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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1977-1984.)
© 1997 American Heart Association, Inc.

Articles

Role of Tyrosine Kinases in Extracellular Matrix–Mediated Modulation of Arterial Smooth Muscle Cell Phenotype

Ulf Hedin; Johan Thyberg; Joy Roy; Alexandra Dumitrescu; ; Phan Kiet Tran

From the Department of Surgery, Karolinska Hospital (U.H., J.R., A.D., P.K.T.), and the Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet (J.T.), Stockholm, Sweden.

	Abstract

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Abstract Fibronectin (FN) promotes the modulation of freshly isolated arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype by interacting with integrins on the cell surface. This process is characterized by a structural and functional transformation of the cells, including a reorganization of the cytoskeleton, the formation of a large secretory apparatus, and the acquisition of proliferative capacity. In this study we have investigated the role of integrin signaling through tyrosine kinases in the structural changes that occur in SMCs during primary culture on FN. A gradual increase in phosphotyrosine staining in focal adhesions and a concomitant increase in tyrosine phosphorylation of proteins including focal adhesion kinase were observed. In contrast, cells seeded on laminin formed few focal adhesions, and tyrosine phosphorylation of proteins was less than in cells cultured on FN. Treatment of cells cultured on FN with the tyrosine kinase inhibitor genistein strongly suppressed focal adhesion formation, cell spreading, and cytoskeletal reorganization. In addition, electron microscopic analysis demonstrated that the phenotypic modulation was slowed down. These results indicate that the ability of extracellular matrix components to promote a change in the phenotypic properties of SMCs depends on the assembly of focal adhesions with associated tyrosine kinase activity.

Key Words: smooth muscle cells • phenotypic modulation • extracellular matrix • focal adhesions • tyrosine phosphorylation

	Introduction

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Smooth muscle cells build up the arterial media and play an important role in the development of atherosclerotic and restenotic lesions.^{1 2 3} As an integral part in these processes, SMCs migrate into the intima, where they proliferate and secrete extracellular matrix components. This activation of the SMCs is usually referred to as a transition from a contractile to a synthetic phenotype and includes prominent changes in cell structure and function.⁴ SMCs go through a similar shift in phenotype when established in culture,⁵ and the culture system has therefore become an experimental model to study the regulation of SMC structure and function.

We have previously shown that extracellular matrix components take part in the control of SMC phenotype in vitro.⁶ A substrate of FN promotes the transition of the cells into a synthetic phenotype during primary culture, whereas the cells are retained in a contractile phenotype when seeded on a substrate of the basement membrane protein LN.^{7 8} The change in SMC phenotype on FN depends on interactions between the cell-attachment sequence in FN, Arg-Gly-Asp (RGD), and the FN-binding integrin ₅ß₁.^{9 10} Adhesion to FN is followed by a progressive spreading of the cells. At the same time, the actin cytoskeleton is reorganized into stress fiber bundles of nonmuscle actin and the expression of smooth muscle–specific -actin is decreased.¹¹ As a result, the net amount of myofilaments is reduced and a widespread endoplasmic reticulum and a large Golgi complex are formed. Functionally, the cells become able to respond to exogenous mitogens and an increased secretory activity is noted.⁶

The signal-transduction mechanisms responsible for the effects of FN and LN on SMC structure and function are unknown. Recent studies have suggested that integrin-mediated signaling involves tyrosine kinase activity associated with focal adhesions.^{12 13 14} FAK, or pp125^FAK, has been suggested to be a central component of this pathway, but integrins may also signal independently of FAK.^{15 16 17 18} Clustering of integrins is followed by phosphorylation of FAK, which becomes able to activate src-like tyrosine kinases and phosphorylate the focal adhesion–associated protein paxillin.^{19 20} Several other integrin-mediated signaling events have also been described, including activation of small GTP-binding proteins, transient elevation of intracellular Ca²⁺, activation of the Na⁺/H⁺ antiporter, phosphatidylinositol turnover, activation of protein kinase C, and induction of mitogen-activated protein kinase activity.^{21 22 23} Recently, adhesion of nontransformed cells and integrin clustering have also been shown to facilitate progression through the G₁-S phase of the cell cycle by promoting cyclin E-CDK2 activity.²⁴

The regulation of SMC phenotype by the extracellular matrix appears to be intimately coupled to the formation of focal adhesions and the accompanying reorganization of the actin cytoskeleton. However, the involvement of integrin-mediated signaling events in this process has not been evaluated previously. Here we studied the induction of tyrosine phosphorylation of proteins during primary culture of SMCs on substrates of FN and LN. In addition, tyrosine kinase activity and the formation of focal adhesions on these substrates was correlated to changes in SMC phenotype, as judged by electron microscopy.

	Methods

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Cell Culture
SMCs were isolated from the aortic media of 4-month-old male Sprague-Dawley rats (Harlan Sprague Dawley, Inc, Indianapolis, Ind) by digestion with 0.1% collagenase (GIBCO) in Ham's medium F-12 supplemented with 10 mmol/L HEPES and TES (pH 7.3), 50 µg/mL L-ascorbic acid, 50 µg/mL streptomycin, 50 IU/mL penicillin (medium F-12), and 0.1% BSA (Sigma Chemical Co). The cells were resuspended in medium F-12/0.1% BSA, seeded in matrix-coated petri dishes, and incubated at 37°C in a humidified atmosphere of 5% CO₂ in air. The cell cultures were examined in a Nikon Diaphot inverted microscope and photographed using Kodak technical pan film.

Preparation of Substrates
Human plasma FN was isolated by adsorptive chromatography on gelatin–Sepharose 4B (Pharmacia) as described.⁷ Mouse Engelbreth-Holm-Swarm (EHS) sarcoma LN was purchased from GIBCO. Culture substrates were prepared by dissolving FN in PBS and LN in Ca²⁺- and Mg²⁺-free PBS at 20 µg/mL and allowing the proteins to adsorb to the bottom of plastic dishes or to glass coverslips for 16 hours at 20°C. The dishes were then rinsed twice with PBS and left in medium F-12/0.1% BSA for 15 minutes before use.

Tyrosine Kinase Inhibitor
Genistein (4',5,7-trihydroxyisoflavone), a specific tyrosine kinase inhibitor,²⁵ was purchased from Research Biochemicals Inc. A 20-mmol/L stock solution was prepared in DMSO and stored in aliquots at -20°C before use. Final dilutions of the drug were made with culture medium. Controls were made to check that the solvent did not affect the cells adversely at the final concentrations reached in the drug treatments.

Immunological Reagents
A rabbit antiserum against the rat integrin ß₁ subunit (anti-ß₁) was prepared as described and was a gift from Staffan Johansson, Uppsala, Sweden.⁹ Mouse monoclonal antibodies against smooth muscle -actin (anti–SMC -actin), vinculin, and phosphotyrosine (clone PT-66) were obtained from Sigma. For immunoblotting, mouse monoclonal antibody against phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology Inc. Mouse monoclonal antibody against FAK and purified FAK from endothelial cells used as positive control were purchased from Transduction Laboratories. Normal rabbit IgG fractions were from Dakopatts, fluorescein- and rhodamine-conjugated goat anti-rabbit IgG from Cappel, normal isotype-matched mouse IgG and fluorescein-conjugated goat anti-mouse IgG from Sigma, and HRP-labeled goat anti-mouse IgG was from Amersham International.

Immunofluorescence Microscopy
Freshly isolated SMCs were cultured on glass coverslips coated with FN or LN. The cells were fixed in 4% formaldehyde in PBS for 10 minutes and permeabilized in 0.2% Triton X-100 in PBS for 3 minutes. After rinsing with PBS, the specimens were exposed to anti–integrin ß₁ (1:100), anti-vinculin (1:400), or anti-phosphotyrosine (clone PT-66; 1:100), followed by the corresponding secondary antibodies (1:40). Primary and secondary antibodies were diluted in PBS/1% BSA, and the exposures lasted for 2 hours at 37°C. For actin staining, fixed and permeabilized cells were incubated with anti–SMC -actin (1:200), followed by the corresponding secondary antibody, and then with 0.5 µg/mL rhodamine-conjugated phalloidin (Sigma) in PBS for 30 minutes. The specimens were mounted in Vectashield (Vector Labs, Inc), studied in a Nikon Labophot fluorescence microscope, and photographed using Kodak Tri-X-pan film.

Immunoblotting and Immunoprecipitation
Freshly isolated SMCs were seeded in matrix-coated petri dishes and incubated for various time intervals as indicated. The cells were rinsed with cold PBS and lysed on ice in 450 µL RIPA buffer (50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 5 mmol/L EDTA; 1% Nonidet P 40; 1% sodium deoxycholate; 0.1% SDS; 1% aprotinin; 50 mmol/L NaF; and 0.1 mmol/L Na₃VO₄). The cells were scraped off the dish and insoluble material was removed by centrifugation. Protein concentration was determined by using a Bio-Rad DC protein assay (Bio-Rad Laboratories) followed by solubilization of the samples in SDS/sample buffer (62.5 mmol/L Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 5% 2-ß-mercaptoethanol; and 0.001% bromphenol blue) and boiling for 4 minutes. Proteins were thereafter separated by SDS–polyacrylamide gel electrophoresis in 7% gels and electrophoretically transferred to presoaked nitrocellulose membranes (Hybond-ECL, Amersham) for 2 hours at 100 V in a Mini Protean II Trans-Blot apparatus with cooling (Bio-Rad). The membranes were blocked with PBS/3% nonfat dry milk for 20 minutes with agitation at 20°C and then incubated overnight with 1 µg/mL anti-phosphotyrosine (clone 4G10) diluted in PBS/3% nonfat dry milk at 4°C. After washing with water, the membranes were incubated with HRP-conjugated anti-mouse IgG for 1.5 hours at 20°C. The membranes were serially washed in water and PBS/0.05% Tween 20, whereafter HRP activity was detected by chemiluminescence (ECL, Amersham). For detection of FAK, the membranes were placed in blocking buffer (10 mmol/L Tris HCl, pH 7.5; 5% nonfat milk; 100 mmol/L NaCl; and 0.1% Tween 20) for 1 hour at 20°C and then incubated overnight at 4°C with 0.25 µg/mL anti-FAK diluted in blocking buffer. The membranes were washed for 30 minutes in 10 mmol/L Tris-HCl, pH 7.5; 100 mmol/L NaCl; and 0.1% Tween 20, followed by incubation with secondary antibody and detection of HRP activity as described above.

For immunoprecipitation, cells were lysed in RIPA buffer and the cell lysates precleared by incubation with protein A (Pharmacia). The samples were thereafter incubated with 4 µg of anti-FAK at 4°C for 16 hours, followed by incubation with rabbit anti-mouse IgG. Immune complexes were precipitated with protein A–Sepharose (Pharmacia) at 4°C for 2 hours and washed with PBS. The precipitate was solubilized in SDS/sample buffer, boiled, and the proteins were separated by SDS–polyacrylamide gel electrophoresis on 7% gels, followed by immunoblotting with anti-phosphotyrosine antibody as described above.

Electron Microscopy and Morphometry
The cells were fixed with 3% glutaraldehyde in 0.1 mol/L sodium cacodylate–HCl buffer (pH 7.3) with 0.05 mol/L sucrose for 1 to 2 hours, scraped off the petri dishes, and transferred to plastic tubes. The specimens were postfixed in 1% osmium tetroxide in 0.1 mol/L sodium cacodylate–HCl buffer (pH 7.3) with 0.5% potassium ferrocyanate for 1 hour at 4°C, dehydrated in ethanol, stained with 2% uranyl acetate in ethanol, and embedded in low-viscosity epoxy resin. Thin sections were cut on an LKB Ultrotome IV, stained with alkaline lead citrate, and examined in a JEOL 100CX electron microscope. To follow the structural transformation of the SMCs, one large section from each culture was scanned without overlapping, and all cells (100 to 200) were registered as being either in a contractile (cytoplasm dominated by myofilaments) or synthetic (cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex) phenotype. This classification method is based on earlier morphometric analyses of rat aortic SMCs in primary culture and the realization that these cells may shift between two major phenotypic states with a distinct morphology.²⁶ For stereological measurements, approximate midsagittal sections through the central parts of the cells (extending from the nucleus toward the periphery) were photographed at a final magnification of 25 000x. Twenty randomly selected cells were included in each group. A test system consisting of an equilateral triangular network with a test line of 12.5 mm (equivalent to 0.5 µm in the cells) was superimposed on the micrographs, and the volume density of the main cytoplasmic organelles was determined by point counting.²⁷

	Results

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Phosphotyrosine Distribution in Cells Cultured on Substrates of FN and LN
The distribution of phosphotyrosine in rat aortic SMCs during primary culture was analyzed by indirect immunofluorescence microscopy. Freshly isolated cells were seeded on a substrate of FN in serum-free medium and fixed after 5, 24, 48, and 96 hours of culture. During this period, a gradual increase in phosphotyrosine staining was noted in focal adhesion–like structures at the cell edges. The staining was weak after 5 hours (Fig 1A), easily detected after 24 hours (Fig 1B), and strong after 48 and 96 hours (Fig 1C and 1D).

View larger version (107K): [in this window] [in a new window]   Figure 1. Indirect immunofluorescence microscopy demonstrating the gradual increase in phosphotyrosine staining (arrowheads) during primary culture of SMCs on a substrate of FN. Freshly isolated SMCs were seeded on FN in medium F-12/0.1% BSA, fixed, and stained with anti-phosphotyrosine antibody after 5 (A), 24 (B), 48(C), and 96 (D) hours as described in "Methods." Bar=10 µm.

The ability of LN to promote assembly of adhesive contacts containing phosphotyrosine was also evaluated. After 4 days, a positive reaction was detected in cells cultured on FN (Fig 2A) but not in cells cultured on LN (Fig 2B). To assess formation of focal adhesions, the cells were labeled with antibodies against vinculin and ß₁ integrin. With both antibodies, staining was found in focal adhesions in cells cultured on FN (Fig 2C and 2E). In contrast, only a diffuse or finely punctate staining was detected in cells cultured on LN (Fig 2D and 2F).

View larger version (118K): [in this window] [in a new window]   Figure 2. Comparison between phosphotyrosine (A and B), vinculin (C and D), and integrin ß1 (E and F) staining in SMCs after 4 days of primary culture on a substrate of FN (A, C, and E) and LN (Lam; B, D, and F). Freshly isolated cells were cultured on the substrates in medium F-12/0.1% BSA, fixed, and stained with the respective antibodies as described in "Methods." Bar=10 µm.

Phosphorylation of Proteins During Primary Culture of SMCs on FN and LN
Tyrosine phosphorylation of proteins in response to adhesion to FN and LN was analyzed by immunoblotting of proteins isolated after 0, 2, 5, 12, 24, 48, and 96 hours of culture. An increased tyrosine phosphorylation was most prominent of proteins in the molecular-weight range of 125, 90, and 75 kD (Fig 3A). In agreement with the focal adhesion–related phosphotyrosine staining, a gradual increase of protein phosphorylation was observed in these bands during the first days of culture, whereas a decreased phosphorylation of two higher-molecular-weight species occurred. Tyrosine phosphorylation of protein bands around 75 kD was increased up to 96 hours of culture, whereas phosphorylation in protein species of 125 and 90 kD peaked after 48 hours of culture (Fig 3A) and then declined. In contrast to cells cultured on FN, tyrosine phosphorylation of proteins isolated from cells cultured on LN was weak, thus confirming the immunocytochemical observations (Fig 3D). Immunoprecipitation with anti-FAK and subsequent immunoblotting with anti-phosphotyrosine showed no or barely detectable phosphorylation of FAK in freshly isolated cells and after 2 hours of adhesion to FN, whereas FAK was phosphorylated after 5 hours and remained in a phosphorylated state up to 96 hours of culture (Fig 3B). The levels of FAK in freshly isolated cells in suspension did not change after adhesion to FN (Fig 3C).

View larger version (48K): [in this window] [in a new window]   Figure 3. Analysis of SMC proteins during primary culture on FN and LN by immunoblotting. A, Immunoblotting with anti-phosphotyrosine (4G10) of proteins isolated from freshly isolated SMCs during primary culture on FN. B, Immunoblotting with anti-phosphotyrosine of samples immunoprecipitated with anti-FAK after primary culture of SMCs on FN. C, Immunoblotting with anti-FAK to demonstrate the presence of FAK after 0 and 48 hours of primary culture on FN with purified FAK used as control (Ctrl). D, Immunoblotting with anti-phosphotyrosine of proteins isolated from SMCs after primary culture on LN. The cells were cultured in medium F-12/0.1% BSA, lysed in RIPA buffer, and proteins were separated by SDS–polyacrylamide gel electrophoresis on 7% gels, transferred to nitrocellulose filters, and subjected to immunoblotting as described in "Methods." Molecular-weight markers are expressed in kilodaltons.

Effects of Genistein on Cell Spreading and the Reorganization of the Actin Cytoskeleton
Freshly isolated SMCs seeded on FN attached within a few hours, and after 2 days most of the cells were elongated with lamellae-like extensions (Fig 5A). Cell spreading was completed after 4 days (Fig 5B), and the cells were similar in appearance after 6 days (Fig 5C). Incubation with increasing concentrations (1 to 40 µmol/L) of the tyrosine kinase inhibitor genistein inhibited phosphorylation of FAK in a dose-dependent manner, with a complete inhibition at 40 µmol/L (Fig 4). In the presence of 40 µmol/L genistein, initial cell attachment was unaffected, and after 2 days the cells were similar in morphology to the controls (Fig 5D). However, genistein prevented further cell spreading, and the cells remained spindle shaped after 6 days of culture (Fig 5E and 5F). Immunocytochemical analysis showed that genistein inhibited the assembly of focal adhesions. After 4 days of culture on FN in the presence of 40 µmol/L genistein, a diffuse staining for phosphotyrosine and vinculin was observed (Fig 6C and 6D), whereas a focal adhesion–like pattern was obtained with both antibodies in control cells (Fig 6A and 6B). The effects of the tyrosine kinase inhibitor on the reorganization of the actin cytoskeleton were studied by double staining with an antibody against SMC -actin and phalloidin. The controls were characterized by a well-defined stress fiber organization and a partial loss of SMC -actin (Fig 7A and 7B). Cells exposed to 40 µmol/L genistein showed an intense staining for SMC -actin, and no distinct stress fibers could be discerned (Fig 7C and 7D). Genestein did not affect SMC morphology during the first 4 days of culture on LN (not shown).

View larger version (181K): [in this window] [in a new window]   Figure 5. Effects of genistein (40 µmol/L; D through F) on the spreading of SMCs in primary cultures compared with control cultures (A through C). The cells were grown on a substrate of FN in serum-free medium, fixed after 1 (A and D), 2 (B and E), and 4(C and F) days of culture, and photographed in the inverted microscope using phase-contrast optics. Bar=50 µm.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of genistein on the tyrosine phosphorylation of FAK. SMCs were cultured for 24 hours on FN in the presence of 0, 1, 10, and 40 µmol/L genistein, and FAK phosphorylation was analyzed by immunoprecipitation and phosphotyrosine immunoblotting as described in "Methods." The control (Ctrl) shows immunoblotting with anti-FAK of purified FAK protein for comparison.

View larger version (104K): [in this window] [in a new window]   Figure 6. Effects of genistein (40 µmol/L; C and D) on the assembly of focal adhesions in SMCs after 4 days of culture on a substrate of FN, as determined by vinculin (B and D) and phosphotyrosine staining (A and C). Untreated cultures are shown in A and B. Bar=10 µm.

View larger version (103K): [in this window] [in a new window]   Figure 7. Effects of genistein (40 µmol/L; C and D) on the cytoskeletal organization in SMCs after 4 days of culture on a substrate of FN. The actin cytoskeleton was visualized by double labeling with SMC -actin (A and C) and rhodamine-phalloidin (B and D). Bar=20 µm.

Effects of Genistein on the Structural Reorganization of the Cells
To further define the effects of the tyrosine kinase inhibitor on phenotypic modulation, SMCs were grown in primary culture on a substrate of FN and analyzed by electron microscopy. Genistein (40 µmol/L) had an inhibitory effect on the conversion of the cells from a contractile to a synthetic phenotype (Fig 8). In its presence, >50% of the cells had a cytoplasm dominated by myofilaments after 4 days, and >30% after 6 days (Fig 9). The effect of genestein on the structural transformation of the cells was dose dependent and maximal at 40 µmol/L (not shown). Quantitative stereological analysis confirmed these observations. After 6 days of culture, cells treated with genistein had a significantly lower volume density of endoplasmic reticulum and the fraction of the cytoplasm occupied by myofilaments was significantly higher than in control cells (Table).

View larger version (188K): [in this window] [in a new window]   Figure 8. Fine structure of SMCs grown on a substrate of FN in serum-free medium for 6 days either without drug addition (A) or in the presence of 40 µmol/L genistein (B). The control cell (A) has adopted a synthetic phenotype, with a prominent endoplasmic reticulum (ER) and Golgi complex (G). The genistein-treated cell (B) remains in a contractile phenotype, and its cytoplasm is for the most part occupied by myofilaments (F). M indicates mitochondria and N, nucleus. Bars=1 µm.

View larger version (22K): [in this window] [in a new window]   Figure 9. Effects of genistein on the conversion of SMCs into a synthetic phenotype in primary cultures. The cells were grown on a substrate of FN in control medium or medium containing genistein (40 µmol/L). On the indicated days, the cells were fixed and processed for electron microscopic analysis as described in "Methods." The results are presented as mean±SD (n=3).

View this table: [in this window] [in a new window]   Table 1. Effects of Genistein on Phenotypic Modulation of SMCs in Primary Culture as Evaluated by Electron Microscopic Stereology

	Discussion

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Components of the extracellular matrix have previously been shown to participate in the regulation of SMC phenotype in vitro.^{6 28 29 30} In vivo, similar changes in SMC morphology have been observed in atherogenesis and in the development of restenotic lesions after reconstructive vascular procedures.^{1 2} Observations made in human lesions and in experimental models indicate that these pathological processes are accompanied by changes in the composition of the extracellular matrix,² including altered expression of collagen, elastin, and proteoglycans,^{31 32} and the expression of adhesive and deadhesive extracellular matrix proteins,³³ such as thrombospondin, tenascin, and osteopontin.^{34 35 36} Possibly, changes in the extracellular matrix of lesions may not only determine the physical properties of atherosclerotic and restenotic lesions but also take part in the control of SMC function.

In this study, the molecular mechanisms by which the extracellular matrix regulates SMC phenotype in vitro were examined by analyzing the role of tyrosine kinase activity in this process. Adhesion of freshly isolated rat aortic SMCs to FN was followed by a progressive increase in tyrosine phosphorylation of proteins with a relative molecular mass of 120 to 130, 90, and 75 kD. Several focal adhesion–related proteins are tyrosine phosphorylated after integrin clustering, such as p130Cas, FAK, and paxillin, which may be present among the observed proteins.^{15 19 20} Immunoblotting with anti-FAK and immunoprecipitation with anti-FAK followed by immunoblotting with anti-phosphotyrosine also demonstrated the phosphorylation of FAK 5 hours after adhesion of SMCs to FN, and FAK thereafter remained phosphorylated. The activation of FAK in response to interaction with FN is consistent with previous observations in subcultured SMCs and other cell types after adhesion to FN.^{37 38} Wilson et al, however, demonstrated a lack of FAK activation in mouse SMCs after adhesion to FN.³⁹ These authors observed an increased phosphorylation of FAK after adhesion of cells to SMC-derived extracellular matrix. Hence, it cannot be excluded that the endogenous formation of a pericellular matrix influences the observed phosphorylation of FAK during primary culture on FN, especially since tyrosine phosphorylation of proteins under these conditions was extended in time and phosphorylation of FAK was considerably delayed compared with subcultured SMCs (Hedin et al, unpublished data, 1997). Results from experiments with FAK-deficient cells also indicate that FAK may be necessary for the turnover of focal adhesions.¹⁸ Possibly, the extended FAK activity in SMCs during primary culture on FN is required for the turnover of focal adhesions during the prolonged cell spreading and rearrangement of the cytoskeleton that take place under these culture conditions.

In agreement with the gradual increase in tyrosine phosphorylation of proteins during primary culture on FN, the assembly of focal adhesions and phosphotyrosine staining were extended over the first 2 to 4 days. Under these conditions, we have previously observed a gradual organization of integrin ₅ß₁, along with a progressive increase in the formation of stress fibers of actin and a rearrangement of SMC -actin.¹¹ In contrast, LN did not promote focal adhesion formation and associated tyrosine kinase activity. In cells cultured on LN, no phosphotyrosine staining was observed in focal adhesions during the first 4 days of culture, and ß₁ integrins showed a diffuse distribution. In addition, tyrosine phosphorylation of proteins was weak in comparison with proteins isolated from cells cultured on FN. The phenotypic modulation of SMCs on LN was previously reported to be delayed and to depend on endogenous synthesis of FN.⁸ Thus, it is conceivable that the inability of LN to promote the transition of SMCs from a contractile to a synthetic phenotype in vitro is related to the lack of focal adhesion formation and associated tyrosine kinase activity.

To examine whether tyrosine kinase activity is required for FN-promoted modulation of SMC phenotype, cell cultures were treated with the tyrosine kinase inhibitor genistein. In the presence of this drug at concentrations that efficiently inhibited FAK phosphorylation, genistein restricted cell spreading and inhibited the organization of ß₁ integrin and vinculin in focal adhesions. No focal adhesion–associated phosphotyrosine labeling was observed, and the reorganization of the cytoskeleton was inhibited. Genestein also caused a significant inhibition of the structural transformation of the cells. The formation of an extensive endoplasmic reticulum was slowed down, and myofilaments were retained in the cytoplasm. Because genestein nonspecifically inhibits protein tyrosine kinases, we cannot exclude the possibility that this drug affected kinase activity involved in the control of SMC phenotype distinct from integrin signaling and focal adhesion formation. On the other hand, the effect of genestein on SMC phenotype was limited, suggesting that non–tyrosine kinase–dependent processes may also be involved in the control of SMC structure. In general, the effect of tyrosine kinase inhibition on SMCs cultured on FN was similar to the behavior of SMCs in response to culture on substrates of LN or type IV collagen.^{8 9 11} Previously, tyrosine kinase inhibitors have been shown not only to restrict focal adhesion formation and cytoskeletal assembly in cultured cells⁴⁰ but also to inhibit the migration of SMCs.⁴¹

Taken together, these results contribute new information by suggesting that the effects of different extracellular matrix proteins on SMC phenotype in vitro are due at least in part to their ability to promote focal adhesion formation, associated tyrosine kinase activity, and rearrangement of the actin cytoskeleton. Whether similar mechanisms regulate SMC phenotype in vivo is not known. However, observations in embryonic vasculature suggest that there is an association between extracellular matrix components, integrin-associated tyrosine kinases, and the functional state of the SMCs. In the embryo, SMCs proliferate and deposit extracellular matrix and thereby build up the arterial media.⁴² Increased expression of FAK mRNA and protein has recently been described in the developing vasculature of mouse embryos.³⁸ In addition, FN is abundant in the extracellular matrix of developing blood vessels of chick embryos, whereas LN is expressed at the stage of vascular maturation.⁴³ Since signals from integrins may regulate cell-cycle progression, this pathway may be a central part of SMC growth control in the vessel wall.^{24 44 45}

	Selected Abbreviations and Acronyms

 

FAK = focal adhesion kinase FN = fibronectin HRP = horseradish peroxidase LN = laminin SMC = smooth muscle cell

	Acknowledgments

 
This study was supported by grants from the Swedish Medical Research Council (06537 and 09100), the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, the Åke Wiberg Foundation, the Swedish Society for Medicine, and Karolinska Institutet. The rabbit antiserum against the rat integrin ß₁ subunit (anti-ß₁) was a gift from Staffan Johansson, Uppsala, Sweden.

	Footnotes

 
Reprint requests to Ulf Hedin, MD, PhD, Department of Surgery, Division of Vascular Surgery, Karolinska Hospital, S171 76 Stockholm, Sweden.

Received April 16, 1996; accepted December 3, 1996.

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