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Cardiovascular Research 2006 71(1):170-178; doi:10.1016/j.cardiores.2006.03.003
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Copyright © 2006, European Society of Cardiology

{alpha}8 Integrin expression is required for maintenance of the smooth muscle cell differentiated phenotype

Ramin Zargham and Gaétan Thibault*

Institut de recherches cliniques de Montréal, Université de Montréal and Department of Experimental Medicine, 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 4 January 2006; revised 10 February 2006; accepted 3 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. VSMC phenotypic changes...
 5. Discussion
 Acknowledgments
 References
 
Objective Vascular smooth muscle cell (VSMC) de-differentiation is a prerequisite for migration from the tunica media to the intima after vascular injury. Integrin cell adhesion molecules participate in VSMC phenotype modulation. {alpha}8β1 Integrin is a differentiation marker of VSMCs and its knockdown heightens migration. In the present study, we examined whether or not {alpha}8 integrin is required for the maintenance of VSMC differentiated phenotype.

Methods {alpha}8 Integrin in rat VSMC was knocked down by short interference RNA (siRNA) targeting {alpha}8 integrin in comparison to a non-silencing siRNA. Cytoskeletal and morphological changes in VSMC were examined by immunofluorescence staining. The expression of phenotype-dependent markers was analyzed by immunoblotting.

Results {alpha}8 Integrin gene silencing evoked drastic changes in characteristics of the VSMC differentiated phenotype, including VSMC morphology, actin fibre organization, focal adhesion assembly and the expression of phenotype-dependent markers in favor of de-differentiation. Then, we investigated whether or not phenotype modulation induced by {alpha}8 integrin gene silencing could be reversed by an inducer of VSMC differentiation. Transforming growth factor-β (TGF-β) failed to upregulate smooth muscle-myosin heavy chain as well as the assembly of parallel actin fibres in VSMCs transfected by siRNA-{alpha}8. In addition, TGF-β-induced vinculin localization at the tip of the cells was impaired by {alpha}8 integrin gene silencing.

Conclusion These data suggest that {alpha}8 integrin expression is required for maintenance of the VSMC differentiated phenotype, a state that is crucial for non-motile VSMCs.

KEYWORDS Angioplasty; Atherosclerosis; Cell differentiation; Cytoskeleton and smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. VSMC phenotypic changes...
 5. Discussion
 Acknowledgments
 References
 
Contraction, the primary function of vascular smooth muscle cells (VSMC), is required to maintain vascular tone and integrity by VSMC interaction with the surrounding extracellular matrix (ECM) [1]. In mature arteries, VSMCs exist in a differentiated state with contractile ability. They have a spindle-like shape with parallel actin stress fibres and express markers associated with contractile function. After vascular injury, however, VSMCs in the tunica media convert to a de-differentiated phenotype, resulting in their migration from there to the intima. In the de-differentiated state, their unique functional and morphological properties are lost, and the expression of SMC-specific differentiation markers disappears [1]. Therefore, factors in the arterial wall must instruct VSMCs to maintain their differentiated phenotype [2].

Like many other cell types, VSMC differentiation is regulated by cell-to-ECM contact [3]. The signal mechanism of VSMC phenotype modulation and migration through the ECM involves new adhesive contacts. Cell–ECM interaction is mainly mediated through the integrin family of cell adhesion receptors. Integrins are heterodimeric proteins consisting of 1{alpha} and 1β subunits [4]. There are more than 24 known integrin combinations composed of 18{alpha} and 9β subunits. Different integrin-matrix adhesion structures vary in composition, signaling and tension-bearing properties [5]. VSMC integrins anchor the cytoskeleton to the ECM, thereby allowing functional contraction of the vessel wall. VSMC phenotype modulation is associated with the expression of an altered set of integrins [6].

We have previously demonstrated an association between {alpha}8β1 integrin and the differentiated phenotype of rat VSMCs. Moreover, {alpha}8 integrin gene silencing heightened VSMC migration [7]. However, the reason why {alpha}8 integrin knockdown facilitated migration was unclear.

The strong expression of {alpha}8 integrin in differentiated VSMCs and its downregulation in the de-differentiated state led us to hypothesize that {alpha}8 integrin is required for maintenance of the VSMC phenotype. Therefore, its absence might be modulating VSMCs to adapt a phenotype that is more appropriate for migration.

In the present study, we provide evidence that maintenance of the VSMC differentiated phenotype is dependent on the presence of {alpha}8 integrin. Investigations to identify the factors regulating VSMC differentiation and migration may provide novel insights into their contribution to neointima formation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. VSMC phenotypic changes...
 5. Discussion
 Acknowledgments
 References
 
2.1 Antibodies and reagents
Antiserum to the {alpha}8 integrin subunit (A8-2) was generated as described elsewhere [8]. Antibodies against GAPDH and smooth muscle myosin heavy chain (SM-MHC) were from Abcam (Cambridge, UK), antibodies against integrin subunits ({alpha}1, {alpha}2, {alpha}5, and {alpha}V) were from Chemicon (Temecula, CA), anti-Kruppel-like factor 4 (KLF4) was from CeMines (Golden, CO), anti-smoothelin was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-SM {alpha}-tropomyosin, anti-SM {alpha}-actin and anti-calponin were from Sigma (Oakville, ON). Alexa Fluor 488-conjugated secondary antibody and TOTO-3 were from Molecular Probe (Eugene, OR). Other secondary antibodies were from Chemicon. TRITC-phalloidin was from Sigma. Short interference RNA (siRNA) against {alpha}8 integrin and siRNA targeting the firefly luciferase gene were synthesized by Dharmacon (Lafayette, CO) [7]. Lipofectamine 2000 was from Invitrogen Life Technologies (Carlsbad, CA). Transforming growth factor-β (TGF-β) was purchased from Fitzgerald (Concord, MA).

2.2 VSMC culture
The carotid arteries of male Sprague–Dawley rats were excised to isolate VSMCs, as described previously [7]. Only cells from passages 0 to 1 were used for {alpha}8 integrin knockdown while passages 5 to 6cells were used for TGF-β stimulation after {alpha}8 integrin knockdown. They were rendered quiescent by 48-h serum deprivation before assay. 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 Western blotting
VSMC extracts were prepared in lysis buffer (0.05mol/L HEPES, 0.15mol/L NaCl, 1% Nonidet P-40, 1nmol/L MgCl2, 1mmol/L CaCl2). Protein concentrations were measured by bicinchoninic assay [9]. Equal amounts of proteins (5µg) were separated by SDS-PAGE according to the method of Laemmli [10] in a Mini-Protean II cell system (Bio-Rad Laboratories). Using prestained calibration protein standards (Life Technologies, Burlington, ON), electrophoresis was run until the red protein marker (~60–65kDa) reached the end of the gel. Immediately after SDS-PAGE, proteins were transferred to Hybond ECL (Amersham Canada Ltd.) in Tris-glycine buffer containing 20% methanol. The nitrocellulose membrane was saturated for 60min in blotting buffer (0.05M NaPO4, pH 7.4, 0.154M NaCl, 0.05% Tween 20) in the presence of 5% calf serum. The membrane was then incubated for 90min in the presence of appropriate diluted antibodies. After the membrane was washed, antibody binding was visualized either by anti-mouse (or anti-rabbit) Ig antibody coupled to peroxidase or by streptavidine–peroxidase conjugate, depending on the primary antibody. Peroxidase activity was revealed by chemiluminescence with ECL (Amersham Canada Ltd.).The bands on the films were quantified by AlphaEase software (Alpha Innotech, San Leandro, CA) and normalized to GAPDH as a loading control.

2.4 Transfection of cultured VSMC by siRNA-{alpha}8
Transfection of cultured VSMCs by siRNA was described elsewhere [7]. Firefly luciferase GL2 siRNA from Dharmacon served to evaluate the nonspecific effects of irrelevant siRNA as a control. Carotid VSMCs in 12-well plates were transfected with 2µg/well siRNA by Lipofectamine 2000. As demonstrated in our previous report [7] and here in Fig. 1A, there was no significant difference in the basal starting levels of detected protein between irrelevant siRNA transfected VSMCs and without any transfection. Thereby, only irrelevant siRNA was used as control. Moreover, the optimal {alpha}8 integrin downregulation (50% to 70%) in our conditions was observed 48-h post-transfection.


Figure 1
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  		Fig. 1 Expression of VSMC phenotype-dependent markers after {alpha}8 integrin gene silencing. Cultured, quiescent VSMCs were transfected with siRNA-{alpha}8 for 48h. Western blotting analysis of an equal amount of sample proteins showed that {alpha}8 integrin was significantly downregulated compared to a non-silencing siRNA and or without siRNA treatment (A). Gene silencing of {alpha}8 integrin decreased the expression of smoothelin, calponin, SM-MHC, SM {alpha}-tropomyosin and {alpha}1 integrin (B) whereas it increased the expression of KLF4, {alpha}5, {alpha}2, and {alpha}v integrin subunits (C). Bar graphs indicate the relative ratios of the band intensity of the detected proteins normalized to GAPDH level as loading control. siRNA-luciferase treatment served as control (Ctrl) for siRNA-{alpha}8. *P<0.05. The graphs are representative of three independent experiments. The results are means±S.E.M. n=4 wells per treatment group.

 
2.5 Immunofluorescence and confocal laser scanning microscopy
Immunofluorescence staining was used to study F-actin, vinculin and cell morphology. After fixation with 4% paraformaldehyde for 15min and permeabilization with 0.2% Triton X-100 in phosphate buffer solution for 10min, the slides were incubated with blocking buffer (5% milk, 0.1% Tween 20 in Tris buffer solution). To visualize vinculin, the primary antibody was deployed with a secondary anti-mouse antibody conjugated to Alexa Fluor 488. The slides were incubated with TRITC-phalloidin to detect F-actin while TOTO-3 was employed to detect nuclei. Omission of primary antibodies and staining with an irrelevant mouse immunoglobulin of the same isotype served as negative controls. Labeled cells were examined under a Zeiss confocal microscope, and images were obtained with Zeiss Lasersharp software.

2.6 Analysis of {alpha}8β1 integrin by 125I-echistatin–integrin complex
This pharmacological assay is based on the property of the venom peptide echistatin to bind with high affinity to RGD-dependent integrins (which recognize arginine–glycine–aspartate motif on the surface of their ligands) present in cell extracts. The 125I-echistatin–integrin complexes are resistant to sodium dodecyl sulfate (SDS) and can thus be separated and identified, according to their molecular mass, by SDS-polyacrylamide gel electrophoresis (PAGE). This procedure is described in detail elsewhere [11]. VSMCs were solubilized in ice-cold lysis buffer in 0.05mol/L HEPES, pH 7.4, containing 0.15mol/L NaCl, 1% Nonidet P-40, 1mmol/L MgCl2, 1mmol/L CaCl2, 5mmol/L MnCl2, and protease inhibitors. After centrifugation, protein samples (20µg) were incubated with 5µL of 125I-echistatin (250,000cpm) in a total volume of 20µL in the presence of 5mmol/L MgCl2. After 90min of incubation, 5µL of sample buffer (without thiol-reducing reagent) was added, and samples (without heating) were separated by SDS-PAGE. The gels were stained, dried, and exposed to X-OMAT AR film for visualization of the 125I-echistatin–integrin complexes.

2.7 Statistical analysis
Data are expressed as means±S.E.M. Each experiment was repeated 3 times, and representative results are shown. Values were subjected either to Student's t-test, 1-way or 2-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. VSMC phenotypic changes...
 5. Discussion
 Acknowledgments
 References
 
3.1 Expression of phenotype-dependent markers of VSMC after {alpha}8 integrin gene silencing
The expression level of contractile (differentiation) markers in VSMCs is a useful paradigm in the analysis of SMC phenotypic transitions [12]. Fig. 1A shows that there is a significant decrease (60%) of {alpha}8 integrin after RNA interference compared to an irrelevant siRNA and cells treated without any siRNA. To examine the effect of {alpha}8 integrin knockdown on the VSMC phenotype, the expression of several phenotype-dependent markers was analyzed by Western blotting. Markers including smoothelin, SM-MHC, calponin, {alpha}1 integrin and {alpha}-tropomyosin [13–15] were taken as differentiation markers, while KLF4, {alpha}5, {alpha}2 and {alpha}v integrin [6,16] were adopted as markers of the de-differentiated phenotype. RNA interference targeting {alpha}8 integrin resulted in lower expression of the differentiation markers (Fig. 1B) while expression of the de-differentiation markers was increased (Fig. 1C).

3.2 VSMC morphological changes after {alpha}8 integrin gene silencing
Another hallmark of the VSMC differentiated state is the assembly of actin cytoskeleton into parallel and elongated stress fibres. Immunofluorescence staining under confocal microscopy showed disassembly and aggregation of actin stress fibres around the perinuclear region after {alpha}8 integrin gene silencing (Fig. 2). In addition, VSMC change from a spindle-like to a polygonal shape was observed. It has been shown that the recruitment of actin fibres to the cell membrane and stabilization of stress fibres are achieved via a complex of interacting focal adhesion (FA) components, including vinculin and talin. Such complexes couple integrins to the actin stress fibres [17]. Moreover, recruitment of vinculin to the adhesion sites leading to FA assembly is another characteristic of differentiated VSMCs [18]. Fig. 3 shows that {alpha}8 integrin gene silencing led to the disassembly of FAs, revealed by decreased vinculin localization at the tips of the cells and its relocalization along with actin fibres around the perinuclear region. The above results suggest that {alpha}8 integrin is required for FA and stress fibres formation which may contribute to the differentiated VSMC phenotype.


Figure 2
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  		Fig. 2 Disassembly of actin stress fibres by {alpha}8 integrin gene silencing. Representative images of primary VSMCs transfected by non-silencing siRNA used as a control (A) or siRNA-{alpha}8 integrin (B) are shown. Confocal images reveal F-actin distribution in control VSMCs. Note the presence of parallel stress fibres crossing the VSMC body (A) in the controls, whereas VSMCs transfected by siRNA-{alpha}8 show the presence of disorganized perinuclear bundles of actin (B). In addition, the VSMCs were changed from a spindle-like to a polygonal shape after {alpha}8 integrin gene silencing. Nuclei stained in blue with TOTO-3. The results are representative of 3 independent experiments.

 

Figure 3
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  		Fig. 3 Disassembly of FAs by {alpha}8 integrin gene silencing. TRITC-phalloidin and anti-vinculin antibody after siRNA transfection in VSMCs showed vinculin, a marker of mature FAs, at the tip and edge of elongated control VSMCs (A and C). In contrast to control cells, in siRNA-{alpha}8 integrin treated cells, vinculin was localized in the perinuclear region while its lower expression was observed at the tip of polygonal VSMCs (B and D). Upper panels: vinculin; lower panels: merged images with actin fibres. The results are representative of 3 independent experiments.

 

   4. VSMC phenotypic changes by TGF-β after {alpha}8 integrin gene silencing
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. VSMC phenotypic changes...
5. Discussion
 Acknowledgments
 References
 
TGF-β is known to induce SMC differentiation in a variety of cell types [19]. In VSMCs, it has been demonstrated that TGF-β stimulation leads to actin stress fibres assembly [20] and the expression of differentiation markers, including SM-MHC [21]. To induce VSMC differentiation by TGF-β, less differentiated cells were required. While freshly isolated (passages 0–1) VSMCs in the cell culture are still differentiated, upon serial passages they exhibit a less differentiated phenotype [22]. Therefore, VSMC differentiation of passages 1, 3, 5 and 8 was evaluated by the detection of {alpha}8β1 and {alpha}vβ3 integrins levels.

To detect both integrins at the same time, we performed a radioligand-binding assay based on the interaction of 125I-echistatin with RGD-dependent integrins. As illustrated in Fig. 4, 170 to 220kDa SDS-stable complexes formed between 125I-echistatin and integrins. The higher molecular weight complex was previously identified by immunoblotting, immunoneutralization, and RGD affinity chromatography as containing {alpha}8β1 integrin [11]. Fig. 4 shows a decrease of {alpha}8β1 integrin at higher passages (especially passages 5 and 8). Moreover, a contrary trend was seen in the third band, identified as {alpha}vβ3 integrin. The results herein revealed that passage-5 VSMCs exhibited a less differentiated phenotype and were thus chosen to study the effects of TGF-β. TGF-β stimulation of these VSMCs for 24h upregulated SM-MHC, as illustrated in Fig. 5A. However, preincubation with siRNA-{alpha}8 for 18h before the addition of TGF-β completely abolished the ability of TGF-β to upregulate SM-MHC in VSMCs. Fluorescence staining showed that elongation and assembly of actin stress fibres as well as the spindle-like shape of VSMCs induced by TGF-β (Fig. 5C, G) also were suppressed after {alpha}8 integrin gene silencing (Fig. 5E, I). Fig. 6 demonstrates that TGF-β stimulation of VSMCs increased vinculin localization at the edge of the cells along with the tip of the stress fibres. However, in VSMCs transfected by siRNA-{alpha}8 integrin, the above changes could not be observed by TGF-β stimulation, and vinculin aggregated in the perinuclear region. These results disclosed that {alpha}8 integrin gene silencing could induce the phenotype modulation of VSMC, suggesting an important role of {alpha}8 integrin in the stability and organization of stress fibres and FAs.


Figure 4
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  		Fig. 4 Analysis of RGD-dependent integrins in VSMCs after different passages. {alpha}8β1 integrin was analyzed by autoradiography with 125I-echistatin in rat VSMC extracts from passages 1, 3, 5 and 8. Reduced levels of the first band, identified as {alpha}8β1 integrin at passages 5 and 8, were observed, while the third band, {alpha}vβ3 integrin, showed increased intensity at later passages.

 

Figure 5
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  		Fig. 5 Phenotypic modifications by TGF-β after {alpha}8 integrin gene silencing. TGF-β stimulation of passage-5 VSMCs upregulated SM-MHC which was impaired in cells pretreated with siRNA-{alpha}8 integrin. SM-MHC was analyzed by Western blotting. *P<0.05 vs. Ctrl. +P<0.05 vs. TGF-β+siRNA-{alpha}8 integrin. The graphs are representative of three independent experiments. The results are means±S.E.M. n=3 wells per treatment group (A). Passage-5 VSMCs grown on coverslips were transfected with siRNA-{alpha}8 integrin or non-silencing siRNA. After 24h, each group was treated with or without TGF-β for another 24h. VSMCs transfected with non-silencing siRNA alone served as negative controls. Transfected cells were processed for immunofluorescence microscopy 48h after transfection and analyzed by confocal microscopy. Cells were stained with TRITC-phalloidin. TGF-β-stimulated VSMCs exhibited an increase of spindle shape with elongated stress fibres traversing the cells (C and G), compared to non-treated TGF-β cells (B and F). However, the above changes induced by TGF-β were not observed in VSMCs transfected by siRNA-{alpha}8 integrin (D and H). Note that {alpha}8 integrin gene silencing disintegrates actin fibres to short and disorganized fibres paralleled by polygonal shaped VSMCs (E, I) which were not rescued by TGF-β treatment (D and H). Photomicrographs are 20 x (B–E) and 63 x (F–I) magnification. The results are representative of 3 independent experiments.

 

Figure 6
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  		Fig. 6 TGF-β induced formation of FA assemblies in VSMCs after {alpha}8 integrin gene silencing. Immunofluorescence microscopy was performed using anti-vinculin antibody and Alexa Fluor 488-conjugated anti-mouse IgG (A to D) in combination with TRITC-phalloidin staining for actin fibres (E to H). TGF-β stimulated VSMCs exhibited strong localization of vinculin, a marker of mature FA, at the tip of the actin fibres in the edge of VSMCs (B and F) compared to the controls (A and E). However, {alpha}8 integrin knockdown induced lower localization of vinculin in FA sites and increased its localization in the perinuclear region, even in the presence of TGF-β (C and G). VSMCs transfected with siRNA-{alpha}8 integrin without TGF-β stimulation served as positive controls (D, H). The yellow-colored vinculin plaques in merged images (lower panels) represent co-localization of the protein with related actin fibres. Images are representative of cells observed in 3 independent experiments.

 
4.1 Increased expression of G-actin after {alpha}8 integrin gene silencing
Up to now, the molecular mechanism of phenotype modulation after actin fibres disassembly in VSMCs is poorly understood. However, it has been proposed that breakdown of F-actin and increase of its building blocks (G-actin) inhibit factors required for the transcription of differentiation markers [23]. Interestingly, an increased intracellular presence of SM {alpha}-actin after {alpha}8 integrin gene silencing was detected by Western blotting (Fig. 7). This indicates that the increase of G-actin may represent a possible mechanism of decreased expression of differentiation markers induced by {alpha}8 integrin gene silencing.


Figure 7
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  		Fig. 7 G-actin expression after {alpha}8 integrin gene silencing. Following {alpha}8 integrin gene silencing, Western blotting analysis was used to detect SM {alpha}-actin expression. The bar graph shows an increase of SM {alpha}-actin after {alpha}8 integrin gene silencing. The graphs are representative of three independent experiments. The results are means±S.E.M. *P<0.05 vs. control. n=4 wells per treatment group.

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. VSMC phenotypic changes...
 5. Discussion
Acknowledgments
 References
 
ECM–actin interaction by integrin cell adhesion molecules is likely to play an important role in VSMC phenotype regulation [24,25]. Actin fibres in mature VSMCs emanate from distinct areas of the plasma membrane known as focal adhesions (FAs) where clusters of integrins bind to ECM proteins. Some integrins are expressed more intensely in mature VSMCs including {alpha}1β1, {alpha}8β1 and {alpha}7β1, whereas in de-differentiated VSMCs, an altered set of integrins, including {alpha}5β1, {alpha}2β1 and {alpha}vβ3are expressed [6]. Our previous study [7] showed that {alpha}8β1 integrin expression is associated with the differentiated phenotype of VSMCs in the tunica media of arteries. In addition, RNA interference targeting the {alpha}8 integrin subunit resulted in heightened VSMC migration in vitro. However, the reason why {alpha}8 integrin gene silencing can affect VSMC migration is unclear. It is assumed that de-differentiation of VSMCs after vascular injury is a prerequisite for VSMC migration from the tunica media to the intima, leading to the genesis of neointima. Therefore, we hypothesized that the presence of {alpha}8 integrin is required for maintenance of the VSMC differentiated phenotype, whereas its downregulation can induce de-differentiation, which is crucial for VSMC migration.

The effect of {alpha}8 integrin knockdown on VSMC phenotype was examined in the present study. Our data indicate that {alpha}8 integrin expression is indeed required for maintenance of the VSMC differentiated phenotype. In addition, our results revealed that even TGF-β stimulation failed to reverse the less differentiated phenotype of VSMC to a differentiated phenotype when {alpha}8 integrin expression was turned off.

VSMC phenotypic modulation is associated not only with changes in the level of expression of contractile proteins, but also with the reorganization of actin fibres and FAs. Along with phenotype modulation, the expression of FA components at FA sites is no longer obvious and is more evenly distributed throughout the cell. Therefore, the anchorage of actin fibres to FAs is disrupted, leading to perinuclear aggregation of short bundles. Consequently, loss of contractile forces results in loss of contractile protein expression.

To ascertain whether or not {alpha}8 integrin negative regulation of VSMC migration is due to its effect on the VSMC phenotype, all the above criteria were examined after {alpha}8 integrin expression was turned off. This strategy evoked the downregulation of differentiation markers of VSMC and the upregulation of de-differentiation markers. The expression of SM-specific genes depends on the existence of stress fibres [26]. Moreover, one of the most important characteristics of phenotype-modulated VSMCs is the disorganization of stress fibres. Our data indicate that {alpha}8 integrin gene silencing results in stress fibres disintegration in a non-directional fashion into discrete aggregates. Most of the stress fibres fragmented into short bundles, moved to the perinuclear region and formed a network-like structure. Thus, it is possible that the downregulation of {alpha}8 integrin may cause stress fibres to dissociate from adhesion plaques and subsequently disintegrate.

Another hallmark of the VSMC differentiated phenotype is the development of mature FAs from focal complexes which are associated with the accumulation of different sets of integrins on the cell membrane. FAs are more elongated than focal complexes. Integrins involved in FA attach to bundles of actin stress fibres that traverse the cell [27] and recruit vinculin to FAs which are required to transmit force [28]. The possible role of vinculin in the maintenance of stress fibres integrity has long been a subject of speculation [29]. The existence of FAs and stress fibres is closely correlated. They are absent from many migratory cells and prominent in the least motile cells [30,31]. It has been shown that tension forces-induced integrin upregulation in the membrane leads to the recruitment of adhesion structural proteins and the elongation of adhesion sites in the direction of force [32]. There is also an association between the reduction of vinculin levels, reflecting decreased adhesion and increased cell motility with modulation of SMC to the synthetic phenotype [24]. {alpha}8 Integrin gene silencing, however, may induce the breakdown of stress fibres through disassembly of FAs.

Immunofluorescence staining of primary VSMCs demonstrated that vinculin is detected at the ends of elongated stress fibres where adhesion plaques are located. However, {alpha}8 integrin knockdown showed a lower expression of vinculin in the focal contacts. Dispersal of vinculin throughout the cell was paralleled by the aggregation of disrupted fragments of stress fibres in the perinuclear region. Alteration in the localization of vinculin reflected the changes in SMC function, from the non-motile contractile cell to a more motile synthetic cell [18,30].

It is known that freshly isolated VSMCs upon several passages exhibit a less differentiated phenotype [22]. While primary cultured VSMCs exhibit strong {alpha}8β1 integrin expression, {alpha}8β1 integrin significantly decreased and {alpha}vβ3 increased in higher passages (Fig. 4).

Cultured VSMCs present a high degree of plasticity and reversibility of their phenotype by the re-expression of differentiation markers. TGF-β can revert the phenotype of less differentiated VSMCs by the upregulation of differentiation markers. Therefore, we stimulated passage-5 VSMCs with TGF-β to examine the hallmarks of VSMC phenotype. TGF-β stimulation upregulated SM-MHC, a known differentiation marker, in these cells as well as the assembly of actin fibres to more elongated parallel fibres and the assembly of FAs. However, in the presence of siRNA-{alpha}8 integrin, TGF-β stimulation failed to induce the aforementioned changes in VSMCs. These observations support our hypothesis that {alpha}8 integrin downregulation can shut down the mechanisms responsible for the VSMC differentiated phenotype. However, it raised a question about actin fibre disassembly by {alpha}8 integrin could result in the alteration of phenotype-dependent markers.

Previous reports have documented that the increase of SM {alpha}-actin availability inside the cytoplasm, due to actin stress fibres breakdown into its building blocks, sequesters co-factors required for serum response factor (SRF) activation [23]. SRF is a transcription factor responsible for the expression of SMC differentiation genes. SRF activation is dependent on the binding of its co-activator, megakaryocytic acute leukemia (MAL). Decreased G-actin availability associated with actin polymerization results in the nuclear accumulation of MAL, where it co-activates the transcription of genes containing SRF sites in their promoters [23]. Interestingly, {alpha}8 integrin gene silencing increased the amount of SM {alpha}-actin in VSMCs, as detected by Western blotting, probably via the de-polymerization of stress fibres. Although this mechanism may explain how {alpha}8 integrin downregulation can affect VSMC phenotype characteristics, the mechanism of {alpha}8 integrin involvement in the assembly of stress fibres and maturation of FAs remain to be elucidated. In addition, the possibility still exists that {alpha}8 integrin downregulation acts as a relay on the function and/or expression of other proteins, which may then play a crucial role in G-actin polymerization.

One way to investigate the mechanisms by which {alpha}8 integrin is involved in VSMC phenotype modulation is to determine whether it interacts with and affects signaling pathways previously shown to regulate VSMC differentiation. For instance, it is well documented that RhoA activity is critical for controlling VSMC differentiation [26] and the assembly of actin stress fibres and FAs [33]. The expression of VSMC differentiation genes is thought to be regulated by RhoA-mediated actin polymerization [26,34]. Furthermore, it has been shown that RhoA regulates SRF-dependent cardiac gene expression through cross-talk with the β1 integrin signaling pathway via an organized actin cytoskeleton [35]. Thus, further investigation regarding the physical and functional interaction between {alpha}8 integrin and RhoA is warranted to elucidate the mechanism of {alpha}8 integrin regulation of the VSMC differentiated phenotype.

Altogether, this study aimed to address the question as to why {alpha}8 integrin knockdown leads to heightened VSMC migration, a process which requires phenotype modulation. One explanation, as indicated by the present investigation, is that {alpha}8 integrin downregulation could modulate VSMC characteristics in favor of a phenotype which is more appropriate for VSMC migration.

In summary, our results revealed the important role of {alpha}8 integrin in the maintenance of VSMC differentiated state, which is implied in the process of neointima formation. Although downregulation of some other integrins, including {alpha}7 [36] and {alpha}1 [6], has been shown to be associated to the VSMC de-differentiation, to the best of our knowledge this is the first report to demonstrate that gene silencing of an integrin is able to induce a de-differentiated VSMC phenotype.

These data suggest that changes in VSMC phenotype-dependent markers as well as disassembly of FAs and stress fibres induced by {alpha}8 integrin knockdown may play an important role in promoting VSMC migration. Further clarification of the mechanism of differentiated phenotype maintenance should shed significant insight into the pathobiology of neointimal lesion development, thus facilitating the development of novel therapies of vascular occlusive disorders.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. VSMC phenotypic changes...
 5. Discussion
 Acknowledgments
References
 
This study was supported by the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Quebec and the Natural Science and Engineering Research Council of Canada. The technical assistance of Dominic Filion is greatly appreciated.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. VSMC phenotypic changes...
 5. Discussion
 Acknowledgments
 References
 

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