19]. Migration studies using cells not expressing the {alpha}vβ3 integrin were used as a negative control. In all experiments, abciximab was studied in parallel with LM609, which is known to block the binding of {alpha}vβ3, and inhibit cell adhesion to Vn.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
The rat osteopontin (Opn) was kindly donated by Smithkline–Beecham, USA. Human vitronectin (Vn) was purchased from Life Technologies. A monoclonal antibody against smooth muscle cell {alpha}-actin (1A4) was purchased from Sigma Bioscience. The c7E3 Fab was kindly donated by Centocor, USA. Mouse monoclonal antibodies against human {alpha}vβ3 integrin (MAB1976, clone LM609), {alpha}vβ5 integrin (MAB1961) and vitronectin (MAB1945) were purchased from Chemicon International, FITC-conjugated F(ab')2 fragment of rabbit anti-mouse immunoglobulin (F0313) was purchased from Dako. Mouse IgG1 ({kappa}) antibody against trinitrophenol, (03000D, now-03191D) was used as a control irrelevant antibody in both the adhesion and migration studies. It is marketed as a control antibody by Pharmingen, and was used instead of pooled IgG, which may have contained antibodies that interacted with SMCs. It is referred to as control IgG. Mouse anti-human integrin {alpha}5 monoclonal antibody (33220D) was also purchased from Pharmingen, and was used in the adhesion assays as a second control as c7E3 Fab is not thought to interact with this integrin sub-unit.

2.2 Cell culture
Human vascular SMCs were obtained from primary explant culture of fragments of saphenous vein or aortic punch biopsy obtained from patients undergoing surgery as described previously [20]. The investigation conforms with the principles outlined in the declaration of Helsinki (Cardiovascular Research 1997;35:2–3). Briefly, the endothelium was removed by gentle scraping, and the adventitia was dissected away. The remaining tissue was cut into small pieces, and incubated in DMEM with 20% foetal calf serum (FCS), pyruvate (0.11 g/l) and gentamicin at 37°C, 5% CO2. SMCs growing out of explant were sequentially cultured in 10% FCS-containing medium. SMCs of two–six passages were used in the study. They were characterised by their typical hill and valley morphology at confluence, and by their staining with antibodies against SMC {alpha}-actin.

2.3 Adhesion assays
The adhesion experiments were carried out using cells derived from the saphenous vein from one subject. These studies were performed twice, using four wells per set of conditions. Ninety-six-well plates were pre-coated with 50 µl (100 nmol/l) of Vn or Opn at 4°C overnight, blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at 37°C and rinsed with PBS. Uniform coating with Vn was confirmed by indirect immunofluorescence using mouse antibodies against Vn and FITC-conjugated F(ab')2 fragment of rabbit anti-mouse immunoglobulin. Adhesion assays were conducted as described previously with some modifications [12,20]. Confluent cells were resuspended in serum-free DMEM with 0.25% BSA. Cells in suspension (104 cells in 100 µl) were allowed to adhere to pretreated 96-well plates for 1 h at 37°C (5% CO2). When antibodies were used, the cells were preincubated with antibodies for 30 min at 37°C and allowed to adhere in their presence. Antibodies were used at a concentration of 10 µg/ml, except for the varied dose studies, where concentrations were from 0.5 to 25 µg/ml. After adhesion the medium was removed by gentle suction, and non-adherent cells were removed by washing twice with PBS. The adhesion was quantified using a CyQUANTTM nucleic acid fluorescence assay kit (Molecular Probes). Calibration curves showed the fluorescence reading to be proportional to the cell number.

In further studies, the experiments performed with cells derived from saphenous vein were repeated using aortic SMCs in order to confirm the results with arterial cells.

2.4 Migration assays
The initial results of the adhesion experiments led us to investigate SMC migration, this time focusing the effects of c7E3 Fab and LM609 on migration to Vn and Opn. PDGF was also included as it is one of the important chemotactic growth factors present in vivo, and we wished to find out whether c7E3 Fab was able to influence migration towards it.

Migration studies were conducted as described previously [11,20] with some modifications. Confluent cells were resuspended in serum-free DMEM with 0.25% BSA. Cells in suspension (5x104 cells per 100 µl) were added to the upper compartment of a Transwell plate (Costar, 8-µm polycarbonate membrane). The lower compartment contained 600 µl of DMEM+0.25% BSA, with Vn (20 µg/ml), Opn (20 µg/ml), or PDGF-BB (10 ng/ml), added when appropriate. The concentrations of Vn, Opn and PDGF-BB used were determined in preliminary experiments of SMC migration in the absence of antibodies, where all three were found to induce migration in a dose-dependent manner with a plateau being achieved at higher concentrations. The concentrations selected were within the plateau range. The plates were incubated at 37°C, 5% CO2 for 6 h. The upper compartment of the chamber insert was rinsed with PBS, and the cells were fixed in 4% paraformaldehyde at room temperature for 20 min. Cells on the upper surface of the membrane were removed by gentle wiping. The inserts were incubated with 20 µg/ml of propidium iodide (Molecular Probes) in the presence of ribonuclease A (0.2 mg/ml) for 30 min at 37°C to stain cell nuclei. The membranes were mounted on glass slides and the number of migrating cells was averaged over five x200 power fields per membrane. All experiments were performed at least twice, using three inserts for each set of conditions.

2.4.1 Preliminary migration studies and immunoprecipitation
The early observation that the cells from one particular saphenous vein did not migrate significantly to Opn led to experiments being performed to characterise the integrin expression of the cells. Immunoprecipitation was carried out on these cells, and compared to immunoprecipitation of SMCs from a saphenous vein culture that did migrate to Opn.

SMCs were grown overnight in methionine-lacking MEM supplemented with 10% FCS containing 20 µCi/ml of 35S-methionine (Amersham). Cells were washed three times with PBS, and dissolved and scraped in RIPA buffer, containing 10 mM Tris–Cl, pH 7.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% NP40, 1% sodium deoxycholate, protease inhibitors cocktail (Sigma), followed by centrifugation to remove cellular debris. Protein extracts containing equal amounts of 35S-labelled proteins (107 cpm) were diluted up to 400 µl with RIPA buffer and incubated with 5 µg of antibodies against integrins β1, {alpha}5, {alpha}vβ3 and {alpha}vβ5. Immune complexes were precipitated with 75 µl of 50% suspension of protein G-beads on ice for 1 h. The beads were preincubated with 1% BSA to reduce non-specific binding. Immune complexes were resolved by 8% SDS–PAGE and detected by autoradiography on X-OMAT film (Kodak) according to standard procedures [21].

Cells derived from these two saphenous vein sources were also compared for their ability to migrate to Vn and PDGF.

2.4.2 The influence of antibodies on SMC migration
The effect of c7E3 Fab, LM609 and control IgG on the migration of cells from each of these two saphenous vein sources was compared. The cells were preincubated with antibodies for 30 min at 37°C and allowed to migrate in their presence. Antibodies were used at a concentration of 10 µg/ml, except for the studies evaluating antibody dose curves, where concentrations were selected between 0.5 and 25 µg/ml.

Finally, the antibody experiments were repeated using cells from an aortic source that were similar in behaviour and integrin expression to those saphenous cells able to migrate to Opn.

2.5 Statistical analysis
All data are represented as mean±standard deviation. Comparison of the effect of antibodies within a treatment group was performed using one-way ANOVA. Comparison of the effect of c7E3 Fab (versus no antibody) in different experiments was performed using two-way ANOVA. All analyses were performed using SPSS version 9. P-values were adjusted for multiple testing, but statistical significance was interpreted with caution.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 SMC adhesion
The experiments set out to determine the effect of c7E3 Fab on SMC adhesion. Several commercial antibodies inhibiting cell adhesion were included in this study in parallel with c7E3 Fab. The antibody LM609, known to inhibit cell adhesion to Vn by blocking {alpha}vβ3 integrin, was used a positive control of inhibition of cell adhesion. The antibody against {alpha}vβ5 integrin is known to inhibit cell adhesion to Vn. The two antibodies (IgG and anti-{alpha}5) were included as negative controls.

The adhesion experiments described in Sections 3.1.1 and 3.1.2 (below) were carried out using SMCs derived from saphenous vein from one subject (Figs. 1 and 2Go).


Figure 1
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  		Fig. 1 Dose-dependent inhibition of SMC adhesion to Vn (A) and Opn (B) by anti-{alpha}vβ3 antibodies c7E3 Fab and LM609. Adhesion in the presence of antibodies control IgG, c7E3 Fab and LM609 are shown in white, black and spotted bars, respectively. Cell adhesion is expressed as a percentage of control cell adhesion in the absence of antibodies. SMC adhesion to untreated plastic was 29.5±2.0% and 30.9±2.6% in A and B, respectively. Bars represent the means±S.D. The inhibition was statistically significant compared with control starting from 1 µg/ml of antibodies for Vn and 0.5 µg/ml of antibodies for Opn (*P<0.05).

 

Figure 2
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  		Fig. 2 Antibody inhibition of SMC adhesion to Vn and Opn. Adhesion to Vn and Opn is shown in dark grey and light grey, respectively. Antibodies against {alpha}5 and {alpha}vβ5 integrin subunits were used in addition to c7E3 Fab. All antibodies were used at a concentration of 10 µg/ml. Cell adhesion is expressed as a percentage of control cell adhesion in the absence of antibodies. SMC adhesion to untreated plastic was 28.1±3.5%. Bars represent the means±S.D. *P<0.05 compared to adhesion without antibodies present.

 
3.1.1 The adhesion of smooth muscle cells to vitronectin and osteopontin is inhibited by c7E3 Fab and LM609 (anti-{alpha}vβ3) antibodies
Antibodies LM609 and c7E3 Fab inhibited SMC adhesion to Vn and Opn in a dose-dependent manner (Fig. 1A and B). The maximal level of inhibition of SMC adhesion to both Vn and Opn was observed in the range of antibody concentrations from 2 to 25 µg/ml, therefore we used antibody concentration 10 µg/ml in further experiments. c7E3 Fab decreased SMC adhesion to Opn to a greater extent than to Vn (Figs. 1 and 2Go) (P = 0.002). Neither antibody reduced adhesion to the levels seen on untreated plastic wells (which was approximately 25–30% of adhesion seen on wells treated with ECM proteins). Neither c7E3 Fab or LM609 reduced SMC adhesion to untreated plastic (not shown). Neither control IgG nor anti-{alpha}5β1 antibodies reduced adhesion significantly (P = 0.5 and 0.3 respectively for Vn, and P = 0.75 and 0.08 respectively for Opn).

Using 10 µg/ml of antibody, cell adhesion on vitronectin was reduced to 69.2±3.3% (P<0.001) by c7E3 Fab, and 71.1±7.6% (P<0.001) by LM609, when compared to adhesion without antibody inhibition (Figs. 1and 2Go.). There was no significant difference between the effect of c7E3 Fab and LM609 (P = 0.5).

Adhesion on osteopontin was reduced to 52.5±4.8% (P<0.001) by c7E3 Fab, and 50.1±4.8% (P<0.001) by LM609 (Fig. 2). Again, there was no significant difference between the effect of c7E3 Fab and LM609 (P = 0.4).

Combining c7E3 Fab with LM609 in the same experiment (10 µg/ml of each) did not inhibit adhesion to a greater extent than either antibody alone (Fig. 2) (P = 0.3 for Vn and 0.1 for Opn).

3.1.2 The adhesion of smooth muscle cells to vitronectin and osteopontin is further inhibited by anti-{alpha}vβ5 antibodies
Anti-{alpha}vβ5 integrin antibody reduced adhesion on both Vn and Opn to a similar degree to c7E3 Fab or LM609 (Fig. 2.) However, whereas the addition of LM609 to c7E3 Fab showed no further reduction in SMC adhesion, the combination of anti-{alpha}vβ5 integrin antibody with c7E3 Fab had an additive effect on SMC adhesion to Vn and Opn (P<0.001 compared with c7E3 Fab alone for Opn and Vn). c7E3 Fab and anti-{alpha}vβ5 integrin antibody combined reduced cell adhesion almost to the levels seen on plastic wells that had not been treated with ECM proteins, suggesting that these antibodies act via different integrins.

3.1.3 Adhesion of SMCs derived from an arterial source is also reduced by abciximab and LM609
We also compared the effect of LM609 and c7E3 Fab on SMCs derived from aorta (from another patient) with the results obtained with the saphenous vein cells (Table 1). A similar degree of inhibition of aortic SMC adhesion was seen with the antibodies (P<0.001 for c7E3 Fab and LM609, compared with adhesion with no antibody present). No difference was seen between the influence of LM609 and c7E3 Fab (P = 0.7 for Vn and 0.5 for Opn).


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  		Table 1 Comparison of the effect of antibodies on adhesion of saphenous vein and aortic SMCsa

 
3.2 SMC migration
3.2.1 Immunoprecipitation demonstrated varied expression of the {alpha}vβ3 integrin
The results of the Western Blot from the immunoprecipitation experiments can be seen in Fig. 3. A faint band can be seen corresponding to β3 at approximately 108 Kda in the Western blot of cells derived from the saphenous vein culture that migrated in response to Opn (B), but not in the other (A). All the cells were grown under identical conditions. For the purposes of our experiments, the cells not expressing the {alpha}vβ3 integrin acted as a useful negative control, and will be described as {alpha}vβ3–. Those expressing {alpha}vβ3 are described as {alpha}vβ3+.


Figure 3
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  		Fig. 3 Integrin expression in two different saphenous vein cell lines (A and B). Autoradiographs of the SDS–PAA dried gels are shown. The cells able to migrate to Opn are represented on the right (B). 35S-labelled proteins in SMC extracts were immune precipitated with antibodies against integrins indicated on the top of each lane and resolved by SDS–PAGE. The positions of molecular weight markers are shown on the left. The positions of integrin subunits are marked on the right. The bands of {alpha}v and β5 integrin subunits are much stronger on panel B, than on panel A. The faint band of β3 subunit is noticeable on panel B, but not on panel A.

 
3.2.2 Vn-, Opn- and PDGF-induced SMC migration was dependent on the expression of the {alpha}vβ3 integrin
Migration of {alpha}vβ3+ and {alpha}vβ3– cells to Opn, Vn and PDGF was assessed (Fig. 4.). The migration of {alpha}vβ3+ SMCs was increased in the presence of all three agents, although Vn and PDGF induced {alpha}vβ3+ cell migration to a greater extent than Opn. SMC migration was influenced by {alpha}vβ3 integrin expression, with all three agents inducing migration of {alpha}vβ3+ SMCs to a higher degree than the level of migration of {alpha}vβ3– SMCs. Opn did not significantly induce migration of {alpha}vβ3– SMCs, compared to background migration levels without Opn present (P = 0.3). Expression of the {alpha}vβ3 integrin influenced migration towards ECM proteins much more than PDGF-induced migration.


Figure 4
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  		Fig. 4 Opn-, Vn- and PDGF-induced migration of {alpha}vβ3 expressing ({alpha}vβ3+) and {alpha}vβ3 non-expressing ({alpha}vβ3–) SMC lines (B and A in Fig. 3, respectively). The migration of {alpha}vβ3+ SMC line (B) is shown in dark grey, and the migration of {alpha}vβ3– cells (A) is shown in light grey. The horizontal axis shows migratory stimuli. The concentrations used were 20 µg/ml for Vn and Opn, and 10 ng/ml for PDGF. The vertical axis represents the cell number per high power field. Bars represent the means±S.D. *P<0.05 compared with migration of {alpha}vβ3+ SMC. SMC migration in the absence of migratory stimuli was between four and seven cells per high power field.

 
3.2.3 c7E3 Fab and LM609 (anti-{alpha}vβ3) antibodies inhibited migration of {alpha}vβ3+ smooth muscle cells to Opn, Vn, and PDGF. Inhibition was dose dependent
As observed in the SMC adhesion experiments, inhibition of migration of {alpha}vβ3+ SMCs by c7E3 Fab and LM609 was dose dependent, with maximal inhibition being achieved at concentrations above 5 µg/ml (data not shown). Control IgG did not significantly affect SMC migration.

At a concentration of 10 µg/ml, both antibodies c7E3 Fab and LM609 significantly reduced SMC migration to PDGF, Opn and Vn (Fig. 5). The inhibitory effect on SMC migration to Opn was most marked, with c7E3 Fab reducing migration to just 20.3±6.1% of migration levels without antibody present (P<0.001). c7E3 Fab reduced Vn-induced migration to 51.6±8.3% of migration in the absence of antibodies (P<0.001). The inhibitory effect of c7E3 Fab on migration to PDGF was less than the effect on Vn- and Opn-induced SMC migration, but remained significant (P = 0.004). In each experiment, there was no significant difference seen between the effects of LM609 or c7E3 Fab (P = 0.06 for Opn, P = 0.08 for Vn, P = 0.6 for PDGF).


Figure 5
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  		Fig. 5 Antibody inhibition of migration of {alpha}vβ3+ SMC line towards Opn, Vn or PDGF. The vertical axis represents a percentage of SMC migration with no antibody present. All antibodies were used at a concentration of 10 µg/ml. SMC migration in the presence of control IgG (white), c7E3 Fab (black) and LM609 (spotted) is shown. Bars represent the means±S.D. *P<0.05 compared to migration with no antibody present. Migration without Opn, Vn or PDGF was between 5 and 8% of induced migration.

 
3.2.4 Migration of cells that did not express {alpha}vβ3 was not influenced by c7E3 Fab
Migration of {alpha}vβ3– cells to Vn and PDGF was not inhibited by c7E3 Fab (P = 0.8 and P = 0.9 respectively), in keeping with the hypothesis that c7E3 Fab inhibits SMC migration via this integrin alone.

3.2.5 Migration of {alpha}vβ3+ SMCs derived from an arterial source behaved in a similar manner to {alpha}vβ3+ saphenous vein SMCs
SMCs derived from aorta were also assessed. They were found to behave similarly to {alpha}vβ3+ saphenous vein-derived cells (Table 2). Immunoprecipitation (not shown) confirmed their expression of the {alpha}vβ3 integrin. c7E3 Fab and LM609 both significantly reduced their migration to Opn, Vn and PDGF, with the greatest effect being seen on Opn-induced migration, and the least on PDGF-induced migration. c7E3 Fab reduced Opn-induced migration to just 25.5±5.2% of migration in the absence of antibody, but this remained significantly greater than non-induced migration in the absence of Opn (P<0.001).


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  		Table 2 Comparison of the effect of antibodies on migration of {alpha}vβ3+ saphenous vein and aortic SMCsa

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Restenosis following successful angioplasty remains a major limitation to the long term success of the procedure. The introduction of stenting has reduced the vessel wall remodelling and elastic recoil that contribute to the process [22], but restenosis still occurs due to neointimal formation, and in-stent restenosis is particularly difficult to treat [23]. It is known that restenosis involves the migration of SMCs across the basement membrane to the intima, where they proliferate [5]. The migration process involves degradation of old ECM, cell detachment, migration, and formation of new cell adhesions and deposition of new ECM. Interestingly, ECM in atherosclerotic vessels contains proteins such as Vn, Opn and thrombospondin, which are not detectable in normal blood vessels [16–18,24]. Vascular damage exposes SMCs to serum containing high concentrations of Vn (0.25–0.4 mg/ml). Opn and especially Vn have been shown to induce SMC migration [9–12,16,17].

The present study has shown that LM609 and c7E3 Fab both act via the {alpha}vβ3 integrin to inhibit SMC adhesion and migration to Vn and Opn. The concentration of c7E3 Fab used in the experiments was 10 µg/ml, which is similar to the therapeutic plasma concentration of c7E3 Fab, of around 6 µg/ml [25].

The adhesion experiments showed that c7E3 Fab and LM609 inhibited SMC adhesion to both Opn and Vn coated wells. The two antibodies consistently inhibited adhesion to a similar degree, suggesting they are likely to act via the same integrin, but neither could prevent adhesion completely. Combining these two antibodies in the same experiment did not have any additional effect, again compatible with them acting via the same pathway. The combination of c7E3 Fab and an antibody against the {alpha}vβ5 integrin, however, reduced SMC adhesion to Vn and Opn to a greater extent than either antibody alone. Similar findings have been reported previously with melanoma M21 cells, with this combination of antibodies preventing significant adhesion in vitro [4]. Previous studies have shown that {alpha}vβ1 integrin is also involved in SMC adhesion to Opn [12]. This integrin is likely to be expressed in SMCs used in this study, as both {alpha}v subunit and β1 subunit are detectable (Fig. 3A and 3B). In this study, however, {alpha}vβ3 and {alpha}vβ5 integrins were mostly responsible for SMC adhesion to Opn and Vn.

The inhibitory effect of c7E3 Fab on SMC migration was much higher than on SMC adhesion to both Vn and Opn, which would be expected, as cell migration occurs through multiple cycles of cell attachments and detachments. It is also consistent with previously published data demonstrating that the {alpha}vβ3 integrin contributes to SMC migration to a greater extent than adhesion [12]. The inhibitory effect of c7E3 Fab and LM609 was greater for Opn than Vn, suggesting that the relative role of {alpha}vβ3 integrin in the mediation of these cell functions varies depending on ECM substrate.

It has frequently been observed that expression of the {alpha}vβ3 integrin varies in human vascular SMCs in culture and in vivo [12,26,27]. The chance identification of a culture of saphenous vein derived SMCs that did not express the {alpha}vβ3 integrin ({alpha}vβ3–) enabled further confirmation of the role of this integrin and c7E3 Fab in SMC migration. While these {alpha}vβ3– SMCs acted as a useful negative control, they probably play little role in the pathology following vessel wall injury. The {alpha}vβ3 integrin is up-regulated following vascular injury and plays a major role in SMC migration in vivo in postangioplasty events [7,8,16]. It is therefore expected that SMCs following vascular injury in vivo would behave in a similar way to the {alpha}vβ3+ cells in our experiments. Opn did not induce migration of {alpha}vβ3– SMCs significantly, although Vn and PDGF did still induce some migration. From this, we can conclude that {alpha}vβ3 integrin is the major integrin responsible for migration to Opn, although anti-{alpha}vβ3 integrin antibody does not abolish Opn-induced migration. Vn- and PDGF-induced migration of SMCs appears to involve not only anti-{alpha}vβ3 integrin, but other cellular receptors as well. Vn and PDGF-induced migration of {alpha}vβ3– SMCs was not inhibited by c7E3 Fab, consistent with the hypothesis that c7E3 Fab acts via this integrin.

It is interesting to note that SMC migration to PDGF was only slightly reduced by LM609 and c7E3 Fab. The published data on the role of {alpha}vβ3 integrin on PDGF-induced SMC migration are inconsistent. Itoh et al. [28] showed that both anti-β1 integrin and anti-{alpha}vβ3 integrin antibodies inhibited SMC migration to PDGF, whereas other studies found that the inhibitory effect of anti-β1 integrin antibodies on SMC migration is small or negligible compared to the effect of anti-{alpha}vβ3 integrin antibody LM609 [13,14]. It is feasible that the inhibitory effect of anti-integrin antibodies would vary according to the expression of different integrins in studied cell lines.

Our data on SMC migration to Vn also indicate other receptors may be involved. Recently, urokinase-type plasminogen activator receptor uPAR and plasminogen activator inhibitor PAI-1 were identified as Vn-binding proteins in several cell types [29,30]. These proteins recognise the same site on Vn as integrins and, therefore, compete with integrins for Vn binding. Both uPAR and PAI-1 are involved in SMC migration [30–32]. Furthermore, an involvement of the plasminogen activator system in SMC migration after coronary angioplasty may have important implications for the outcome of post-angioplasty events and needs further investigation. c7E3 Fab may exert beneficial effects via pathways other than those demonstrated in these experiments. Its role in platelet inhibition reduces the thrombus load, potentially limiting platelet degranulation and release of growth factors and chemoattractants, such as PDGF. Recent studies have implicated {alpha}vβ3 integrin in SMC apoptosis. VanderZee et al. [33] reported that anti-{alpha}vβ3 integrin antibody LM609 administered after angioplasty reduced intimal thickening in rabbit model by inducing apoptosis in SMCs. Since both anti-{alpha}vβ3 integrin antibodies c7E3 Fab and LM609 had very similar effect on SMCs, it would be expected that c7E3 Fab is able to induce apoptosis in SMCs by blocking {alpha}vβ3 integrin.

Overall, the inhibitory effect of c7E3 Fab on SMC adhesion and migration (present study), on SMC proliferation [7], and an involvement of {alpha}vβ3 integrin-mediated apoptosis in restenosis demonstrate a convincing role for c7E3 Fab in the inhibition of SMC functions implicated in the restenotic process. However, other pathways are involved. The diversity of in vivo mechanisms may ensure that the effects in vitro are not translated into therapeutic benefit. Although the EPIC trial [34] and some provisional 6-month angiographic results for the EPISTENT trial [35] suggested a role in reducing restenosis, the ERASER study, designed to investigate prospectively the effect on in-stent restenosis by c7E3 Fab, failed to show a reduction in intimal hyperplasia at 6 months [36]. Recently published primate experiments have shown that a high systemic dose of c7E3 Fab, which would not be tolerated in man, can reduce neointimal formation [37]. It is feasible that adjustments in the dose and mode of administration of c7E3 Fab may be required in order to maximise clinical potential. Integrin inhibition may be required for up to 2 weeks, as this is the period during which the {alpha}vβ3 integrin is upregulated after vascular injury [7,8,16]. Local drug delivery of c7E3 Fab, perhaps loaded onto a stent, may be the way forward to ensure prolonged administration and to achieve higher local concentrations. Our research [38] demonstrates that c7E3 Fab can be adsorbed onto polymer-coated stents. The antibody elutes slowly in vitro, penetrating underlying SM layers, and significantly inhibits platelet deposition. The use of such loaded stents could influence SMC activity within them.

Time for primary review 33 days.


   Acknowledgements
 
This work was funded by grants CH/92021 and RG95/167 from British Heart Foundation.


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

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