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Cardiovascular Research 1998 39(2):475-484; doi:10.1016/S0008-6363(98)00079-0
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen

L. Xie, G.F. Clunn, J.S. Lymn and A.D. Hughes*

Department of Clinical Pharmacology, Imperial College School of Medicine, St Mary's Hospital, London W2 1NY, UK

* Corresponding author. Tel.: +44-171-8866562; Fax: +44-171-8866145; E-mail: a.hughes@ic.ac.uk

Received 12 May 1997; accepted 19 February 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 
Objective: Fibrinogen is an independent risk factor for cardiovascular disease. This study has investigated the role of intracellular Ca2+ ([Ca2+]i) and tyrosine phosphorylation in the attachment of human and rat-derived cultured vascular smooth muscle cells to fibrinogen. Methods: Cells were cultured from human saphenous vein segments (HVSMC) and from an established rat aortic cell line (A7r5). [Ca2+]i was measured using fura-2 and adhesion was studied using pre-coated 96 well polystyrene plates. Results: Fibrinogen increased [Ca2+]i in both cell types. In A7r5 cells fibrinogen-induced increases in [Ca2+]i were partially inhibited by a peptide containing the amino acid sequence Arg-Gly-Asp (RGD) which interferes with binding to integrins. In contrast RGD increased [Ca2+]i in HVSMC, but did not inhibit responses to fibrinogen. Ni2+, an inorganic calcium channel blocker largely abolished the rise in [Ca2+]i, but blockers of voltage-operated calcium channels failed to affect [Ca2+]i responses to fibrinogen in either cell type. Genistein, an inhibitor of tyrosine kinase inhibited fibrinogen-induced rises in [Ca2+]I, while daidzein, an inactive analogue, was without effect. Adhesion of cells to fibrinogen was concentration- and time-dependent. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both cell types. Adhesion of cell to fibrinogen was inhibited by chelation of [Ca2+]i with BAPTA-AM, inhibition of Ca2+ entry by Ni2+, and inhibition of tyrosine kinases by genistein, but heparin had no effect on adhesion. Conclusions: Vascular smooth muscle cells attach to fibrinogen in part through RGD-type interactions. Activation of tyrosine kinase(s) and a subsequent rise in [Ca2+]i appear to be important signals mediating the response to fibrinogen.

KEYWORDS Fibrinogen; Vascular smooth muscle; Intracellular calcium; Tyrosine kinase; Adhesion; Integrin; Human; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 References
 
Fibrinogen is a ~340 kDa glycoprotein present in human plasma at concentrations around 3 mg/ml [1]. Elevated plasma fibrinogen levels have been demonstrated to be a powerful independent risk factor for cardiovascular disease, an effect often attributed to changes in blood viscosity or thrombogenesis [2–5]. However recently, fibrinogen has also been shown to have a direct influence on cells in the vascular wall [6–8], and there is considerable evidence that fibrinogen can penetrate the arterial wall particularly following injury [9–11]or in areas of perturbed intimal shear stress [12, 13].

The directional migration of arterial smooth muscle cells from the internal elastic lamina into the intimal layer is thought to play a key role in the response to injury and in the pathogenesis of atherosclerosis and restenosis [14, 15]. Several defined growth factors, cytokines and extracellular matrix components which are released at the sites of lesion have been implicated in the regulation of vascular smooth muscle cells and other lesion-associated cells [14]. Extracellular matrix components in the vascular wall may play an active signalling role as well as acting as a scaffold in the process of smooth muscle cell adhesion and migration [16]. Morphological and immunohistochemical studies have demonstrated the presence of fibrinogen and fibrin in atherosclerotic lesions and the transformation of fibrinogen to fibrin within the arterial wall accompanies the progression of atherosclerosis [9, 11, 17].

Fibrinogen has been reported to bind to and activate platelets via integrin {alpha}IIbβ3 [18, 19](also termed platelet glycoprotein IIb/IIIa). However, although a number of integrins have been described, there are no reports indicating the presence of integrin {alpha}IIbβ3 on vascular smooth muscle cells, and the site responsible for binding fibrinogen has not been clearly defined. Many integrins interact with their ligand, at least in part, by binding to the sequence Arg-Gly-Asp (RGD) [16]which is present in a number of matrix proteins including fibrinogen, fibronectin, thrombospondin, vitronectin, laminin and collagen (type 1). Activation of integrins by proteins containing RGD sequences is associated with integrin clustering which appears required for full signal transduction and can be blocked by short peptides containing the RGD motif.

In platelets fibrinogen has been reported to evoke a number of intracellular signals including tyrosine phosphorylation of several proteins [20–22]with subsequent phosphorylation of other proteins such as pp125FAK (FAK) being dependent on a rise in intracellular Ca2+ concentration ([Ca2+]i), activation of protein kinase C (PKC) and cytoskeletal organization[23]. Similar effects of integrin activation have also been reported in some other non-muscle cell types (reviewed in [24–26]). The nature of the intracellular signal transduction pathways activated by fibrinogen in vascular smooth muscle is unknown.

We have postulated that integrins on vascular smooth muscle cells may bind to an RGD sequence in fibrinogen, and that, as has been described in platelets, a rise in [Ca2+]i in conjunction with activation of tyrosine phosphorylation may mediate some of the effects of fibrinogen. A rise in [Ca2+]i serves as a second messenger for many cellular processes in vascular smooth muscle cells and is necessary for excitation–contraction coupling and cell movement [27, 28]. Therefore in the present work, we investigated the adhesion of cultured A7r5 and human vascular smooth muscle cells derived from saphenous vein to fibrinogen and the importance of RGD interactions in this process. We also investigated the possible role of increased tyrosine phosphorylation and changes in [Ca2+]i as potential intracellular mediators of the action of fibrinogen.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
A rat arterial smooth muscle cell line (A7r5) [29]and human cultured saphenous vein smooth muscle cells (HVSMC) were used. The investigation conforms with the principles outlined in the Declaration of Helsinki. A7r5 cells were grown to confluence in 80 cm2 flasks containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), while HVSMC were grown from segments of human saphenous vein surplus to coronary artery bypass graft surgery, using an explant technique as described previously [30, 31]. Both cell types were routinely grown in DMEM buffered to pH 7.4 with 25 mM HEPES and supplemented with foetal calf serum (15% (v/v) for HVSMC, 10% for A7r5 cells), 4 mM L-alanyl-glutamine (Glutamax-I), penicillin (100 U/ml), streptomycin (100 µg/ml) and gentamicin (25 µg/ml). Cell cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37°C. HVSMC at passage 3 and A7r5 between passages 10 and 30 were employed for all experiments.

2.2 [Ca2+]i measurements
In order to measure [Ca2+]i, a flask containing ~106 cells was incubated with DMEM containing 5 µM fura-2AM at 37°C for 90 min [32]. After loading cells were washed with physiological saline (PSS) to remove excess dye, briefly trypsinized, centrifuged and resuspended in about 20 ml PSS comprising (mM): NaCl 127, KCl 5.9, MgCl2 1.2, CaCl2 1, glucose 14 and HEPES 10.6 adjusted to pH 7.4 with NaOH. All experiments were performed at 37°C. 2.5 ml aliquots of the cell suspensions were placed in a stirred quartz cuvette and illuminated by excitation wavelengths of 340 nm (F340) and 380 nm (F380) with emission measured at 510 nm using a Deltascan spectrofluorimeter (Photon Technology International, USA). Background fluorescence consisting of cell autofluorescence and light scatter (≤10% total fluorescence) were subtracted from readings and experiments were calibrated and [Ca2+]i concentration calculated as previously described [32]based on the determination of maximal (Rmax) and minimal (Rmin) fluorescence ratios and the ratio (β) between the maximal and minimal fluorescence at 380 nm in the presence of 2 mM EGTA and 1 mM Ca2+ respectively, using 50 µM digitonin to permeabilize the cells. As previously described [33], genistein caused small reductions in F340 and F380, and a small overall fall in ratio. We have previously reported that genistein does not alter the affinity of fura-2 for Ca2+, and readings were calibrated as previously described [33]. In experiments employing Ni2+, following permeabilization with digitonin, Ni2+ induced a marked quench of fluorescence at both wavelengths and it was therefore not possible to perform calibrations in these studies. Data from these experiments were therefore calibrated using values for Rmin, Rmax and β derived from other aliquots of the same cell suspension prepared at the same time. Application of Ni2+ prior to permeabilization induced no quenching of F340 or F380 indicating that there was no detectable influx of Ni2+ into cells in the absence of digitonin.

2.3 Adhesion
Adhesion assays were conducted in flat-bottomed 96 well polystyrene plates (611F96 from Bibby Sterilin Ltd) essentially as described by Adams and Lawler [34]. Plates were coated overnight at 4°C by incubation with Tris buffered saline (150 mM NaCl, 50 mM Tris.Cl, 2.5 mM CaCl2, pH 7.5) containing fibrinogen. After coating, wells were rinsed with Tris buffered saline and blocked with Tris buffered saline containing heat-denatured bovine serum albumin (BSA; 1 mg/ml) for 30 mins to prevent non-specific attachment. Cells were briefly trypsinized, centrifuged, and resuspended in DMEM containing 1 mg/ml BSA. 100 µl of the cell suspension (containing 104 cells) was added to each well and adhesion was allowed to proceed for 2 h (or time specified) at 37°C. Non-adherent cells were removed by washing and the attached cells were fixed in 3.7% formaldehyde, stained with 5% Giemsa then counted with a light microscope at 200x magnification. The average of 4 randomly selected fields was used as the representative count for an individual well. Experiments were carried out in quadruplicate.

2.4 Materials
Bovine serum albumin (Sigma, Dorset, UK) was denatured by heating to 90°C for 5 min prior to use. Stock solutions of amlodipine (a gift from Pfizer International, USA), BAPTA-AM (Calbiochem, Nottingham, UK), daidzein (Calbiochem, Nottingham, UK), fura-2AM (Molecular Probes, USA), genistein (Sigma, Dorset, UK) were made up in dimethylsulphoxide (DMSO) and the final concentration of DMSO used did not exceed 0.1%, and had no effect on responses. Fibrinogen (Calbiochem, Nottingham, UK), heparin (a gift from B. Molloy), Synthetic RGD peptide (H-Gly-Arg-Gly-Asp-Ser-Pro-OH) and control peptide (H-Gly-Arg-Gly-Glu-Ser-Pro-OH) (Novabiochem, UK), human recombinant platelet derived growth factor-BB homodimer (PDGF-BB) (GIBCOBRL), vasopressin (Sigma, Dorset, UK) and verapamil (Sigma, Dorset, UK) were made up in distilled H2O. Other chemicals were obtained from Sigma (Dorset, UK) and were freshly prepared prior to use.

2.5 Statistics
Data are means±SEM of n observations. Statistical comparisons were made using a unpaired Student's t-test: p<0.05 was considered statistically significant. Concentration-response data were analysed by fitting data to a hyperbolic function by non-linear regression analysis using a macro written in Excel (Microsoft, USA) and EC50, the concentration inducing 50% of the maximum response calculated.


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 References
 
3.1 Effect of fibrinogen on [Ca2+]i in vascular smooth muscle cells
Resting [Ca2+]i was 67±7 nM (n=32) and 219±15 nM (n=73) in HVSMC and A7r5 cells. Fibrinogen (0.05–3 mg/ml) increased [Ca2+]i in both HVSMC and A7r5 (Fig. 1). EC50 for HVSMC and A7r5 cells were 0.3 and 0.6 mg/ml respectively. The increase in [Ca2+]i induced by fibrinogen was 15% of that induced by vasopressin (100 nM) in A7r5 cells (vasopressin was without effect in HVSMC), but comparable to that induced by a near maximal concentration of PDGF-BB (10 ng/ml) in both cell types. Rises in [Ca2+]i in response to fibrinogen, were relatively delayed in comparison to those seen in response to vasopressin in A7r5 cells (Fig. 1B), occurring after a lag of 30–120 s and sometimes showed a biphasic character. In A7r5 cells the effect of fibrinogen on [Ca2+]i was inhibited by RGD peptide (0.1 mM); the peak rise in [Ca2+]i ({Delta}[Ca2+]i) being reduced by 48% (Fig. 2). The effect of fibrinogen was not affected by preincubation with control peptide (0.1 mM). In A7r5 cells RGD peptide had no effect on resting [Ca2+]i and had no significant effect on responses to vasopressin. In contrast in HVSMC, preincubation with RGD peptide had no significant effect on the rise in [Ca2+]i in response to fibrinogen (0.6 mg/ml) (Fig. 2). In HVSMC addition of RGD peptide (0.1 mM) alone consistently caused a small elevation in [Ca2+]i ({Delta}[Ca2+]i=30±15; n=7, p<0.05) unlike in A7r5 cells.


Figure 1
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  		Fig. 1 Effect of fibrinogen on [Ca2+]i in cultured human saphenous vein cells (HVSMC) and A7r5 cells. (A) Representative traces showing rises in [Ca2+]i in suspensions of HVSMC and A7r5 cells in response to fibrinogen. [Ca2+]i was determined on the basis of fluorescence using the dye fura-2 as described in Section 2. Fibrinogen (0.6 mg/ml) was added to the cell suspension for the period indicated by the bar. (B) Representative traces showing rises in [Ca2+]i in suspensions of A7r5 cells in response to fibrinogen (F) and vasopressin (V). Fibrinogen (0.6 mg/ml) or vasopressin (100 nM) was added to the cell suspension for the period indicated by the bar. (C) Concentration-response relationships for the rise in [Ca2+]i induced by fibrinogen in HVSMC ({circ}) and A7r5 cells (bullet). Data are expressed as {Delta}[Ca2+]i (nM), i.e. peak increase in stimulated [Ca2+]i less resting [Ca2+]i and are expressed as mean ± SEM of 3–11 separate experiments.

 

Figure 2
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  		Fig. 2 The effects of inhibitors on the rise in [Ca2+]i induced by fibrinogen and vasopressin in (A) HVSMC and (B) A7r5 cells. Cells were prepared as described in Section 2and loaded with fura-2. F=fibrinogen (0.6 mg/ml) + vehicle, Dz=Daidzein (50 µM), G=genistein (50 µM), R=RGD peptide (0.1 mM), E=control peptide (0.1 mM), V=vasopressin (100 nM). Data are expressed as {Delta}[Ca2+]i (nM), i.e. peak increase in stimulated [Ca2+]i less resting [Ca2+]i and are expressed as mean ± SEM of n separate experiments as shown below the column legends. *=p<0.05.

 
Activation of integrins has previously been reported to be linked to increased tyrosine phosphorylation of cellular proteins in some cell types. The possible role of tyrosine kinases in these responses of vascular smooth muscle cells was therefore examined. An inhibitor of tyrosine kinases, genistein (50 µM) inhibited the rise in [Ca2+]i in response to fibrinogen in both HVSMC and A7r5 cells. Genistein had no significant effect on the response to vasopressin (100nM) in A7r5 cells (Fig. 2) or angiotensin II (1 µM) in HVSMC (data not shown). Daidzein (50 µM), an inactive analogue of genistein failed to affect responses to fibrinogen in HVSMC (Fig. 2A). Although qualitatively similar, the control responses of HVSMC to fibrinogen in these latter studies tended to be larger than those seen in earlier studies. The reason for this is unclear, but may reflect differences between cells cultured from different individuals.

The source of the rise in [Ca2+]i in response to fibrinogen was examined using inhibitors of Ca2+ entry. Ni2+ (2mM), an inorganic calcium channel blocker reduced resting [Ca2+]i from 79±2 nM to 31±1 nM (n=4) and 151±30 to 45±4 nM (n=25) in HVSMC and A7r5 cells respectively. In the presence of Ni2+ fibrinogen (0.6 mg/ml) caused a reduced transient increase in [Ca2+]i, while the sustained component of the response seen in the absence of Ni2+ was greatly attenuated. Preincubation with 2 mM Ni2+ reduced peak {Delta}[Ca2+]i by 63% and 55% in HVSMC and A7r5 cells respectively (Fig. 3) and responses to vasopressin (100 nM) and PDGF-BB (10 ng/ml) were similarly inhibited by Ni2+ in A7r5 cells (Fig. 3B). Amlodipine (5 µM), an inhibitor of voltage-dependent calcium channels, failed to block the rise in [Ca2+]i in response to fibrinogen in either cell type (Fig. 3) and similar results were observed in both HVSMC and A7r5 cells using verapamil (10 µM; n=2 and 3 respectively), consistent with earlier studies which failed to demonstrate voltage-dependent calcium channels in either cell type under the conditions of cell culture used for these studies [32, 35]. Experiments were not conducted in the absence of extracellular Ca2+ as these conditions have been reported to inhibit matrix–integrin interactions [36, 37]and so effects on Ca2+ influx could not be differentiated from effects on ligand binding, However the effect of the intracellular Ca2+ chelator BAPTA-AM was examined.Preincubation with BAPTA-AM (100 µM) reduced resting [Ca2+]i in A7r5 from 162±15 nM (n=29) to 125±12 nM (n=17) although this fall was not statistically significant. Following pre-exposure to BAPTA-AM rises in [Ca2+]i in response to vasopressin, PDGF and fibrinogen were virtually abolished (Fig. 4).


Figure 3
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  		Fig. 3 Effect of calcium entry blockade on the increases in [Ca2+]i induced by stimulants in A) HVSMC and B) A7r5 cells. Cells were prepared as described in Section 2and loaded with fura-2. Fibrinogen (0.6 mg/ml F), platelet-derived growth factor-BB (10 ng/ml; BB) or vasopressin (V; 100 nM) were added 2 min after addition of Ni2+ (2 mM; Ni) or amlodipine (5 µM; Aml) to the cell suspension. Data are expressed as peak {Delta}[Ca2+]i (nM) and are means±SEM of n observations as indicated by the number in parenthesis below the column label. *=p<0.05.

 

Figure 4
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  		Fig. 4 The effect of BAPTA-AM on the increase in [Ca2+]i induced by agents in A7r5 cells. Cells were pre-incubated with BAPTA-AM (100 µM; BA) for 3 h prior to loading with fura-2AM (5 µM). Cell suspensions loaded with fura-2 were prepared as described in Section 2and the effect of fibrinogen (F; 0.6 mg.ml), platelet-derived growth factor BB (BB, 10 ng/ml) or vasopressin (V; 100 nM) examined in BAPTA-AM or vehicle (DMSO) pre-treated cells. Data are means ± SEM, n is shown in parenthesis below the columns. *=p<0.05.

 
3.2 Adhesion of vascular smooth muscle cells to fibrinogen
Fibrinogen (0.01–3 mg/ml) promoted the adhesion of HVSMC and A7r5 cells (Fig. 5A). The concentrations of fibrinogen in the coating solution which induced 50% of maximum adhesion were 0.03 and 0.18 mg/ml for HVSMC and A7r5 cells respectively. Adhesion and spreading of cells to fibrinogen was found to be time-dependent; adhesion reached a plateau at ~1 h (Fig. 5B), at which time cells were also well spread. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both HVSMC and A7r5 cells (Fig. 6A and B). Control peptide at concentrations up to 1 mM had no effects on the adhesion of either cell type. Adhesion to fibrinogen was largely blocked by chelation of [Ca2+]i following pre-incubation of cells with 100 µM BAPTA-AM for 3 h (Fig. 7). This concentration of BAPTA-AM completely abolished cell spreading. Increasing the concentration of BAPTA-AM to 300 µM had little additional inhibitory effect on adhesion of HVSMC (78±5% inhibition; n=6). In the presence of Ni2+, an inorganic calcium channel blocker, adhesion of both cell types was inhibited in a concentration-dependent manner over the range 1–10 mM (Fig. 8). At concentrations above 1 mM, Ni2+ completely inhibited cell spreading, although adhesion was not completely blocked by Ni2+ even at 10 mM. The tyrosine kinase inhibitor, genistein (50 µM), also inhibited cell adhesion and spreading (Fig. 7), but heparin (0.1–1 mg/ml), a glycosaminoglycan which inhibits adhesion to some extracellular matrix proteins such as thrombospondin-1 [34]had no significant effect on cell adhesion or spreading on fibrinogen.


Figure 5
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  		Fig. 5 Attachment of HVSMC ({circ}) and A7r5 cells (bullet) to fibrinogen coated wells. (A) Concentration-dependence of cell attachment to fibrinogen. Wells were coated with fibrinogen at the concentrations specified as described in Methods. Attached cells were counted after 2 h. (B) Time-dependence of cell attachment to fibrinogen. Wells were coated with fibrinogen (1 mg/ml) as described in methods and the number of attached cells were counted at the times indicated. All data represent the mean±SEM of 3–7 separate experiments conducted in quadruplicate.

 

Figure 6
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  		Fig. 6 Effects of RGD peptide on attachment to polystyrene wells coated with 1 mg/ml fibrinogen. Attached cells were counted after 2 h. (A) Effect of RGD peptide (1 mM; R) and control peptide (1 mM; E) on attachment of HVSMC to 1 mg/ml fibrinogen-coated wells. All data represent the mean±SEM of 6 separate experiments conducted in quadruplicate. *=p<0.05. (B) Concentration-dependent inhibition of attachment to fibrinogen in A7r5 by RGD peptide (0.1–1 mM; {square}) and control peptide (0.1–1 mM; {circ}). All data represent the mean±SEM of 4 separate experiments conducted in quadruplicate.

 

Figure 7
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  		Fig. 7 Inhibition of attachment of (A) HVSMC and (B) A7r5 cells to polystyrene wells coated with 1 mg/ml fibrinogen by heparin (Hep 0.1 and 1 mg/ml), BAPTA (100 µM; BA), and genistein (100 µM; G). All data represent the mean±SEM of 6 separate experiments conducted in quadruplicate, *=p<0.05.

 

Figure 8
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  		Fig. 8 Effect of Ni2+ on the attachment of HVSMC ({circ}) and A7r5 cells (bullet) to 1 mg/ml fibrinogen-coated wells. Adhesion was determined as described in Methods. Counts represent average number of cells/high power (200x) field. Data are expressed as mean±SEM of 3–6 experiments conducted in quadruplicate.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
References
 
In this study, we investigated the effect of fibrinogen on [Ca2+]i and adhesion of HVSMC and A7r5 cells, a cultured cell line derived from embryonic rat aorta. The results indicate that fibrinogen elevates [Ca2+]i and is an effective adhesion-promoting protein in both cell types at concentrations below those found in plasma [1]. The concentrations of fibrinogen in the coating solution are similar to those previously reported to induce adhesion of bovine foetal aortic smooth muscle cells [7]and platelets [38]. Fibrinogen has previously been reported to act as a chemotactic agent in bovine foetal aortic cells [8]and also induces migration in A7r5 cells (unpublished data). In view of the importance of changes in [Ca2+]i to cell adhesion and migration our findings suggest that an increase in [Ca2+]i, secondary to activation of tyrosine kinase(s) may also be involved in these actions of fibrinogen. Although the concentrations of fibrinogen inducing a rise in [Ca2+]i are broadly similar to those used in the coating solutions it should be borne in mind that these two measures represent liquid and solid-phase assays. A number of factors complicate direct comparison of such concentration-response data. Coating of plastic surfaces by proteins such as fibrinogen is saturable and generally conforms to a Langmuir isotherm. Previous studies [7, 39]indicate that coating with fibrinogen achieves a maximum surface density of around 0.5–2 µg cm–2 which is close to the theoretical maximum for a closely packed monolayer of fibrinogen molecules attached ‘end-on’ and similar to the maximum surface density achieved with other proteins such as fibronectin and thrombospondin [40, 41]. This has been calculated to correspond to 2.8x106 molecules of fibrinogen/100 mm2 (i.e. the approximate surface area of a smooth muscle cell) or to a surface layer concentration of 350 mgml–1 [39]. However, such calculations should be viewed with caution as far as estimates of affinity are concerned. Solid phase-based estimates of affinity (or avidity) are complicated by issues such as restricted diffusion, steric hindrance and altered protein affinity as a result of altered presentation [42]. Hence, we have not attempted to directly relate concentration-response data from studies of [Ca2+]i to adhesion studies.

A rise in [Ca2+]i is recognized to be an important intracellular signal in smooth muscle. In addition to its well recognised role in initiating the contraction of differentiated smooth muscle [28], it is also involved in actin regulation, integrin recycling and cell motility [27, 43]. In these studies fibrinogen caused a sustained elevation of [Ca2+]i. In both cell types the rise in [Ca2+]i in response to fibrinogen probably involves release of intracellular calcium stores and Ca2+ influx, since Ni2+, which blocks extracellular calcium influx through both receptor- and voltage-operated calcium channels [32], attenuated the rise in [Ca2+]i to similar extents. However the more pronounced second phase in the rise in [Ca2+]i seen in some experiments using A7r5 cells may indicate subtle differences in patterns of Ca2+ mobilization between the two cell types. Blockers of voltage-operated calcium channels were without effect in both cell types indicating that influx of Ca2+ occurs through channels other than the voltage-dependent calcium channel. The identity of the channel(s) responsible for the fibrinogen-induced rise in [Ca2+]i is unknown at present, though a number of Ca2+ influx pathways sensitive to inorganic blockers and insensitive to blockers of voltage-dependent calcium channels have been described in vascular smooth muscle cells [32, 44, 45].

Preincubation with a chelator of [Ca2+]i, BAPTA-AM, virtually blocked the rise in [Ca2+]i in response to fibrinogen, PDGF-BB and vasopressin. BAPTA-AM also largely inhibited adhesion indicating that a rise in [Ca2+]i plays an important role in this process. However a limited degree of adhesion still occurred following BAPTA-AM, although spreading was completely inhibited. Similarly Ni2+ which inhibited the rise in [Ca2+]i, also partially inhibited adhesion and blocked spreading completely, consistent with the latter process being more dependent on the rise in [Ca2+]i.

In platelets and other non-vascular cells fibrinogen has been reported to act through binding to integrins. Integrin subunits {alpha}IIb, {alpha}m, {alpha}v, β2 and β3 have been reported to play roles in adhesion to fibrinogen via an RGD-type interaction [46–49], while {alpha}vβ1 and {alpha}vβ5 integrins have been reported not to bind fibrinogen [50, 51]. Our data indicate that an RGD-type interaction, i.e. integrin binding is at least partially involved in the adhesion of smooth muscle cells to fibrinogen. The failure of an RGD peptide to completely inhibit adhesion to fibrinogen in HVSMC is similar to a recent report in human fibroblasts [52], but contrasts with an earlier study using bovine aortic smooth muscle cells, where adhesion to fibrinogen and vitronectin was completely inhibited by RGD peptide. Surprisingly, in the same study cell adhesion to fibronectin, which also contains an RGD sequence, was almost unaffected by RGD peptide [8]confirming that non-RGD interactions can mediate adhesion of vascular smooth muscle cells to RGD-containing matrix proteins. Similarly, RGD peptide has also been reported to inhibit only partially migration of human vascular smooth muscle cells in response to vitronectin, despite near complete inhibition by an antibody to {alpha}vβ3 integrin [53]. A number of integrin isoforms have been described on vascular smooth muscle cells under a variety of conditions, including {alpha}1, {alpha}2, {alpha}3 {alpha}5, {alpha}8, {alpha}9, {alpha}v, β1, β3 and β4 [54–58], however it is uncertain which integrin subunits are responsible for binding fibrinogen in HVSMC. Further work using different integrin subunit antibodies needs to be done to clarify this.

The role of RGD binding sites in the ability of fibrinogen to increase [Ca2+]i is less clear. Soluble fibrinogen increased [Ca2+]i in both HVSMC and A7r5 cells. In A7r5 cells RGD peptide partially inhibited fibrinogen-induced rises in [Ca2+]i, while in HVSMC RGD peptide alone caused a small but consistent rise in [Ca2+]i. This observation is similar to previous studies in fibroblasts [59]and osteoclasts [60, 61]where addition of RGD-type peptides alone increased [Ca2+]i. However the importance of this mechanism to the action of fibrinogen in HVSMC is uncertain since RGD peptide failed to inhibit [Ca2+]i responses to fibrinogen. These observations may indicate at least two distinct binding sites for fibrinogen on smooth muscle cells, one RGD-dependent, another RGD-independent, both linked to an increase in [Ca2+]i. Since some integrin-mediated binding has been reported to involve non-RGD-type sequences this observation does not exclude integrins from mediating this action of fibrinogen.

Many studies have demonstrated that many growth factors, including PDGF, epidermal growth factor, basic fibroblast growth factor, can induce an increase in [Ca2+]i by binding to receptor-linked tyrosine kinases. In part this involves activation of phospholipase C-{gamma} which leads to production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, with IP3 stimulating release of [Ca2+]i from the sarcoplasmic reticulum [28]. In addition there is also evidence that tyrosine kinases can activate Ca2+ influx into vascular smooth muscle [62–64], though the pathways involved in this action remain to be fully defined. Our data have shown that genistein, a selective inhibitor of tyrosine kinases almost completely blocked adhesion, spreading and the rise in [Ca2+]i in response to fibrinogen. Preliminary studies with A7r5 cells have demonstrated increased tyrosine phosphorylation of a number of proteins in A7r5 cells following exposure to fibrinogen (Hughes and Lymn unpublished data). The identity of these proteins and their role in fibrinogen-induced responses is currently under study. Nevertheless our results support the notion that an integrin or a related receptor linked to tyrosine kinase(s) is involved in the increase in [Ca2+]i which plays a key role in the subsequent adhesion, spreading and chemotaxis of vascular smooth muscle cells.

In conclusion, direct actions of fibrinogen on vascular smooth muscle cells may constitute an additional pathway by which this agent may act as a risk factor for cardiovascular disease. The ability of fibrinogen to increase [Ca2+]i and induce the adhesion and migration of smooth muscle cells through activation of tyrosine phosphorylation could play a role in the pathological response of the vasculature in regions of damage or thrombosis and contribute to atheroma formation at these sites.

Time for primary review 24 days


   Acknowledgements
 
We are grateful to Ms K Gallagher for technical assistance with cell culture and Dr J Adams for advice regarding the cell adhesion assay. Surgical material was kindly supplied by the theatre staff of St Mary's Hospital, London, UK. L.X. was in receipt of a British Council China Fellowship and GC was supported by a grant from the British Heart Foundation. Work in this study was also supported in part by an educational grant from Pfizer International, USA.


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

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