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Circulation Research. 1997;80:627-637

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(Circulation Research. 1997;80:627-637.)
© 1997 American Heart Association, Inc.

Articles

Fibroblast Growth Factor-2 Potentiates Vascular Smooth Muscle Cell Migration to Platelet-Derived Growth Factor

Upregulation of {alpha}2ß1 Integrin and Disassembly of Actin Filaments

J. Geoffrey Pickering, Shashi Uniyal, Carol M. Ford, Thu Chau, Mary Ann Laurin, Lawrence H. Chow, Christopher G. Ellis, Jonathan Fish, , Bosco M. C. Chan

From the John P. Robarts Research Institute and London Health Sciences Centre, Departments of Medicine (Cardiology) (J.G.P., C.M.F., M.A.L., L.H.C.), Medical Biophysics (J.G.P., C.G.E., J.F.), Biochemistry (J.G.P.), and Microbiology and Immunology (S.U., T.C., B.M.C.C.), University of Western Ontario, London, Canada.

Correspondence to J. Geoffrey Pickering, London Health Sciences Centre, 339 Windermere Rd, London, Ontario N6A 5A5, Canada. E-mail gpickrng{at}rri.uwo.ca


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering ß1 integrin–mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of {alpha}2ß1, {alpha}3ß1, and {alpha}5ß1 integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding {alpha}2ß1 integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen–coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2–induced elongation and migration on collagen were inhibited by a blocking anti-{alpha}2ß1 antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle {alpha}-actin–containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of {alpha}2ß1 integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of {alpha}2ß1 integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.


Key Words: smooth muscle cell • integrin • collagen • actin • fibroblast growth factor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Migration of vascular SMCs is an important means by which the arterial intima thickens during the course of vascular diseases.1 2 The mechanisms mediating SMC migration are incompletely understood, but potential regulators include chemotactic cytokines and growth factors3 as well as ECM molecules.4 5 6 7 A number of physical changes to the cell and surrounding ECM are also features of cell migration, including restructuring of the actin cytoskeleton,8 dynamic attachment to and release from the ECM,9 10 and local turnover of the ECM.11 12

FGF-2 (basic FGF) is a potent mitogen for SMCs13 14 and a proven mediator of SMC proliferation after injury to the rat carotid artery.15 A role for FGF-2 in mediating SMC migration has also been suggested by in vivo studies. Injection of FGF-2 following gentle deendothelialization of the rat carotid artery significantly stimulated SMC migration from the media to the intima. Furthermore, SMC migration induced by balloon disruption of the arterial media was abrogated by a blocking antibody to FGF-2.16 However, the mechanism by which FGF-2 mediates SMC migration is unclear. Unlike PDGF, which is a potent chemoattractant for SMCs,3 17 FGF-2 has no,3 or at best weak,17 18 chemotactic activity for SMCs. A potential role for FGF-2 in modulating the interactions between SMCs and the ECM has been suggested16 but not explored.

Integrins are cell surface receptors that serve to bridge the ECM with the cell cytoskeleton.19 In SMCs, integrins of the ß1 family appear to be the dominant integrins responsible for cell adhesion to the ECM, and several ß1 integrins have been identified on SMCs.10 20 21 22 23 24 25 Migration of SMCs has been noted to be dependent on ß3 integrins,26 27 but a number of recent studies have also implicated ß1 integrins in SMC migration. Antisera against ß1 integrin blocked fibronectin-promoted migration of rat SMCs,27 and {alpha}2ß1 integrin has been implicated in PDGF-induced chemotaxis on collagen.25 On the other hand, excessive ß1 integrin–mediated adhesion may impair SMC migration, as demonstrated by recent studies with a function-activating anti–ß1 integrin antibody.10 28

Expression of ß1 integrins is regulatable by growth factors. Transforming growth factor-ß1 upregulated {alpha}1 and {alpha}5 integrin subunits in rabbit SMCs,29 and PDGF-BB has been reported to increase the {alpha}5 integrin subunit in bovine and rabbit SMCs.29 30 Klein et al31 have demonstrated that FGF-2 increases the expression of {alpha}2ß1, {alpha}3ß1, {alpha}5ß1, and {alpha}6ß1 integrins in microvascular endothelial cells. However, the effect of FGF-2 on ß1 integrin expression in vascular SMCs is unknown.

In the present study, we have examined the mechanism by which FGF-2 controls migration of human SMCs, focusing particularly on the interplay between SMCs and the ECM. Using flow cytometry, we found that FGF-2 upregulates the expression of a number of ß1 integrins, most prominently the collagen-binding {alpha}2ß1 integrin. Associated with this altered integrin repertoire was morphological elongation of the cell and complete disassembly of the actin stress fiber–containing cytoskeleton. Consistent with other reports,3 FGF-2 was not chemotactic for human SMCs. However, SMCs treated with FGF-2 displayed enhanced migration on type I collagen, including augmented chemotaxis to PDGF-BB, that was inhibited by a blocking antibody to {alpha}2ß1 integrin. This {alpha}2ß1 integrin–mediated increase in migration was not accompanied by an increase in adhesion to collagen, suggesting a complex response to FGF-2 designed to selectively enhance motility. The findings indicate that coordinated alterations in the ß1 integrin–cytoskeletal axis may be a critical means by which FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Antibodies and Reagents
mAbs used included the {alpha}1ß1 integrin–specific mAb, Ts2/732 ; the {alpha}2ß1 integrin–specific mAb, BHA2.133 (Chemicon Inc); the {alpha}3ß1 integrin–specific mAb, P1B5 (GIBCO/BRL); an {alpha}5ß1 integrin–specific mAb, HA5; a ß1 integrin subunit–specific mAb, HB1.1(Chemicon Inc); and an {alpha}v integrin subunit–specific mAb, 23C6 (Serotec). Isotype-matched control antibodies were used for flow cytometry and blocking experiments. The F(ab')2 fragment of FITC-conjugated anti-mouse antibody was purchased from Becton-Dickinson.

Human recombinant FGF-2 and PDGF-BB were purchased from GIBCO/BRL. Rat-tail collagen solubilized in acetic acid was used as a source of type I collagen and was prepared as previously described.34 Human fibronectin was isolated from human plasma by gelatin-Sepharose chromatography,35 and EHS tumor–derived laminin was obtained from GIBCO/BRL.

SMC Culture
Primary cultures of human arterial SMCs were initiated by explant outgrowth from unused segments of internal mammary artery retrieved at the time of coronary artery bypass surgery, as previously described.36 37 The identity of vascular SMCs was confirmed morphologically and by positive immunostaining for smooth muscle {alpha}-actin (clone 1A4, Dako). Cells were grown in medium (M199, GIBCO/BRL) supplemented with the designated concentration of FBS. All experiments were performed using human SMCs in the third or fourth subculture.

Immunofluorescence
SMCs seeded on to multiwell slides were fixed in ice-cold acetone and stained for smooth muscle {alpha}-actin (clone 1A4, Dako) as previously described.36 F-actin stress fibers were also visualized using FITC-conjugated phalloidin (Sigma Chemical Co). Cells were coverslipped using glycerol/PBS (9:1) containing Hoechst 33258 (2.5 µg/mL, Sigma) to identify cell nuclei and evaluated by fluorescence microscopy.

Flow Cytometry
Flow cytometry for detection of integrin expression was carried out by indirect immunofluorescence staining as described previously.33 38 Early confluent SMCs were trypsinized and washed in cold PBS with 1% BSA. Cells were incubated for 30 minutes on ice with control or integrin-specific mAbs at predetermined saturating concentrations. Washed cells were incubated with FITC-labeled anti-mouse F(ab')2 fragment, and fluorescence staining was analyzed using a Becton-Dickinson FacScan.

SMC Adhesion Assay
Cell adhesion was assessed using SMCs labeled with BCECF (Sigma), as described previously.33 39 Briefly, 5x104 BCECF-labeled cells were allowed to adhere to matrix-coated wells for 20 minutes at 37°C. Unbound cells were removed by gentle washing. Bound fluorescence was measured by a Fluorescence Concentrator Analyzer (IDEXX). Bound fluorescence from adhesion to BSA-coated wells served as control for background adhesion. Net fluorescence was determined by subtraction of background and expressed as a percentage of the total fluorescence yielded from 5x104 labeled SMCs minus background fluorescence. For inhibition experiments, mAb BHA2.1 was used at 10 to 20 µg/mL, a concentration based on prior titering to inhibit adhesion of HT1080 cells to collagen.33

SMC Elongation Assay
SMC shape change after seeding was evaluated using a Boyden-type microchemotaxis chamber (Neuroprobe, Cabot John), as previously described.28 Polycarbonate filters (Nucleopore) were precoated overnight at 4°C with 10 µg/mL of either fibronectin or collagen type I in PBS. Control or FGF-2–treated SMCs were washed in PBS and trypsinized, and 2000 SMCs (40 000 cells/mL in DME with 1% BSA) were placed in the upper well. The lower well was filled with the same medium with or without FGF-2. The chamber was incubated at 37°C for 2 hours, and SMCs adherent to the upper surface of the filter were fixed in methanol and stained with hematoxylin. Prior experiments demonstrated that cell migration to the undersurface of the membrane was negligible (<1 cell/HPF) under these conditions for both control and FGF-2–treated SMCs. The lengths of the major and minor axes transecting the nucleus were measured from digitized calibrated images, using morphometry software (Jandel Scientific).

SMC Migration Assay
Bulk migration was measured using a microchemotaxis chamber, as previously described.25 40 Control or FGF-2–treated SMCs were washed in PBS and trypsinized, and 25 000 cells (500 000 cells/mL in DME with 1% BSA) were added to the upper well of the microchemotaxis chamber. Polycarbonate filters with 10-µm pores were precoated with fibronectin or collagen type I as described above. The lower well of the chamber was filled with DME with 1% BSA with or without growth factor. Migration was allowed to proceed for 6 hours at 37°C under 5% CO2. Cells remaining on the upper surface of the filters were mechanically removed, and then filters were fixed in methanol and stained with Harris's hematoxylin. The number of cells that had migrated to the lower surface was determined by counting under high-power microscopy (x40 objective). All conditions for a given experiment were performed in triplicate.

Time-Lapse Video Microscopy
SMC migration speed was evaluated using a digital time-lapse video recording system. Cells incubated with or without FGF-2 were seeded onto culture dishes coated with matrix proteins at 10 µg/mL. Migration was monitored with an inverted microscope (Diaphot 300, Nikon) using a x10 objective and a halogen light source. A CCD video camera (C72, Dage-MTI) attached to the microscope was used to generate video images, which were digitally acquired over a 5- to 8-hour recording period using a Silicon Graphics Indy workstation and custom-written time-lapse software. Ambient temperature was maintained at 37°C by mounting the culture dish in a Styrofoam sleeve with transparent heating elements (MINCO Products, Inc) placed above and below the dish. Cells were incubated in bicarbonate-reduced medium (M199 with Hanks' salts and 25 mmol/L HEPES, GIBCO/BRL) to maintain physiological pH in room air. Migration was measured from digital images by tracking the location of cell centroids at hourly intervals. Migration speed was determined as the sum of hourly distances divided by the total time, as described previously.41

Statistics
Data are expressed as mean±SD. Comparisons were made by t test or ANOVA with Scheffé's post hoc test. Statistical significance was set at P<.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
FGF-2 Increases Expression of ß1 Integrins in SMCs
To evaluate the effect of FGF-2 on ß1 integrin expression in human SMCs, early confluent cells were incubated in starvation medium (M199, 1% FBS) overnight and stimulated with FGF-2 (0 to 25 ng/mL) for 48 hours. Expression of ß1 integrins was examined by performing flow cytometry. As shown in Fig 1Down, the total ß1 integrin cell surface pool, detected using the pan-ß1 integrin mAb HB1.1, was greater in SMCs treated with 25 ng/mL FGF-2 than in untreated control SMCs. However, the effect of FGF-2 on expression of individual {alpha}ß heterodimers differed. FGF-2 produced either no effect or a slight reduction in {alpha}1ß1 integrin level, whereas it increased the expression of {alpha}2ß1, {alpha}3ß1, and {alpha}5ß1 integrins. Expression of these integrins in cells exposed to 10 ng/mL FGF-2 was intermediate, indicating that the effect was dose dependent (data not shown). {alpha}4ß1 integrin was not detected in the cultured human SMCs, similar to the results of Skinner et al.25 Expression of the {alpha}v integrin subunit was detected and was increased by FGF-2 treatment (data not shown).



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  		Figure 1. A, Expression of ß1 integrins in human SMCs cultured in the presence or absence of FGF-2. SMCs were incubated with specific anti-integrin antibodies for 45 minutes, labeled with FITC-conjugated anti-mouse IgG, and analyzed by flow cytometry. Anti-integrin antibodies used were as follows: HB1.1 (anti-ß1), TS2/7 (anti-{alpha}1ß1), BHA2.1 (anti-{alpha}2ß1), P1B5 (anti-{alpha}3ß1), and HA5 (anti-{alpha}5ß1). P3 was used as an isotype-matched control IgG, and the corresponding fluorescence signal appears on the left of each histogram. Of the other two tracings, the thin and the thick lines depict the specific antibody-bound signal from SMCs incubated for 48 hours with vehicle or 25 ng/mL FGF-2, respectively. B, Time course of FGF-2–mediated expression of {alpha}2ß1 integrin by SMCs. Cells were cultured with 25 ng/mL FGF-2 for the designated length of time and harvested simultaneously. Integrin expression was assessed by flow cytometry using mAb BHA2.1, and the net mean fluorescence index (MFI) was determined. Graph is representative of two separate experiments. C, Effect of ECM substrate on FGF-2–mediated expression of {alpha}2ß1 integrin. Cells were seeded onto culture dishes coated with fibronectin, laminin, or type I collagen, and FGF-2 (25 ng/mL) was simultaneously added. After 48 hours, integrin expression was evaluated by flow cytometry. The absolute values for the MFI are plotted. Two further experiments showed the same findings.

The quantitatively greatest change in expression induced by FGF-2 was observed for {alpha}2ß1 integrin; thus, this response was characterized further. The kinetics of the increase in {alpha}2ß1 integrin was evaluated by harvesting SMCs at various times after the addition of 25 ng/mL FGF-2. As shown in Fig 1BUp, increased expression was detected at 12 hours and was maximal by 35 hours.

{alpha}2ß1 integrin is known to bind collagens and laminin.19 To determine if the ability of FGF-2 to increase {alpha}2ß1 integrin surface expression was dependent on specific interaction with the ECM, SMCs were seeded onto dishes coated with either fibronectin, laminin, or type I collagen and stimulated for 48 hours with FGF-2. The relative increase in {alpha}2ß1 integrin expression induced by FGF-2 was not different for the three substrates: 3.7±0.7-fold for cells on fibronectin, 2.2±0.5-fold for cells on laminin, and 2.4±1.3-fold for cells on type I collagen (P=NS). However, in absolute terms, SMCs seeded on collagen expressed more {alpha}2ß1 integrin under both basal and FGF-2–stimulated conditions than did cells seeded on fibronectin or laminin (P<.05) (Fig 1CUp).

FGF-2 Increases SMC Elongation but Not Adhesion to Type I Collagen via {alpha}2ß1 Integrin
As shown in Fig 2Down, neither acute exposure to FGF-2 (25 ng/mL) nor 48 hours of treatment with FGF-2 at this dose had a significant effect on SMC adhesion to type I collagen. This was interesting in light of the observation that FGF-2 increased surface expression of the collagen-binding {alpha}2ß1 integrin. Of note, however, addition of mAb BHA2.1, which blocks {alpha}2ß1 integrin function,33 had no detectable effect on adhesion of SMCs to collagen in either untreated or FGF-2–treated SMCs (Fig 2Down).



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  		Figure 2. Effect of FGF-2 on SMC adhesion to type I collagen. Untreated SMCs and SMCs exposed acutely or for 48 hours to 25 ng/mL FGF-2 were allowed to adhere for 20 minutes in the presence of either control IgG (P3, 10 µg/mL) or mAb BHA2.1 (10 µg/mL). The proportion of adherent cells was determined as described in "Materials and Methods" and was not significantly different among the various conditions.

The effect of FGF-2 on the morphology of SMCs was assessed first by evaluating changes in cell shape over 48 hours. As shown in Fig 3Down, SMCs incubated with 25 ng/mL FGF-2 acquired a distinct spindle-like shape that was evident after 24 hours of treatment and most prominent after 48 hours. This shift to a spindle-like morphology was observed in SMCs growing on collagen type I, fibronectin, or laminin. To quantitatively examine the effect of FGF-2 on SMC elongation, SMCs were treated with FGF-2 (25 ng/mL) for 48 hours and then trypsinized and allowed to spread on matrix-coated filters for 2 hours. The length and width of 80 SMCs were determined for each condition, and data were obtained in triplicate. As shown in Fig 3CDown and 3DDown, the ratio of length to width was significantly higher in SMCs treated with FGF-2, indicating that FGF-2 promoted elongation of SMCs. This effect of FGF-2 was observed for cells seeded on collagen (P<.05) and fibronectin (P<.01). mAb BHA2.1 inhibited the FGF-2–mediated increase in elongation of SMCs on collagen (P<.05) but had no effect on elongation on fibronectin (Fig 3Down). Further evidence for the role of {alpha}2ß1 integrin in mediating shape changes came from visual assessment of the SMCs seeded onto matrix. FGF-2–treated SMCs seeded on collagen with mAb BHA2.1 displayed atypical thin cytoplasmic processes in an arborized pattern, suggesting a failed attempt at reshaping the cell (data not shown).



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  		Figure 3. Effect of FGF-2 on SMC morphology. A and B, Phase-contrast photomicrographs of control cells (A) and cells treated for 48 hours with 25 ng/mL FGF-2 (B). C and D, Ratio of length to width 2 hours after seeding onto type I collagen (C) or fibronectin (D). SMCs were seeded in the presence of either control IgG (P3, 10 µg/mL) or mAb BHA2.1 (10 µg/mL), and measurements were taken from 80 cells for each condition. Data represent mean of triplicate values.

FGF-2 Disassembles the Actin Stress Fiber Cytoskeleton
Actin microfilaments are a major component of the cytoskeleton and are known to interact with integrins on the inner face of the cell membrane. To determine if FGF-2 influenced cytoskeletal organization, and hence its relationship with integrins in focal adhesions, SMCs were fixed and immunostained for smooth muscle {alpha}-actin. As shown in Fig 4Down, there was a gradual loss of smooth muscle {alpha}-actin microfilament bundles traversing the cell body. This was evident by 24 hours and complete in almost all SMCs after 48 to 72 hours. The disassembly of smooth muscle {alpha}-actin–containing stress fibers was seen in cells growing on either collagen or fibronectin. Furthermore, the response did not require {alpha}2ß1 integrin function, as mAb BHA2.1 failed to prevent actin disassembly. Similar results were seen when F-actin was visualized using FITC-phalloidin (data not shown).



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  		Figure 4. Fluorescence micrographs of SMCs cultured on either type I collagen (A through D) or fibronectin (E through H) and immunostained for smooth muscle {alpha}-actin. A and E, Untreated SMCs. B and F, SMCs incubated with 25 ng/mL FGF-2 for 24 hours. C and G, SMCs incubated with 25 ng/mL FGF-2 for 48 hours in the presence of control IgG (P3, 10 µg/mL). D and H, SMCs incubated with 25 ng/mL FGF-2 for 48 hours in the presence of mAb BHA2.1 (20 µg/mL).

FGF-2 Is Not Chemotactic for Human SMCs but Enhances Migration Through Type I Collagen–Coated Membranes and Increases Migration Speed on Type I Collagen
Having identified that FGF-2 alters ß1 integrin expression, SMC shape, and the actin cytoskeleton, we determined whether SMCs incubated with FGF-2 would also manifest altered migratory properties. Control SMCs and SMCs treated for 48 hours with 25 ng/mL FGF-2 were seeded onto ECM-coated porous polycarbonate filters, and migration to the lower surface was evaluated after 6 hours. For these experiments, FGF-2 (0 to 25 ng/mL) was placed in the lower chamber. As illustrated in Fig 5Down, several observations were made. First, there was no evidence for chemotaxis of human SMCs toward FGF-2; ie, for a given set of experimental conditions, the number of cells that migrated to the undersurface of the membrane when FGF-2 was in the lower well was not different than when vehicle alone was in the lower chamber. This lack of chemotaxis to FGF-2 was observed with SMCs seeded on membranes precoated with fibronectin or type I collagen. PDGF-BB (10 µg/mL) served as a positive control for chemotaxis and produced SMC migration when placed in the bottom but not upper well of the chamber (eg, see Fig 8Down). FGF-2 in the upper well also had no effect on migration. A second observation was that SMCs pretreated with FGF-2 migrated through collagen-coated filters in significantly greater numbers than did untreated control SMCs. The number of FGF-treated SMCs that migrated to the lower surface, in the absence of growth factor in the lower chamber, was 3.1±0.8-fold greater than control SMCs (P<.001, n=5 experiments). Similar results were obtained when FGF-2 (1 to 25 ng/mL) was present in the bottom well of the chamber. The third observation was that the FGF-2–induced augmentation of migration was more prominent for SMCs on type I collagen than on fibronectin. Migration through fibronectin-coated membranes was increased 1.8±0.2-fold by FGF-2 pretreatment (P<.05, n=3 experiments), but this increase was significantly less than that for SMCs migrating on collagen-coated membranes (P<.05).



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  		Figure 5. Effects of FGF-2 on SMC migration through type I collagen-coated (top) and fibronectin-coated (bottom) polycarbonate membranes. Before the migration assay, SMCs were incubated for 48 hours with or without 25 ng/mL FGF-2. The migration assay was performed in a chemotaxis chamber with designated concentrations of FGF-2 in the lower well. Results are representative of three experiments.



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  		Figure 8. Effect of FGF-2 on PDGF-induced chemotaxis of SMCs on type I collagen. Untreated SMCs and cells treated for 48 hours with 25 ng/mL FGF-2 were placed in the upper well of the migration chamber together with either control IgG (P3, 10 µg/mL) or mAb BHA2.1 (10 µg/mL). PDGF-BB (0 to 30 ng/mL) was in the lower well, and the number of cells that migrated to the lower membrane surface was counted after 6 hours. *P<.01 vs untreated SMCs incubated with control IgG, untreated SMCs incubated with mAb BHA2.1, and FGF-2 treated SMCs incubated with mAb BHA2.1; {dagger}P<.01 vs untreated SMCs incubated with mAb BHA2.1 and FGF-2–treated SMCs incubated with mAb BHA2.1.

To directly measure the speed of SMC migration, SMC movement was monitored by digital time-lapse video microscopy. The results of image quantification including representative migration paths are shown in Fig 6Down. SMCs preincubated for 48 hours with FGF-2 migrated on collagen at speeds that, on average, were 2.1-fold higher than those of untreated SMCs (19.4±5.2 versus 9.3±3.8 µm/h, P<.05). FGF-2–treated SMCs also migrated on fibronectin faster than did untreated SMCs, although the relative increase was only 1.3-fold (11.2±0.4 versus 8.4±0.8 µm/h, P<.05). These findings thus corroborate those of the chemotaxis chamber assay and implicate augmented crawling speed, likely together with cell shape changes, as components of the enhanced migratory response of SMCs to FGF-2.



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  		Figure 6. Effect of FGF-2 on SMC migration path and migration speed on type I collagen and fibronectin. SMCs treated with FGF-2 (25 ng/mL) or vehicle for 48 hours were monitored by digital time-lapse video microscopy. Migration paths (A through D) depict hourly translocations of eight cells per experimental condition, over a total of 5 hours. Migration speed over the monitoring period (E) was determined for each cell, and the results are the mean of three separate experiments on each of collagen and fibronectin. *P<.05 vs untreated SMCs on collagen; {dagger}P<.01 vs untreated SMCs on fibronectin.

FGF-2–Mediated Enhancement of SMC Migration on Collagen Is Inhibited by Anti–{alpha}2ß1 Integrin Antibody
Although inhibition of {alpha}2ß1 integrin had no significant effect on SMC adhesion to collagen, {alpha}2ß1 integrin function was important for FGF-2–stimulated morphological changes. Therefore, the role of {alpha}2ß1 integrin in mediating the FGF-2–induced increase in SMC migration was evaluated using the blocking mAb BHA2.1. As shown in Fig 7Down, the addition of 10 µg/mL mAb BHA2.1 to the upper well prevented the FGF-2–mediated increase in migration through collagen-coated membrane (P<.01), whereas the isotype-matched control antibody had no effect. The same dose of mAb BHA2.1 had no effect on basal or FGF-2–enhanced migration through fibronectin-coated membranes.



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  		Figure 7. Effect of mAb BHA2.1 (anti–{alpha}2ß1 integrin) on FGF-2–mediated SMC migration on collagen-coated (top) and fibronectin-coated (bottom) polycarbonate membranes. Untreated SMCs and cells treated for 48 hours with 25 ng/mL FGF-2 were placed in the upper well of the migration chamber together with either control IgG (P3, 10 µg/mL) or mAb BHA2.1 (10 µg/mL). Cells that migrated to the lower membrane surface were counted after 6 hours. *P<.01 vs untreated SMCs; {dagger}P<.01 vs FGF-2–treated SMCs in the presence of control IgG.

FGF-2 Enhances Chemotaxis to PDGF via {alpha}2ß1 Integrin
Both FGF-216 and PDGF17 have been found to regulate SMC migration in vivo. Therefore, we next determined if exposure of SMCs to exogenous FGF-2 altered the chemotactic response to PDGF. As illustrated in Fig 8Up, PDGF induced dose-dependent chemotaxis through collagen-coated filters, as has been previously described.3 25 When cells were pretreated for 48 hours with 25 ng/mL FGF-2, the migratory response to 1 and 10 ng/mL PDGF was significantly increased (P<.01). At these doses of PDGF, migration of SMCs not preincubated with FGF-2 was unaffected by the anti–{alpha}2ß1 integrin mAb BHA2.1. However, mAb BHA2.1 blocked the incremental increase provided by FGF-2 treatment (P<.01). Thus, FGF-2 treatment potentiated the chemotactic response to PDGF on collagen, by effectively supplementing an {alpha}2ß1 integrin–independent process with an {alpha}2ß1 integrin–dependent process. The response to high-dose PDGF (30 ng/mL) differed in that FGF-2 did not augment the chemotactic response in this setting. Furthermore, migration of SMCs not preincubated with FGF-2 to high-dose PDGF was partially inhibited by mAb BHA2.1 (P<.01), indicating that an {alpha}2ß1-mediated mechanism already existed.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
A role for FGF-2 in vascular SMC migration has been demonstrated in cell culture42 and in vivo.16 However, the mechanism by which FGF-2 promotes SMC migration is not well defined. A number of studies,3,17,18 including the present report, indicate that FGF-2 provides chemotactic stimulus to SMCs; no, or at best a weak induction of directed migration is therefore unlikely to be a mechanism of action. Induction of endogenous FGF-2 has been implicated in migration to PDGF,42 and Bilato et al18 recently found that FGF-2 was necessary to activate calcium/calmodulin-dependent protein kinase II, which is required for PDGF-mediated chemotaxis. The data presented here provide evidence for a very different mechanism by which FGF-2 promotes SMC migration, namely, by altering the nature of the interaction between SMCs and the ECM. The promigratory effect of FGF-2 was apparent after sustained exposure and was chemokinetic in nature, since a gradient of FGF-2 was not required for the effect. The effect was especially pronounced for SMCs migrating on a substrate of type I collagen. As well, exposure to FGF-2 served to potentiate directed migration of SMCs (chemotaxis) to PDGF-BB on this substrate. This enhanced motility was mediated by {alpha}2ß1 integrin, the expression of which was increased by over 2-fold by FGF-2. Finally, FGF-2–induced motility was also associated with postintegrin alterations in the cytoskeleton, including disassembly of actin stress fibers.

The regulation of cell migration by growth factors is often considered and studied in a time frame of a few hours. SMC chemotaxis, for example, typically is manifested in vitro within 4 to 6 hours.12 18 25 However, SMC migration within the arterial wall follows a more prolonged time course. Indeed, the rationale for examining the consequences of a more prolonged exposure to FGF-2 comes from in vivo studies. After balloon injury to the rat carotid artery, SMC proliferation begins after 24 hours,43 and SMC migration is detectable by 3 days.16 Both responses have been shown to be regulated by FGF-2,15 16 which implies that in this setting SMCs may be exposed to FGF-2 over a matter of days. The fact that FGF-2 binds with low affinity to the ECM is also compatible with sustained delivery of FGF-2 to the SMC, during conditions of active remodeling.44

FGF-2 increased the surface expression of several ß1 integrins, including {alpha}2ß1, {alpha}3ß1, and {alpha}5ß1 integrins. However, the effect was not generalized, since {alpha}1ß1 integrin expression was not increased. The quantitatively greatest increase in expression was seen for {alpha}2ß1 integrin. {alpha}2ß1 integrin is capable of binding collagen types I and IV as well as laminin,19 and there is evidence that expression of {alpha}2ß1 integrin is dependent on SMC phenotype. Specifically, expression of {alpha}2ß1 integrin was not observed in SMCs within the aortic media but was induced after growth in culture, suggesting that {alpha}2ß1 integrin expression is a feature of an "activated" SMC phenotype.25 Although one study did not detect {alpha}2ß1 integrin after injury to the rat carotid artery,45 neoexpression of {alpha}2ß1 integrin has been identified in human SMC tumors.46 The effect of growth factors on {alpha}2ß1 integrin expression by SMCs has not previously been reported, although it is of interest that FGF-2 has been shown to increase {alpha}2ß1 integrin expression in microvascular endothelial cells.31

It was noteworthy that the FGF-2–mediated increase in the expression of {alpha}2ß1 integrin was not associated with increased adhesion to collagen but was associated with cell elongation and increased motility on this substrate. Under basal or FGF-2–stimulated conditions, adhesion to collagen was not inhibited by mAb BHA2.1, an anti-{alpha}2ß1 antibody previously shown to block adhesion to type I collagen.33 Similar results were obtained using another {alpha}2ß1 integrin mAb (data not shown). Skinner et al25 also did not observe a role for {alpha}2ß1 integrin on SMC attachment to type I collagen. On the other hand, Lee et al24 observed a 31% decrease in adhesion of human venous SMCs to type I collagen by an anti–{alpha}2ß1 integrin antibody, implying that the {alpha}2ß1 receptor is capable of at least partly mediating adhesion of SMCs to type I collagen. Other receptors besides {alpha}2ß1 integrin may be involved in binding to type I collagen, including {alpha}1ß1 and {alpha}3ß1 integrins,19 38 and cooperativity has been demonstrated between {alpha}1ß1 and {alpha}2ß1 integrins.24 Therefore, it is conceivable that inhibition of {alpha}2ß1 integrin alone may be insufficient to impair adhesion and that inhibition of multiple receptors is necessary before a functional consequence on adhesion is manifested. Even so, it is of interest that in the face of a 2.4-fold increase in {alpha}2ß1 integrin expression, FGF-2 did not increase SMC adhesion to collagen whatsoever. FGF-2 did, however, promote SMC elongation and migration on collagen in a manner that was dependent on the increase in {alpha}2ß1 integrin. The ability of the cell to modulate its shape and extend over its substrate is fundamental to locomotion and morphogenesis, and a role for {alpha}2ß1 integrin in these processes has been suggested in a few cell lines.47 48 For example, in a mammary epithelial cell line, inhibition of {alpha}2ß1 integrin significantly reduced cell spreading and migration speed as well as the ability of these cells to wrap around collagen fibers.47 The present studies support a similar role for {alpha}2ß1 integrin in human SMCs, specifically in the setting of sustained stimulation by FGF-2.

The ability of FGF-2 to induce integrin-mediated changes in cell shape without augmenting adhesivity may be precisely the scenario required to optimize cell locomotion. Theoretical analysis has suggested that maximum migration speed will occur at an intermediate adhesiveness,49 and subsequent experimental evaluation of SMC migration on fibronectin and type IV collagen has supported this concept.5 Furthermore, using an anti–ß1 integrin antibody that augments the binding affinity of ß1 integrins, Seki et al10 and Koyama et al28 observed enhanced adhesion of SMCs to ECM substrates but decreased SMC migration to PDGF on Matrigel. Thus, if integrin-mediated adhesion is excessive, the cell may be effectively "frozen" in location. Therefore, it appears that utilization of {alpha}2ß1 integrin for shape change (elongation) but not for adhesion is a coordinated response to FGF-2 designed to facilitate motility.

What is the potential mechanism by which a FGF-2–mediated increase in {alpha}2ß1 integrin increases SMC elongation and migration but does not increase adhesion to collagen? Presumably, there are other effects of FGF-2 that effectively dissociate the expression of {alpha}2ß1 integrin from its adhesive function. In this regard, it was noteworthy that incubation of SMCs with FGF-2 led to disassembly of the actin stress fiber network, evidenced by staining for smooth muscle {alpha}-actin and F-actin. It is well established that integrins associate with the actin-based cytoskeleton,19 including the actin stress fibers that traverse the cell. These actin microfilament bundles are structurally linked to integrins in focal adhesion contacts, and their presence is felt to be an important feature of the stationary cell phenotype50 ; eg, actin stress fibers are prominent in corneal fibroblasts attached and flattened over a planar substrate but are absent in fibroblasts migrating in three-dimensional collagen gels51 or in vivo.52 In vivo, actin stress fibers are present in endothelial cells subjected to high shear stress,53 where strong attachment to the underlying ECM is required. Given the association between actin stress fiber network and strong surface adhesion, disassembly of actin stress fibers may be one means by which the cell liberates itself for movement. We speculate that disassembly of actin stress fibers in SMCs by FGF-2 minimizes or even prevents increased adhesivity in the setting of increased integrin expression, by disrupting the intracellular structural network required for stable attachment. Other possible mechanisms underlying the differential response include selective activation of intracellular signaling cascades that mediate migration but not adhesion or selective modulation of integrin function or activation state through conformational changes in specific receptor domains.10 54

Disassembly of actin stress fibers by FGF-2 was observed in cells cultured on collagen as well as on fibronectin, and the process did not require {alpha}2ß1 integrin function. Therefore, this particular effect of FGF-2 does not appear to be specific for a given ECM environment. However, actin disassembly could reflect an overall reduced level of interaction between the cell and the ECM. It has recently been shown that cellular interaction with ECM is necessary for stress fiber attachment to focal adhesion contacts.55 Furthermore, we have recently determined that FGF-2 induces expression of matrix metalloproteinase-1 (collagenase) in human SMCs.56 This raises the possibility that local degradation of collagen, induced by FGF-2, may reduce the available ECM ligands for integrins, with disorganization of actin stress fibers as a downstream consequence. This remains speculative, and whether a similar degradation of fibronectin occurs in response to FGF-2 is not known. However, the interplay between ECM degradation and integrin function has recently been highlighted by the observation that signaling via {alpha}2ß1 integrin induces matrix metalloproteinase-1 expression.57 Other potentially relevant mediators of stress fiber integrity include members of the Rho family of GTP-binding proteins.58 59 Some growth factors, such as PDGF and epidermal growth factor, induce stress fiber assembly via functional Rho.58 The effect of FGF-2 on Rho function is unexplored.

Both FGF-216 and PDGF17 have been shown to mediate SMC migration following balloon injury to the rat carotid artery. Previous studies have indicated that PDGF-mediated release of FGF-2 plays a role in chemotaxis to PDGF.18 42 However, during vigorous vascular remodeling, eg, after mechanical injury, FGF-2 may also be liberated from dead or injured cells as well as from ECM depot sites. The present findings of augmented chemotaxis to PDGF following FGF-2 exposure may be particularly relevant to such circumstances. By endowing the SMC with the capacity to migrate faster on collagen, FGF-2 induced a functional augmentation of PDGF-induced migration. Interestingly, this cooperative effect was dose dependent and apparent when SMCs were exposed to 1 to 10 ng/mL PDGF but not to high-dose PDGF (30 ng/mL). In the absence of FGF-2, SMC migration to the lower concentrations of PDGF was largely independent of {alpha}2ß1 integrin function. However, preincubation of SMCs with FGF-2 effectively supplemented an {alpha}2ß1 integrin–independent process with an {alpha}2ß1 integrin–dependent process. In contrast, migration to high-dose PDGF in the absence of FGF-2 was partly dependent on {alpha}2ß1 integrin, consistent with that reported by Skinner et al.25 Under these circumstances, FGF-2 did not provide an additional effect, likely because the {alpha}2ß1-mediated component of migration had already been recruited. Other effects of PDGF relevant to cell motility have also been shown to depend on PDGF concentration. In particular, the effects of low-dose PDGF (5 ng/mL) on membrane ruffling, actin organization, and tyrosine phosphorylation of focal adhesion kinase and phosphatidylinositol 3-kinase were found to differ from those of high-dose (30 ng/mL) PDGF.60 Furthermore, we have observed that high-dose PDGF induced a small increase in {alpha}2ß1 integrin expression, but this was not detectable at lower concentrations (data not shown).

Although the promigratory effect of FGF-2 was greatest for SMCs on collagen, an enhancement in migration was also seen with SMCs on fibronectin. The mechanism for this response is not presently defined; however, both disassembly of actin stress fibers and cell elongation occurred on fibronectin and may be contributing factors. Furthermore, integrins other than {alpha}2ß1 may be relevant. FGF-2 effected a modest increase in expression of {alpha}5ß1 integrin and the {alpha}v integrin subunit, both of which are capable of interacting with fibronectin.19 A role for {alpha}vß3 integrin in SMC migration and vascular lesion formation has been previously suggested.61 62

In summary, we have determined that FGF-2 increases the speed with which SMCs migrate and have identified two associated subcellular processes: upregulation of {alpha}2ß1 integrin and disassembly of the actin stress fiber network. The former process is necessary for the increase in migration on collagen; the latter may underlie the differential utilization of {alpha}2ß1 integrin for migration but not for adhesion. These coordinated changes in the ß1 integrin–cytoskeletal axis may be a critical means by which FGF-2, and possibly other growth factors, regulate SMC migration in vivo. Furthermore, they provide a unique basis of cooperativity between PDGF and FGF-2 in the control of SMC migration.


*    Selected Abbreviations and Acronyms
 
DME = Dulbecco's modified minimum essential medium
ECM = extracellular matrix
FGF = fibroblast growth factor
HPF = high-power field
mAb = monoclonal antibody
PDGF = platelet-derived growth factor
SMC = smooth muscle cell


*    Acknowledgments
 
This study was supported by grants from the Heart and Stroke Foundation of Canada (Drs Pickering, Chan, and Ellis), the Medical Research Council of Canada (Drs Pickering and Chan), and the Natural Sciences and Engineering Research Council of Canada (Dr Chan). Drs Pickering and Chan are Research Scholars of the Medical Research Council of Canada.

Received September 18, 1996; accepted February 20, 1997.


*    References
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