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Cardiovascular Research 2005 65(4):813-822; doi:10.1016/j.cardiores.2004.11.021
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Copyright © 2004, European Society of Cardiology

{alpha}8β1 Integrin expression in the rat carotid artery: involvement in smooth muscle cell migration and neointima formation

Ramin Zargham and Gaétan Thibault*

Institut de recherches cliniques de Montréal (IRCM), Université de Montréal and McGill University, 110 avenue des pins ouest, Montréal, Québec, Canada H2W 1R7

* Corresponding author. Tel.: +1 514 987 5613; fax: +1 514 987 5585. Email address: thibaug{at}ircm.qc.ca

Received 23 August 2004; revised 5 November 2004; accepted 19 November 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Migration of vascular smooth muscle cells (VSMCs) from the tunica media to the intima is a key event in neointima formation after coronary artery angioplasty. The central dogma in VSMC migration is cell modulation from the contractile to the noncontractile phenotype. Increased {alpha}8β1 integrin expression, observed in situations where the majority of cells are in the contractile phenotype, led us to hypothesize that a decrease of {alpha}8β1 integrin may play an important role in the migratory state of VSMCs.

Methods and results: To test this hypothesis, neointima formation was induced in the left common carotid artery of adult male Sprague–Dawley rats by balloon dilatation. Immunohistochemical and Western blotting analysis showed reduced expression for up to 4 weeks of both the {alpha}8 and β1 integrin subunits as well as smooth muscle {alpha}-actin in the tunica media following balloon injury. Moreover, ex vivo culture of carotid VSMCs revealed diminished {alpha}8 integrin expression in the platelet-derived growth-factor-dependent migratory state with an increase in the angiotensin-II-induced contractile state. To ascertain the functional role of {alpha}8 integrin in VSMC migration and proliferation, {alpha}8 gene expression was reduced by nearly 70% by short interference RNA (siRNA). Decreased {alpha}8 expression resulted in a significant increase of carotid VSMC migration but not of proliferation.

Conclusions: Our results are consistent with those of other studies demonstrating that {alpha}8 integrin could be used as an appropriate differentiation marker. In addition, depressed {alpha}8 integrin expression (after vascular injury or siRNA knockdown) was correlated with heightened cell migratory activity, demonstrating its potential role in neointima formation.

KEYWORDS Arteries; Cell culture; Cell differentiation; Restenosis; Smooth muscle

This article is referred to in the Editorial by N. Uesugi and N. Sakata (pages 766–767) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Vascular injury followed by luminal loss or restenosis is a major complication induced by balloon angioplasty, vascular smooth muscle cells (VSMCs) playing a key role in the pathogenesis of neointima formation [1]. In normal conditions, VSMCs replicate at a very slow rate, and function principally to establish and maintain vascular tone. Quiescent VSMCs of the media are differentiated mature cells that express a set of proteins and adhesion molecules that are characteristic of the contractile phenotype [2,3]. However, VSMCs can also exhibit great plasticity with respect to environmental conditions, showing reversible modulation between a differentiated (contractile) phenotype and a dedifferentiated (noncontractile, proliferative) phenotype [4].

During arterial catheterization, the endothelium layer is removed by balloon dilatation, resulting in loss of this important antithrombogenic layer. In addition to mechanical and stretch injury, thrombosis and its contribution to cytokine, growth factor and other mitogen release activate cells in the injured vessel wall, especially VSMCs in the tunica media. Consequently, activated VSMCs change from the contractile to the highly proliferative, noncontractile phenotype [1]. Transformed VSMCs replicate, then migrate toward the source of stimulation, and produce a neointima. The principle behind cell migration is the interaction between the actin cytoskeleton and the extracellular matrix (ECM) [5]. Cell-ECM adhesion is dynamically regulated by the coordination of adhesion and release events. Activated VSMCs exhibit an altered set of adhesion receptors and modulated actin cytoskeletal reorganization [2,6]. Among cell adhesion receptors, the integrin superfamily, by its interactions with the actin cytoskeleton and the ECM, plays an important role in VSMC migration, proliferation, contractility and survival [2].

Integrins are heterodimeric proteins consisting of 1 {alpha} and 1 β subunit. More than 24 integrins have been described with the identification of 18 different {alpha} and 8 β subunits [7]. It has been shown that some integrins, including {alpha}5β1, {alpha}vβ3, {alpha}vβ5, {alpha}1β1 and {alpha}2β1, are involved in cell migration and proliferation [8]. Some studies have established a link between proliferation and {alpha}vβ3 and {alpha}vβ5 expression after vascular injury [9,10]. {alpha}8β1 integrin is expressed in several cell types, including fibroblasts, VSMCs and mesangial cells of the kidney [11]. Interestingly, it is overexpressed in situations in which the majority of cells display a contractile phenotype, such as cardiac [12,13], lung and hepatic fibrosis [14]. Cell phenotype (fibroblast vs. myofibroblast) dependence on {alpha}8β1 integrin led us to hypothesize that its downregulation may be indispensable for VSMC migration from the tunica media toward the intima in restenosis after balloon angioplasty. Therefore, the main objective of the present study was to understand the role of {alpha}8β1 integrin in neointima formation in a rat model of carotid angioplasty. We examined {alpha}8 integrin expression in that model and in cultured rat carotid VSMCs showing different phenotypes. The study also aimed to define whether {alpha}8 integrin downregulation by RNA interference gene knockdown has a functional influence on VSMC migration.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Antibodies and reagents
Antiserum to the {alpha}8 integrin subunit (A8-2) was generated as described elsewhere [13]. Anti-β1 subunit antibody (130L) was obtained from Dr. R.O. Hynes (Howard Hughes Medical Institute, Cambridge, MA). Anti-smooth muscle (SM) {alpha}-actin was from Sigma (Oakville, ON), mouse anti-GAPDH was from Abcam (Cambridge, UK), anti-{alpha}5 integrin and anti-PCNA were from Chemicon (Temecula, CA). All secondary antibodies were from Chemicon or Molecular Probes (Eugene, OR). Short interference RNA (siRNA) was synthesized by Dharmacon (Lafayette, CO). Lipofectamine 2000 was from Invitrogen Life Technologies (Carlsbad, CA). [Sar1]Angiotensin (Ang) II was purchased from Bachem California (Torrance, CA), and platelet-derived growth factor (PDGF)-BB from Sigma. Culture media, reagents and plates were from Invitrogen Life Technologies. Fetal bovine serum (FBS) was procured from Wisent (St. Bruno, QC). Collagenase type I was from Worthington BR (Freehold, NJ), and elastase, BSA and soybean trypsin inhibitor were from Sigma.

2.2. Induction of neointima formation
Balloon denudation of the common carotid artery endothelium was evoked in male adult Sprague–Dawley rats (350 to 400 g; Charles River Breeding Laboratories, St. Constant, QC). Under isoflurane (Abbott Laboratories, St. Laurent, QC) anesthesia, a neck midline incision was made and, after exposure of the left carotid artery, a 2F Fogarty balloon catheter (Edwards Lifesciences, Mississauga, ON) was inserted into the external carotid branch to the aortic arch, insufflated to produce slight resistance, and withdrawn three times. They were compared with sham-operated controls in which the same procedure was performed, except balloon insertion. Animal housing and experimentation in accordance with Canadian Council on Animal Care and NIH guidelines were approved by the local animal care committee.

2.3. Morphometric analysis
At various time points (7, 14, 21 and 28 days after balloon injury), the rats were anesthetized by injection of ketamine (80 mg/kg, Ayerst, Guelph, ON), xylazine (12 mg/kg, Bayer, Toronto, ON) and heparin (500 IU/500 µL, Dragnon Teknika, Toronto, ON), followed by perfusion in the left ventricle with zinc fixative at a flow rate of 5 mL/min [15]. The carotid artery was dissected, cut into two equal paraffin-embedded segments, and 6-µm sections of carotid rings were stained with hematoxylin. Areas of the intima and the intima-to-media-area ratio were calculated by computerized digital planimetry with a dedicated video microscope and customized software.

2.4. Immunological analysis of tissue sections
For each time point, corresponding tissue sections were deparaffinized and washed with 0.05 mol/L Tris–HCl, 0.15 mol/L NaCl and 0.02% Tween 20 (TBT). After quenching endogenous peroxidase with 0.3% H2O2, nonspecific binding was blocked by incubation in 10% normal goat serum (NGS) for all primary antibodies. The {alpha}8 and β1 integrin subunits, SM {alpha}-actin and PCNA were detected by incubation with their appropriate antibodies in 10% NGS in TBT. After washing, primary antibodies were detected with a horseradish-peroxidase-labeled secondary antibody. Sections were counterstained with hematoxylin (without counterstaining for quantification) and mounted in Permount. Slides were visualized with a Zeiss Axiophot 100 M microscope (Carl Zeiss Microimaging, Thornwood, NY), and digital images were analyzed with Northern Eclipse software (Empix Imaging, Mississauga, ON). Nonspecific staining was detected with omission of the primary antibodies.

For {alpha}8 and β1 subunit and SM {alpha}-actin quantification, microscopic images (5 fields per section) were taken through a realtime XCAP-Lite digital color camera linked to a Zeiss Axioskop 2 Plus light microscope. Color images were transformed into gray scale, and staining (pixel intensity) was recorded.

2.5. Western blotting
Carotid segments from balloon-injured and sham-operated animals were snap-frozen in liquid nitrogen. Frozen tissues were pulverized and diluted in cold 0.05 mol/L HEPES, 0.15 mol/L NaCl, 1% Nonidet P-40, 1 nmol/L MgCl2, 1 mmol/L CaCl2. Protein concentrations were measured by the bicinchoninic assay method [16]. Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis on 10% gels and analyzed by immunoblotting as described previously [12]. The bands on the films were quantified with AlphaEase software (Alpha Innotech, San Leandro, CA) and normalized to GAPDH as a loading control.

2.6. VSMC culture
The carotid arteries of male Sprague–Dawley rats were excised and immersed in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 U/mL) and streptomycin (100 µg/mL). After removal of adipose and connective tissue, smooth muscle cells were dissociated by digestion in collagenase type I (3 mg/mL) for 60 min; then, the adventitia was stripped by rubbing with the back of a thin forceps. Incubation was continued in collagenase type I (2 mg/mL), elastase (0.12 mg/mL), BSA (2 mg/mL) and soybean trypsin inhibitor (0.36 mg/mL) for 70 min. The medium was filtered and centrifuged. The cell pellet was resuspended in culture medium (DMEM containing 10% FBS, L-glutamine, HEPES, penicillin and streptomycin). The cells were first plated onto glass coverslips in the bottom of wells for 3 days to remove endothelial cells by their affinity to glass. After coverslip removing, VSMCs that remained attached to the plastic of the wells were grown in culture medium until they reached 60–80% confluence (4 days). Only cells from passages 0 to 4 were used. During this period, {alpha}8β1 and {alpha}vβ3 integrin expression remained stable. Subsequent passages resulted in lower and higher expressions of {alpha}8β1 and {alpha}vβ3 integrins, respectively, suggesting dedifferentiation of VSMCs. von Willebrand factor serving as a negative marker confirmed the absence of endothelial cells. VSMCs showed 99% purity, estimated by cell morphology and positive immunostaining with SM {alpha}-actin antibody. They were rendered quiescent by 48-h serum deprivation before assay.

2.7. DNA synthesis assay
To measure the synthesis of new DNA, quiescent cells (1 x 105/well) plated in 24-well plates were stimulated by PDGF-BB (20 ng/mL) or Ang II (10–7 mol/L). After 6 h, 1 µCi/50 µL [methyl-3H]-thymidine (Amersham Biosciences, Montreal, QC) was added to each well. Stimulation was continued for another 18 h. The cells were fixed with 5% trichloroacetic acid, followed by solubilization in 200 µL 0.1 N NaOH. Four milliliters of scintillation liquid was added to each sample, and radioactivity was measued in a ß counter (LS6500 Scintillation Counter, Beckman, Indianapolis, IN). The thymidine index was calculated as [total counts (cpm)]/[total protein (µg)].

2.8. Transfection of cultured VSMCs by siRNA
Three 21-nucleotide siRNA sequences specifically targeting rat {alpha}8 integrin were synthesized on the basis of mRNA sequence (AF 148797):


oligo1: AAG GUC AGA UCG AGA UUG UdTdT dTdT UUC CAG UCU AGC UCU AAC A
Target sequence:
AAA AGG UCA GAU CGA GAU UGU
oligo2: AGG UCA GAU CGA GAU UGU GdTdT dTdT UCC AGU CUA GCU CUA ACA C
Target sequence:
AAA GGU CAG AUC GAG AUU GUG
oligo3: GUC AAG AAG AUG CCG UAU A dTdT dTdT CAG UUC UUC UAC GGC AUA U
Target sequence:
AAG UCA AGA AGA UGC CGU AUA

Because firefly luciferase gene is not included in the mammalian genome, a Cy3-modified version of luciferase GL2 siRNA from Dharmacon served to evaluate the nonspecific effects of irrelevant siRNA and as a control for optimization of transfection conditions. Carotid VSMCs were seeded onto 24-well plates and, when they had reached 80–90% confluence, cells were transfected with siRNA using Lipofectamine 2000.

2.9. Migration assay
Cell migration was measured in a Transwell migration apparatus (Becton Dickinson Labware, Franklin Lakes, NJ) with 8-µm pore size fibronectin-coated membranes. Rat carotid VSMCs (2 x 104/200 µL), which had been transfected by siRNA against {alpha}8 integrin for 48 h, were loaded in the upper chamber, whereas 630 µL of cells was loaded in the lower chamber, and incubated for 2 h at 37 °C. PDGF-BB (20 ng/mL) were added to the bottom of the wells. After 14 h, the filters were removed, and cells remaining on the upper surface of the membrane (that had not migrated through the filter) were removed with a cotton swab. Then, the membranes were washed with PBS, and cells adhering beneath the membranes were fixed with 2% glutaraldehyde and stained with crystal violet [17]. The data are reported as the number of VSMCs per 4 random fields/filter. Each experiment was repeated three times.

Migration status of cells was also evaluated in a wound migration assay. Confluent cultures of rat carotid VSMCs were injured by scratching twice with a pipette tip across cells in perpendicular directions creating a wound 800 µm wide. Cells that migrated inside intersection of the wound were counted at 0 and 24 h after crystal violet staining by phase contrast microcopy.

2.10. Statistical analysis
The data were expressed as means ± S.E.M. Each experiment was repeated three times and representative results are shown. All values were subjected to Student's t-test, one-way, or two-way ANOVA followed by the Bonferroni t-test. P<0.05 was considered as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Neointima formation
Withdrawal of the inflated balloon resulted in vascular injury leading to neointima formation. In the present study, neointima formation in balloon-injured arteries was confirmed by histology. Seven days after injury, the inner vessel surface was covered with one or several layers of ovoid, irregular-shaped cells forming the neointima (Fig. 1A–C). The number of neointimal cell layers was increased 14 and 21 days after injury. At 28 days after injury, no further neointimal growth was observed. At this time, the neointima was slightly thinner due to consolidation and regular organization of the cell layers.


Figure 1
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  		Fig. 1 Neointima formation in rat carotid arteries. Hematoxylin-eosin-stained sections show increasing neointimal formation after balloon injury at 1 (1 w), 2 (2 w), 3 (3 w), and 4 weeks (4 w) (A). Neointima formation was measured by the area (B) and the intima-to-media ratio (C). The scale bar represents 100 µm in A, *P<0.001, n=6 rats per group.

 
SM {alpha}-actin being a relevant marker of VSMC differentiation, we examined the expression of this contractile protein during neointima formation. Immunostaining revealed significantly lower levels of SM {alpha}-actin especially in the inner layers of the media of balloon-injured rats [Fig. 2A (black arrow) and B] The level of SM {alpha}-actin approached control level at 4 weeks. Western blot analysis of the whole carotid arteries confirmed the decreased expression of SM {alpha}-actin (Fig. 2C and D).


Figure 2
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  		Fig. 2 SM {alpha}-actin expression in the tunica media of rat carotid arteries. SM {alpha}-actin immunoreactivity was intense the media of sham-operated compared to balloon-injured rats at 3 weeks (3 w) (A, black arrows showing the inner layers). The bar graph shows decreased SM {alpha}-actin in the tunica media after balloon injury (B). Typical immunoblot of SM {alpha}-actin of carotid artery lysates at 1 week (1 w) after balloon injury (C). The bar graph shows quantification of Western blots of SM {alpha}-actin after balloon injury (D). *P<0.05, n=5–6 rats per group. The scale bar represents 100 µm in A.

 
The expression of PCNA, a nuclear marker of cell proliferation, was not significantly increased in the media of balloon-injured arteries at any time points (results not shown), indicating that the period of VSMC proliferation had been terminated and occurred during the first week.

3.2. {alpha}8 and β1 integrin expression
We then examined by immunohistochemistry and Western blotting the expression of {alpha}8β1 integrin during neointima expansion. {alpha}8 Staining was detected on VSMCs in the carotid artery media of sham-operated rats (Fig. 3A). Similar images were obtained for β1 integrin subunit (not shown). Seven days after injury, the intensity of both {alpha}8 and β1 integrin immunostaining was markedly decreased in the tunica media, especially in the inner layers (Fig. 3A, black arrow). Fig. 3B and C illustrate the results of quantitative analysis of temporal decreases in both integrin subunits in the tunica media after balloon injury. Carotid artery lysates were also analyzed by Western blotting (Fig. 3D and E). {alpha}8 Integrin protein was downregulated at 1, 2 and 4 weeks after balloon injury. These data disclose a relationship between {alpha}8 integrin downregulation and phenotype modulation, as observed with SM {alpha}-actin, of the tunica media during neointima formation.


Figure 3
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  		Fig. 3 {alpha}8 and β1 Integrin expression in the tunica media of rat carotid arteries. Immunohistochemical staining demonstrates a decrease of {alpha}8 integrin intensity in the media at 3 weeks (3 w) in balloon-injured arteries (A, black arrows showing the inner layers). Similar analysis was performed in sections immunostained for β1 integrin (images not shown). Quantification of immunostained intensity reveals a decrease of {alpha}8 (B) and β1 (C) integrins in the tunica media during the time course after balloon injury. Five micrograms of protein from rat carotid artery lysates on the days indicated (1 to 4 weeks) were loaded per lane and electrophoresed on sodium dodecyl sulfate–polyacrylamide gel, followed by immunoblotting with anti-{alpha}8 integrin antibody. Typical immunoblots of {alpha}8 integrin at 1 (1 w), 2 (2 w) and 4 weeks (4 w) after balloon injury are shown. A cardiac fibroblast extract was used as a positive control (ctl) (D). The bar graph shows decreased {alpha}8 integrin after balloon injury in tissue lysates (E). The results are means ± S.E.M. The scale bar represents 100 µm in A. *P<0.05. n=5–6 rats per group.

 
3.3. {alpha}8 Integrin expression regulation in cultured carotid VSMCs
To assess {alpha}8 integrin expression in a proliferative state, VSMCs were isolated from rat carotid arteries and stimulated with PDGF-BB, an inducer of mitogenesis and migration [18,19]. Stimulation of VSMCs by Ang II favored a nonproliferating contractile phenotype [20,21]. DNA synthesis was associated with decreased {alpha}8 integrin in replicative VSMCs (PDGF treatment), whereas {alpha}8 integrin was increased in the low-replication state (Ang II treatment) compared to serum-free cultures (Fig. 4).


Figure 4
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  		Fig. 4 {alpha}8 Expression in different phenotypes of cultured VSMC. Quiescent VSMCs from the rat carotid artery were stimulated with 20 ng/mL PDGF-BB or 10–7 M Ang II. Mitogenesis was estimated by measuring [3H]thymidine incorporation into DNA after PDGF-BB and Ang II stimulation (A). Following stimulation, Western blotting analysis was used to detect {alpha}8 integrin expression (170 kD) (B). Data were normalized to GAPDH (35 kD) as a loading control. A decrease of {alpha}8 integrin in the proliferative state induced by PDGF-BB and an increase in the contractile nonproliferative state induced by Ang II were observed (C). The results are means ± S.E.M. * P<0.05 vs unstimulated control. n=6 wells per treatment group.

 
Among three different siRNA oligos tested against {alpha}8 integrin, transfection with oligo 1 and 3 produced the best knockdown results (Fig. 5). Between 70% and 80% of the cells were efficiently transfected as observed by comparing the number of VSMCs that were immunofluorescent by Cy3-tagged luciferase siRNA to the number of total cells present. Forty-eight hours after transfection, only 30% of {alpha}8 integrin protein was still apparent. No effect on β1 integrin subunit expression was detected, but {alpha}5 subunit expression was increased following {alpha}8 knockdown (Fig. 5C and D). To ascertain the efficiency of {alpha}8 integrin knockdown, the half-life of {alpha}8 integrin protein was measured in the presence of 20 µM cyclohexamide. Cyclohexamide-treated VSMCs showed that {alpha}8 integrin half-life was around 20 h, in agreement with the reduction of {alpha}8 integrin expression by siRNA.


Figure 5
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  		Fig. 5 {alpha}8 Integrin downregulation by siRNA in VSMCs. Primary VSMCs were transfected with siRNA oligos (1 µg/well) targeting {alpha}8 integrin. At 48 h posttransfection, 5 µg of proteins was employed for Western blotting to detect {alpha}8 integrin (A). The bar graph shows that oligos 1 and 3 were effective in {alpha}8 integrin knockdown (B). The bar graph shows that siRNA oligo 1 against {alpha}8 integrin has no effect on β1 subunit expression (C), but increases {alpha}5 subunit as detected by Western blotting (D). The results are means ± S.E.M. *P<0.05 vs. siRNA-luciferase. n=4 wells per treatment group.

 
To assess the effect of {alpha}8 integrin downregulation on VSMC proliferation, DNA synthesis was measured in both {alpha}8 subunit knockdown and control VSMCs. A similar growth response to PDGF-BB was observed whether or not {alpha}8 integrin expression was downregulated (Fig. 6).


Figure 6
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  		Fig. 6 Downregulation of {alpha}8 integrin and modulation of DNA synthesis in VSMCs. VSMCs were stimulated for 24 h by PDGF-BB 48 h after transfection by siRNA oligo 1. Thymidine incorporation assay was performed. The data indicate that VSMC DNA replication was not modulated by {alpha}8 integrin downregulation in response to PDGF. siRNA-luciferase as well as lipofectamine treatment served as controls for siRNA-{alpha}8. Serum-free cultured VSMCs were controls for PDGF stimulation. *P<0.05 vs. serum-free. n=4 wells per treatment group.

 
To determine if {alpha}8 integrin downregulation had an effect on VSMC migration, a Transwell migration system was used. A significant increase in VSMC migration through the membranes was seen in cells transfected by siRNA against {alpha}8 integrin (Fig. 7). Wells without PDGF-BB treatment served as negative controls. Interestingly, {alpha}8 knockdown increased cell migration toward the bottom of the wells, even in the absence of PDGF-BB. To confirm further these results, a wound closure assay was performed (Fig. 7C). In the absence of PDGF, {alpha}8 siRNA-treated VSMC migrated faster toward the source of the wound.


Figure 7
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  		Fig. 7 Migration of rat carotid VSMCs after {alpha}8 integrin downregulation. Migration was stimulated by the addition of PDGF-BB (20 ng/mL) to the lower chamber. Some wells were used as controls without PDGF addition. VSMCs were allowed to migrate for 14 h. Representative images of crystal violet-stained cells that migrated to the lower surface of the membrane are shown for siRNA-luciferase and siRNA-{alpha}8 oligo 1 (A). Counts of cells on the lower face of the membrane reveal a significant increase of motility in siRNA-{alpha}8-transfected VSMCs vs. siRNA-luciferase and without any siRNA (B). {alpha}8 Integrin downregulation by siRNA, even in the absence of PDGF-BB in the lower chamber, shows also heightened migration (B). In a wound assay, {alpha}8 downregulatation by siRNA-{alpha}8 oligo 1 results in an increased cell number in the wound intersection (C). The data are means ± S.E.M. of each experiment performed in triplicate. *P<0.05 vs. siRNA-luciferase minus PDGF, **P<0.005 vs. siRNA-luciferase plus PDGF. n=4 wells per treatment group (4 fields/well).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
VSMC migration from the tunica media toward the tunica intima and their accumulation within the intima have been recognized as key events in restenosis after angioplasty [1]. VSMC migration implies, as an initial step, the rearrangement of actin–ECM interaction leading to changes in cell morphology [5]. During this rearrangement, the role of integrins, as well as their protein ligands, is crucial as dynamic mediators of the actin cytoskeleton attachment to the ECM.

However, each integrin may play a different role in cell adhesion, proliferation and migration. {alpha}vβ3 Integrin has a stimulating effect, not only in VSMC proliferation but also in their migration and ECM invasion [22]. In addition, it has been demonstrated that {alpha}vβ3 plays a major role in PDGF-stimulated migration [23]. In restenosis, {alpha}5β1, {alpha}1β1 and {alpha}vβ3 integrin expression was particularly increased at the luminal edge of the neointima where the cells are proliferating [24–26]. In addition, {alpha}vβ3 and {alpha}vβ5 integrin expression was also slightly enhanced in the media early after vessel injury [26]. These results emphasize phenotype regulation and potential functional role of the different {alpha} subunits according to the cell state. In pathological situations, such as cardiac, hepatic and lung fibrosis, increased {alpha}8β1 integrin in myofibroblasts with contractile abilities led us to test whether it is representative of the differentiated phenotype. We also reasoned that {alpha}8β1 integrin should be decreased during neointima formation.

Acute vascular injury usually begins from the intima layer, and later it can invest other layers depending on time, severity of the lesion, responsiveness and functional connectivity among the wall tissues. Although recent observations have demonstrated that the vascular wall is composed of phenotypically heterogeneous subpopulations of endothelial cells, VSMCs and fibroblasts, neointima formation is considered as a VSMC-dependent response. The most important source of participating VSMCs in neointima formation is from the tunica media. We showed a reduced SM {alpha}-actin expression in the tunica media after injury (Fig. 2). As previously reported by Kocher et al. [27], this decrease lasted much longer than the proliferation period which was usually detected in the first week following balloon dilatation [28]. Reductions of {alpha}8 integrin and its partner β1 integrin in this situation (Fig. 3), together with SM {alpha}-actin, suggest a cell population that had partially lost its contractile abilities. The decrease of {alpha}8 integrin in the whole artery up to 4 weeks after balloon injury in rats probably corresponds to the period in which the cells are still in a semisynthetic, but nonproliferating, phenotype [29]. To our knowledge, the present study is the first to report decreased expression of a specific integrin during neointima formation. {alpha}8β1 Integrin is also expressed in human tissues [11]. However, to our knowledge, no reported study indicates that a similar pattern of {alpha}8β1 integrin expression occurs during restenosis.

During the course of neointima thickening, several ECM proteins have been documented to be secreted, namely, vitronectin, osteopontin, tenascin and thrombospondin [24,26,30,31]. These proteins belong to a group, called matricellular proteins, that are not directly involved in tissue structures but rather behave as modulators of cell functions [32]. They bind several growth factors and induce de-adhesion favoring migration. They all possess a RGD motif and are thus able to interact with {alpha}vβ3, {alpha}5β1 as well as with {alpha}8β1 integrin. We suggest that their interactions with {alpha}vβ3 and {alpha}5β1 integrins engage the migratory process whereas they may prevent strong adhesion of the fibrillar proteins to {alpha}8β1 integrin. Bieritz et al. [33] have recently demonstrated that {alpha}8-deficient mesangial cells from knockout mice migrate more on fibronectin and vitronectin than {alpha}8-expressing cells, indicating that {alpha}8β1 integrin is involved in adhesion rather than migration. The intracellular signaling mechanisms involved need however to be further clarified.

The behaviour of cultured VSMCs from the tunica media of the rat carotid artery allows a better understanding of {alpha}8 integrin expression in different pathological states. PDGF-BB is a growth factor that plays a more important role in VSMC migration than proliferation during neointima formation [34]. It has been shown that stimulation with PDGF-BB strongly decreases contractile components, including SM {alpha}-actin, SM myosin heavy chain and SM22{alpha} in VSMCs [35]. It was therefore used in our experiments to induce a noncontractile phenotype. On the other hand, it has been noted that Ang II was unable to increase the proliferation rate in growth-factor-deprived cells [20,21]. In addition, it augmented SM {alpha}-actin and other contractile proteins in VSMCs [36]. Therefore, Ang II was used to induce a contractile phenotype. The correlation between {alpha}8 integrin downregulation and proliferation of VSMCs induced by PDGF-BB as well as its upregulation by Ang II, which induces the contractile state in VSMCs (Fig. 4), suggest that {alpha}8 integrin is a marker of the VSMC differentiation. As a differentiation marker, {alpha}8 integrin expression should be negatively involved in VSMC migration and proliferation. By using RNA interference, {alpha}8 integrin protein was reduced by 70% after 48 h of transfection (Fig. 5). In spite of {alpha}8 integrin downregulation by siRNA, no difference in the PDGF-dependent VSMC proliferation rate was observed (Fig. 6). However, {alpha}8 integrin knockdown also resulted in an increase of PDGF-dependent and -independent migration (Fig. 7). Therefore, {alpha}8 integrin knockdown appears to dissociate the mitogenic effect of PDGF from its migratory action. {alpha}8β1 Integrin may be involved in formation of mature focal adhesions rather than of focal complexes that are associated with cell migration [37]. In consequence, its lower expression may displace the cell equilibrium in favor of migration. In addition, whether or not activation of {alpha}8β1 integrin, through affinity and/or valency regulation [38], occurred was not investigated. Of note, an increase of {alpha}5 subunit was observed after {alpha}8 downregulation, suggesting that phenotype modulation can affect expression of other integrins that may also participate in regulating cell migration.

In summary, the data presented here indicate that {alpha}8 integrin expression is related to the presence of a differentiated phenotype of VSMCs. Although the precise mechanism by which {alpha}8 integrin inhibits VSMC migration is not yet clear, its absence appears to facilitate migration. Finally, given the importance of VSMC differentiation and migration during blood vessel development and cardiovascular pathophysiology, further studies to identify the mechanisms regulating {alpha}8 integrin-dependent changes in VSMC differentiation may provide novel insights into their contribution to neointima formation.


   Acknowledgements
 
This study was supported by the Canadian Institutes of Health Research and by the Heart and Stroke Foundation of Quebec. The scientific expertise of Dr. Martin G. Sirois of the Institut de cardiologie de Montréal is acknowledged. The technical assistance of Geneviève Lapalme, Christian Charbonneau, and Annie Vallé is greatly appreciated.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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