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Cardiovascular Research 1999 43(1):210-218; doi:10.1016/S0008-6363(99)00006-1
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Copyright © 1999, European Society of Cardiology

Activated platelets and leucocytes cooperatively stimulate smooth muscle cell proliferation and proto-oncogene expression via release of soluble growth factors

Plinio Cirilloa, Paolo Golinoa,*, Massimo Ragnia, Carmine Battagliaa, Francesco Pacificob, Silvestro Formisanob, Chiara Buonoa, Mario Condorellia and Massimo Chiarielloa

aDivision of Cardiology, University of Naples ‘Federico II’, Naples, Italy
bDepartment of Molecular and Cellular Biology and Pathology, University of Naples ‘Federico II’, Naples, Italy

* Corresponding author. Tel.: +39-81-7462216; fax: +39-81-7462229 golino{at}CDs.unina.it

Received 24 September 1998; accepted 25 November 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Background: Previous studies indicate that platelets and leucocytes might contribute to the development of neointimal hyperplasia following arterial injury. The present study was aimed at further investigating the role of platelets and leucocytes, alone or in combination, in promoting vascular smooth muscle cell (SMC) proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression, such as c-fos. Methods: SMCs from rabbit aortas, made quiescent by serum deprivation, were stimulated with either activated platelets, leucocytes, or both, separated from SMCs by a membrane insert. SMC proliferation was evaluated by measuring the incorporation of 3H-thymidine. The relative contribution of different platelet-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, R68070 [GenBank] , a TxA2 receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet derived growth factor (PDGF) receptor antagonist. The role of different leucocyte sub-populations (neutrophils and monocytes+lymphocytes) was also determined in additional experiments. Results: SMC proliferation was significantly increased by activated platelets to 360±9% of control values (P<0.05). This effect was reduced by ketanserin, R68070 [GenBank] , BN 52021 or trapidil. Whole leucocytes, neutrophils or lymphocytes+monocytes also increased SMC proliferation with respect to control experiments. Simultaneous stimulation of SMCs by platelets and whole leucocytes was associated with a significant greater increase in SMC proliferation as compared to SMC stimulated with platelets or leucocytes alone. c-fos expression, almost undetectable in unstimulated SMCs, was markedly increased by activated platelets or leucocytes. Conclusions: Activated platelets promote SMC proliferation in vitro via release of soluble mediators, including serotonin, thromboxane A2 PAF and PDGF; activated leucocytes also induce a significant SMC proliferation and exert an additive effect when activated together with platelets; SMCs stimulated with activated platelets and leucocytes show an early expression of the proto-oncogene c-fos.

KEYWORDS Smooth muscle cell proliferation; Platelets; Leucocytes; c-fos


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Vascular smooth muscle cell (SMC) proliferation almost invariably occurs in response to vessel injury, and is thought to be one of the key events responsible for the occurrence of restenosis in patients after successful coronary angioplasty [1]. However, despite an extensive number of studies examining the clinical, morphologic, and technical factors associated with an increased risk of restenosis, our understanding of the problem still remains incomplete. Previous studies have shown that the arterial injury caused by angioplasty leads to activation and local adhesion of platelets and leucocytes [2–6]. Both platelets and leucocytes, once activated, can release a number of growth factors, all of which may in turn stimulate SMC proliferation and migration [1]. Indeed, experimental studies in animal models have suggested a pathophysiological role for platelets as well as for leucocytes in contributing to the occurrence of restenosis [7–11]. Although these studies directly support the importance of platelets and leucocytes in modulating SMC growth and migration, the mechanisms through which these effects are obtained are incompletely understood. In particular, little is known about the relative contribution of different platelet and leucocyte-derived growth factors, and the molecular mechanisms involved in this process. Thus, the aim of the present study was to further investigate the role of platelets and leucocytes, alone or in combination, in promoting vascular SMC proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression, such as c-fos, the induction of which is one of the earliest events associated with cell proliferation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Vascular SMC isolation and culture
SMCs were isolated from the thoracic aortas of New Zealand white rabbits by nonenzymatic dissociation as previously described [12]. Briefly, thoracic aortas were dissected and cleaned of connective tissue under sterile conditions. Vascular rings of about 2 cm in length were cut longitudinally and the endothelium was removed by gently rubbing the inner layer of the vessel with a sterile blade. The muscular layers obtained were cut into 2-mm strips, placed onto 100-mm tissue culture dishes with the intimal side oriented toward the bottom of the culture dish and covered with a small volume of Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% heat-inactivated calf serum (FCS), 100 units per ml penicillin and 100units per ml streptomycin. The vessels were maintained in a humidified incubator (95% air–5% CO2) at 37°C. DMEM was replaced after 24 h and then every three days. After about 7 days, SMCs started to migrate from vessel segments to the surrounding bottom of the culture dish. Confluent primary cultures were removed from culture dishes by brief exposure to 0.3% trypsin/0.02% EGTA (ethyleneglycol-bis-(β-aminoethylether)N,N,N',N'-tetraacetic acid) and passaged at a 1:3 split ratio. Cells between the second and seventh passage were used for all studies.

At confluence, when examined with phase-contrast microscopy, the cells displayed the typical hill-and-valley appearance of SMC cultures and grew to form multilayers. In addition, cells were identified by immunocytochemical staining of smooth muscle {alpha}-actin using a specific monoclonal antibody labeled with fluoresceine (Sigma Chemicals, St. Louis, MO). Nearly 100% of the cells showed a positive fluorescence.

2.2 Experimental set up
After harvesting, SMCs were washed with PBS, manually counted in a hemocytometer and subcultured into 24-well culture plates in DMEM containing 10% FCS for 24 h at an initial density of 5x104 cells per well. An equal number of SMCs was plated in each well to exclude variation due to differences in cell number. Twenty-four hours later, SMCs were made quiescent by incubation in fresh DMEM containing 0.1% FCS for 72 h [13]. After 72 h of serum deprivation, a plastic cylinder, closed at the bottom with a microporous membrane (0.2 µm diameter, TranswellTM), Becton Dickinson, Bedford, MA) was inserted into each well. The height of the transwell was such that it did not reach the bottom of the well containing the SMC layers, leaving them undisturbed (Fig. 1). A platelet and/or leucocyte suspension was added into each transwell. Platelets and leucocytes were then activated as described below in Section 2.4. This set-up permitted free passage of soluble mediators through the microporous membrane to the SMC culture medium, but did not allow direct cell–cell interaction (Fig. 1).


Figure 1
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  		Fig. 1 Schematic representation of the experimental set up used in the present study. Smooth muscle cells were stimulated with either activated platelet, leucocytes, or both; cell–cell interactions were not allowed, whereas the passage of soluble mediators were permitted through the microporous membrane (see Section 2.2 for further details).

 
2.3 Isolation of platelets and leucocytes
On the day of the experiment, venous blood was obtained from healthy donors who had not taken any medication for at least 2 weeks before the studies. For isolating platelets, blood was collected into Vacutainer tubes containing 3.8% sodium citrate (9:1 v/v) and centrifuged at 120xg for 20 min at room temperature to obtain platelet-rich plasma (PRP). PRP was removed and centrifuged again at 1000xg for 5 min to obtain platelet-poor plasma (PPP). The platelet count in the PRP was adjusted to 300 000/µl by dilution with PPP as needed. The PRP was kept at room temperature and used within 1 h.

Peripheral blood leucocytes from healthy donors were prepared by Ficoll–Hypaque gradient centrifugation from heparinized venous blood followed by dextran sedimentation, as previously described [14]. Briefly, blood samples were mixed with 6% dextran (1/1, v/v) and the red blood cells were allowed to settle at room temperature for 90 min. The white cell rich supernatant was placed over standard Ficoll–Hypaque separation media and centrifuged at 400xg for 30 min at room temperature. The gradient obtained after centrifugation allowed the isolation of neutrophils (pellet) from monocytes/lymphocytes (upper interface). Any contaminating red cells were lysed with 1 vol of H2O for 30 s followed by addition of 2.7% saline. Leucocytes were then washed in Hanks balanced salt solution, resuspended in phosphate-buffered saline (PBS), counted and immediately used. The neutrophil count was adjusted to 1500 cells per µl, while the monocyte/lymphocyte count was adjusted to 1000 cells per µl. The purity of the neutrophil and lymphocyte/monocyte preparations was checked by microscopy and found to be >99% and >95%, respectively.

2.4 Experimental protocol
To investigate the effects of activated platelet on SMC proliferation, 500 µl of PRP were added to each transwell (see Section 2.2) and platelets were activated with 60 µg of collagen (from calf skin, monomeric, acid-soluble form; Sigma cat.885-1) under mechanical stirring. After 30 min, transwells containing PRP with activated platelets were removed from culture wells and SMCs were maintained in a humidified incubator (95% air–5% CO2) at 37°C for the later assessment of the degree of cell proliferation. Since SMCs were kept under conditions of serum deprivation, any proliferating activity should be related to activated platelets. To determine the relative contribution of the different mediators released by activated platelets, transwells were filled with PRP and platelets were activated as above. Five min after platelet activation, either one of the following compounds were added to the SMC culture medium: ketanserin, a selective serotonin 5-HT2 receptor antagonist [15]; 68070, a drug with simultaneous Thromboxane A2 synthase and receptor-inhibiting properties [16]; BN52021, a selective Platelet Activating Factor receptor antagonist [17]; Trapidil, a Platelet Derived Growth Factor (PDGF) receptor antagonist [18]; all antagonists together. Each antagonist was added to a final concentration of 0.1 mM. The concentration of the different antagonists was chosen on the basis of preliminary experiments which showed that this concentration caused a maximal effect on inhibition of proliferation in response to the respective agonist alone.

In another set of experiments, the effects of whole leucocytes and of different leucocyte subtypes in promoting SMC proliferation was investigated. Five hundred µl of the following suspensions were added to the transwells: (a) whole leucocytes; (b) neutrophils; (c) monocytes together with lymphocytes. A concentration of 1 µM of N-formylmethionyl-leucyl-phenylalanine (fMLP) or 0.2 mg/ml of Zymosan was used to activate neutrophils or monocytes/lymphocytes, respectively, while the whole leucocyte suspension was activated with a mixture of fMLP (1 µM)+Zymosan (0.2 mg/ml). After 30 min of stimulation, transwells with leucocyte suspensions were removed from culture wells and SMCs were maintained as described above to evaluate cell proliferation.

To determine the contribution of oxygen free radicals released by activated neutrophils in promoting SMC proliferation, transwells were filled with a neutrophil suspension as above. Five min after neutrophil activation, superoxide dismutase (SOD, 1000 U/ml) and catalase (2000 U/ml), were added to SMC culture medium. After 30 min of stimulation, transwells with neutrophil suspension were removed from culture wells and SMCs were maintained as described above to evaluate cell proliferation.

Finally, to study the role of platelets and leucocytes together in promoting SMC proliferation, in another set of experiments, SMCs were simultaneously challenged with platelets and whole leucocytes activated as described.

2.5 Evaluation of SMC proliferation
The growth effects of blood cells on SMC proliferation were quantified by measuring the extent of 3H-thymidine incorporation into cell DNA. After the 30-min stimulation period with platelets and/or leucocytes, SMCs were incubated at 37°C for 24 h. Thereafter, SMCs were pulsed with 1 µCi 3H-thymidine and after 24 additional hours, the culture medium was discarded, cells were rinsed twice with PBS and lysed with 0.2% perchloric acid. The contents of the wells were aspirated and wells rinsed an additional time with PBS, and the acid-precipitated cellular material was solubilized with 0.5 ml 0.01 N sodium hydroxide–0.1% sodium dodecyl sulphate. The content of each well was added to 7 ml of Optifluor and radioactivity was measured with a Beckman beta-scintillator. Incorporation of 3H-thymidine was expressed as counts per minute per well. Negative control experiments included SMCs not challenged with platelets or leucocytes and incubated under conditions of serum deprivation, SMCs challenged with non-activated platelets and/or leucocytes, and SMCs incubated with collagen, fMET or Zymosan. Positive control experiments included SMCs incubated with 10% FCS.

To verify that the observed increase in 3H-thymidine incorporation is actually related to an increase in the absolute number of SMCs, additional control experiments were performed. Cells were grown in 100-mm Petri dishes. At confluence, cells were washed with PBS and harvested with 0.3% trypsin/0.02% EGTA diluted in PBS, as described above. Cells were manually counted in a hemocytometer before and 48 h after serum deprivation (negative control), or after stimulation with 10% FCS or activated platelets, as described above.

2.6 Proto-oncogene expression
To determine whether activated platelets and leucocytes may induce the expression of the early growth response gene c-fos, quiescent SMCs were stimulated with platelets, leucocytes or both, as described above. Thirty min of stimulation were followed by an hour of incubation at 37°C in 95% air–5% CO2. SMC mRNA was then extracted with the guanidine thiocyanate method of Chomczynski and Sacchi [19]. Equal aliquots of RNA were denaturated and separated by electrophoresis on 1.2% agarose–formaldehyde gels. Following transfer of the RNA to nitrocellulose paper (Hybond; Amersham International), blots were prepared and prehybridized in 50 mM Tris, pH 7.6, 0.8 M NaCl, 0.5% sodium dodecyl sulphate (SDS), 5xDenhardt’s solution, 40% deionized formamide, 10% dextran sulphate, 0.1% sodium pyrophosphate, and 75 g/ml of salmon sperm DNA for at least 4 h at 42°C. Hybridization was performed at 42°C for 20 h using the same buffer containing a 32P-labeled fos probe,1.06 kb Pst 1 fragment, corresponding to v-fos. v-fos is the viral homologue of the fos gene present in the cellular DNA. To remove the unhybridized probe, filters were washed twice for 15 min in 200 ml of 2xsodium saline citrate (SSC), 0.2% sodium dodecyl sulphate (SDS) at room temperature and then twice in 0.2 X SSC, 0.2% SDS at 50°C for 15 min. Autoradiography of the resulting Northern Blot was performed using Kodak XAR-5 films sensitized by preflashing with Du Pont Cronex intensifying screens at –70°C for 2 days.

SMCs stimulated by adding to culture wells, fresh medium supplemented with 10% FCS, served as positive proliferation control, while RNA extracted from serum-deprived cells served for the evaluation of c-fos steady-state RNA levels.

2.7 Statistical analysis
All values are expressed as mean±SE. For SMC proliferation studies, six different experiments for each experimental condition (including positive and negative controls) were performed. For proto-oncogene expression studies, 24 wells from each experimental condition were pooled together and used for one Northern Blot analysis. A total of three different blots were performed. A one-way ANOVA was used to evaluate differences in cell proliferation among different experimental conditions, followed, whenever a F value was found significant, by a Students t-test for unpaired observations with the Bonferronis correction. A P-value <0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of activated platelets and leucocytes on SMC proliferation in vitro
The effects of activated platelets and leucocytes on SMC proliferation were assessed on SMCs made quiescent by serum deprivation. Thus, any proliferative effect seen under the experimental conditions used in the present study should be ascribed to the presence of activated platelets and/or leucocytes. SMCs not stimulated with platelets and/or leucocytes and kept under conditions of serum deprivation served as negative controls for each set of experiments. All results are expressed as a percent of 3H-thymidine incorporation with respect to these negative control experiments.

Fig. 2 illustrates the effects of activated platelets and of different platelet-derived mediators on SMC proliferation, as assessed by 3H-thymidine incorporation. Collagen-activated platelets caused a marked increase in 3H-thymidine incorporation in SMCs up to 360±29% of the value observed in quiescent cells (Fig. 2A). Similar results were obtained using washed platelets resuspended in PBS (data not shown). Addition of unstimulated platelets (no collagen) or collagen alone (no platelets) into the transwells did not significantly increase 3H-thymidine incorporation with respect to the negative control experiments (Fig. 2A).


Figure 2
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  		Fig. 2 Panel A: Effects of activated platelets on smooth muscle cell (SMC) proliferation, evaluated by measuring the incorporation of 3H-thymidine and expressed as a percent of 3H-thymidine incorporation in negative control experiments (serum-deprived cells). Activated platelets caused a significant increase in SMC proliferation, which averaged 360±29% of the values obtained in the negative control experiments. Addition of collagen alone, or non-activated platelets did not cause a significant increase in SMC proliferation with respect to the negative control experiments. The effects of 10% fetal calf serum (FCS) is shown for comparison. *=P<0.01 vs. negative controls; **=P<0.005 vs. negative controls. Panel B: Effects of different platelet-derived mediators on 3H-thymidine incorporation in smooth muscle cell in culture. Ketanserin, a serotonin 5-HT2 receptor antagonist, R68070, a thromboxane A2 receptor and synthase inhibitor, and BN52021, a platelet-activating factor receptor antagonist, resulted in a similar reduction in 3H-thymidine incorporation to about 50% of the values obtained with activated platelets. Addition of trapidil, a PDGF receptor antagonist, resulted in a greater reduction in of 3H-thymidine incorporation with respect to the values obtained with the other antagonists used.When all antagonists were added together, an even more pronounced antiproliferative effect was observed. *=P<0.01 vs. activated platelets; #=P<0.05 vs. R68070, ketanserin, and BN52021; **=P<0.05 vs. other groups.

 
The relative role of different platelet-derived mediators in promoting SMC proliferation was evaluated in other experiments by adding either ketanserin, a selective serotonin 5-HT2 receptor antagonist, R68070 [GenBank] , a drug with simultaneous TxA2 synthase and receptor inhibiting properties, BN52021, a selective PAF receptor antagonist, or Trapidil, a PDGF receptor antagonist. Ketanserin, R68070 [GenBank] , and BN52021 resulted in a similar reduction in 3H-thymidine incorporation to about 50% of the values obtained with activated platelets (P<0.01 vs. activated platelets, Fig. 2B). Interestingly, addition of trapidil, a PDGF receptor antagonist, resulted in an even greater reduction in 3H-thymidine incorporation with respect to the values obtained with the other antagonists used (P<0.05 vs. R68070 [GenBank] , ketanserin, and BN52021, Fig. 2B), suggesting a more prominent role of PDGF in stimulating SMC proliferation compared to the other platelet-derived mediators. Of note is the finding that when all antagonists were added together, an even more pronounced antiproliferative effect was observed (Fig. 2B).

In another set of experiments, the effects of activated whole leucocytes on SMC proliferation were evaluated. Activated leucocytes also caused a significant increase in of 3H thymidine incorporation up to 210±21% of the values observed in negative control experiments(Fig. 3A). Similar results were obtained when SMCs were stimulated with neutrophils alone or with monocytes+lymphocytes (Fig. 3A). When both platelets and leucocytes were used to stimulate SMCs, a marked increase in 3H-thymidine incorporation was observed, which was significantly greater than that observed with either cell type alone (Fig. 3A).


Figure 3
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  		Fig. 3 Panel A: Effects of activated whole leucocytes, alone or in combination with platelets, and of different leucocyte subtypes (neutrophils; monocytes/lymphocytes) on smooth muscle cell proliferation. Activated leucocytes caused a significant increase in of 3H-thymidine incorporation, up to 210% of the values observed in negative control experiments. Similar results were obtained when smooth muscle cells were stimulated with neutrophils alone or with monocytes+lymphocytes. Whole leucocytes in combination with activated platelets resulted in an additive effect with respect to either cell population alone. *=P<0.05 vs. activated platelets and leucocytes alone. Panel B: Effects of the oxygen free radical scavengers, superoxide dismutase (SOD) and catalase, on neutrophil-induced SMC proliferation. SOD and catalase significantly reduced SMC proliferation induced by activated neutrophils, indicating that these cells stimulate SMC proliferation via release of oxygen free radicals. *=P<0.05 vs. neutrophils.

 
The role of oxygen free radicals released by activated neutrophils in promoting SMC proliferation was evaluated by adding SOD and catalase to culture medium. SOD and catalase significantly reduced 3H-thymidine incorporation as compared to SMCs stimulated with neutrophils alone in the absence of scavengers (P<0.05, Fig. 3B).

Absolute cell counts revealed that under conditions of stimulation with 10% FCS, the SMC number increased from 49 166±4676 to 105 333±3808, while activated platelets increased the SMC number from 55 416±6064 to 90 765±4545. Under conditions of serum deprivation, SMC number did not change significantly from 47 083±4445 to 43 750±3987 after 48 h. Thus, the observed increase in 3H-thymidine incorporation under our experimental conditions is indeed related to an increase in SMC absolute numbers.

3.2 Effects of activated platelets and leucocytes in proto-oncogene expression
The mRNA transcripts for the c-fos gene were almost undetectable in unstimulated, quiescent SMCs (negative controls). In contrast, addition of serum to the medium (positive controls) resulted in a marked increase in c-fos mRNA levels (Fig. 4). Interestingly, activated platelets or leucocytes induced an increase in c-fos mRNA levels which paralleled the increase in 3H-thymidine incorporation (Fig. 4). Of note is the finding that stimulation with platelets and leucocytes together caused a greater increase in c-fos mRNA levels with respect to those observed with leucocytes alone (P<0.05; Fig. 4).


Figure 4
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  		Fig. 4 Results of three different Northern blot analysis performed on mRNA extracts obtained from smooth muscle cells stimulated with activated platelets and/or leucocytes using a specific probe for c-fos. The results are expressed as a percent density scan with respect to the positive control experiments. The mRNA transcripts for the c-fos gene were almost undetectable in unstimulated, fetal calf serum (FCS)-deprived smooth muscle cells (FCS 0.1%). In contrast, addition of serum to the medium resulted in a marked increase in c-fos mRNA levels (FCS 10%). Activated platelets and leucocytes together induced an increase in c-fos mRNA levels (platelets+ leucocytes) which were significantly greater than the levels observed with platelets or leucocytes alone. The insert shows a typical Northern blot experiment. *=P<0.05 vs. leucocytes.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The main findings of the present study are: (1) Activated platelets promote SMC proliferation in vitro via release of soluble mediators, including serotonin, thromboxane A2platelet-activating factor (PAF), and platelet-derived growth factor (PDGF); among these, PDGF seems to exert the most significant proliferating activity. (2) Activated leucocytes (both neutrophils and monocytes+lymphocytes) also induce a significant SMC proliferation and exert an additive effect when activated together with platelets. (3) SMCs stimulated with activated platelets and leucocytes show an early expression of the proto-oncogene c-fos . The intensity of c-fos expression is correlated with the degree of SMC proliferation.

That platelet-derived mediators might play an important role in contributing to the development of neointimal hyperplasia following arterial injury is not a novel concept. For example, previous studies have shown that serotonin [20], thromboxane A2 [21], PAF [22], ADP [20], and PDGF [23], promptly released upon platelet activation, can all stimulate SMC growth in vitro. Other studies have demonstrated that within 30 min of balloon injury, platelet factor IV, one of the constituents of the {alpha}-granules, can be detected throughout the intima and media [24]. Because platelet factor IV and other platelet-derived substances, like PDGF, serotonin, and ADP reside in the same granule population and are secreted in response to the same stimuli, it is likely that these substances also enter the vessel wall after balloon injury [24]. Prompted by these indirect observations, several studies have been conducted to determine whether antiplatelet interventions might be able to reduce the extent of the neointimal hyperplasia that follows balloon angioplasty. While some of these studies have shown that activated platelets play an important role in the pathophysiology of neointimal hyperplasia [8,9], others have reported negative results [25]. In addition, clinical studies employing different antiplatelet regimens have consistently shown failure of these treatments in reducing the incidence of restenosis after successful coronary angioplasty (for a review see Ref. [26]).

A possible explanation for this discrepancy might be found in the data from the present study. Indeed, we have shown that collagen-activated platelets (a stimulus that did not exert any effect on SMC proliferation per se), were capable of inducing a marked proliferation of SMCs in culture. This phenomenon occurred through the release of several soluble growth factors, since the experimental set up used in the present study did not allow a direct cell-to-cell interaction, but it only permitted free passage of soluble mediators. The relative contribution of some of the substances released upon platelet activation on SMC proliferation was evident when specific receptor antagonists were added to the culture medium. When SMCs were stimulated with activated platelets in the presence of either ketanserin, R68070 [GenBank] , or BN52021, a similar reduction in SMC proliferation was observed. Addition of trapidil, a triazolopyrimidine with PDGF receptor antagonistic properties, resulted in a significant greater decrease in SMC proliferation, as compared to the other antagonists used, indicating a more prominent role for PDGF in modulating this phenomenon. More important, however, is the observation that when all antagonists were added together, an even more marked decrease in SMC proliferation was observed, demonstrating that activated platelets stimulate SMC growth through the release of several different mediators, each of which separately contributes to stimulate cell growth. Thus, if one interferes with only one of these platelet-derived mediators, SMC growth might be left practically unaffected; this might explain why clinical trials employing these types of pharmacological interventions have consistently reported negative results.

Another possible cellular source of growth factors that might be involved in the pathophysiology of neointimal hyperplasia following angioplasty is represented by circulating leucocytes. Circulating leucocytes are known to activate and accumulate at the site of vascular injury both in experimental models and in the clinical setting; furthermore, certain leucocyte subtypes, such as monocytes and lymphocytes, are known to contain numerous growth factors in their cytoplasm, including PDGF [27], Epidermal Growth Factor [28], basic Fibroblast Growth Factor [29], Transforming Growth Factor type [30], PAF [31], Tumor Necrosis Factor [32], and interleukin-1 [33]. In addition, neutrophils may also be involved in triggering cell proliferation, even though these cells do not contain growth factors in their cytoplasm. Indeed, neutrophils are capable of producing large amounts of active oxygen species, which have been shown to stimulate SMC growth and proto-oncogene expression [34]. However, the finding of the present study that SOD and catalase inhibit neutrophil-induced SMC proliferation by about 50% indicate that other neutrophil-related mediators might be involved in this phenomenon. More direct evidence supporting the role of circulating leucocytes in promoting neointimal hyperplasia following arterial injury have been recently reported by our group and by others. In those studies, administration of a monoclonal antibody against the leucocyte CD11/CD18 adhesion complex or against its ligand expressed on the membrane of vascular cell, ICAM-1, was associated with a significant reduction in leucocyte infiltration into the arterial wall, ultimately leading to a reduction in neointimal hyperplasia [10,11].

Data from the present study extend these previous observations and indicate that both neutrophils and monocytes/lymphocytes are able to promote SMC proliferation in vitro although probably through different mechanisms. More interestingly, however, the present study indicates that an additive interaction exists between platelets and leucocytes in this phenomenon. Indeed, when SMCs were challenged with both activated platelets and leucocytes, a significant higher degree of cell proliferation was observed, as compared to platelets or leucocytes alone.

Finally, the present study has focused on the molecular mechanisms responsible for platelet and leucocyte-induced SMC proliferation. Northern blot analyses of mRNA isolated from SMCs challenged with activated platelets and leucocytes showed that an early expression of the c-fos proto-oncogene occurs in these cells. The proto-oncogenes were first identified as unexpected sequences of vertebrate genomic DNA corresponding to the acutely transforming genes of RNA tumor viruses [35,36]. A set of proto-oncogenes, whose prototypes are c-fos, c-myb, and c-jun, encode for proteins that are rapidly induced by growth stimuli, are found within the cell nucleus, and may be obligatory for proliferation and other growth factor effects to occur [35]. Transient expression of these proto-oncogenes is triggered by diverse trophic signals such as growth factors, physiological stress, and activity-dependent events [36,37]. Most importantly, it has been shown that interventions that introduce complementary ‘antisense’ oligonucleotides into cells, which bind and decrease the abundance of ‘sense’ mRNA transcripts, can markedly inhibit expression of selected oncogene proteins. For example, blocking c-myb expression by antisense techniques inhibits neointimal hyperplasia in balloon-injured rat carotid arteries [38], thus demonstrating the importance of proto-oncogene expression in leading to the formation of neointimal lesions following arterial damage. In our study, the activation of the nuclear oncogene c-fos occurred in SMCs challenged with activated platelets, leucocytes, or both. Although SMCs stimulated with both activated platelets and leucocytes showed a c-fos expression greater than that observed in SMCs stimulated with leucocytes alone, but not with platelets alone, the degree of c-fos expression roughly paralleled that of SMC proliferation, indicating a relationship between the two phenomena. To our knowledge, our study is the first to report that activated platelets and leucocytes may induce c-fos expression in SMCs in culture and that this event is correlated with the subsequent SMC proliferation.

3.1 Limitations of the present study
A few limitations should be taken into account when considering the results of the present study. First, the effects of the single antagonists on SMC proliferation per se were not tested. It cannot be excluded, therefore, that some or all of the antagonists used may reduce SMC proliferation through different, aspecific mechanisms other than inhibition of the respective growth factor. Second, addition of the various antagonists might have reduced platelet release via a partial inhibition of platelet aggregation. However, this possibility seems unlikely. In fact, the different antagonists were added 5 min after addition of collagen. It is known that after 5-min platelet addition, aggregation in vitro is complete and irreversible.

In conclusion, the data of the present study indicate that platelet and leucocyte-induced SMC proliferation in vitro does not simply depend on a single growth factor, but rather on the simultaneous action of different growth factors, including but not limited to, PDGF, serotonin, thromboxane A2, PAF, and oxygen free radicals. It is therefore likely that in vivo, these and perhaps other factors not investigated in the present study may act in concert, amplifying the action of each other, ultimately leading to a maximized effect on cell growth. This also implies that rather than interfering with only one growth factor, a better strategy should be employed when attempting to inhibit neointimal hyperplasia in the clinical setting. Further studies in this direction are warranted.

Time for primary review 28 days.


   Acknowledgements
 
This work was presented in part at the XIXth annual meeting of the European Society of Cardiology, Stockholm, Sweden, August 1997.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Shirotani M., Yui Y., Kaway C. Restenosis after coronary angioplasty: pathogenesis of neointimal thickening initiated by endotelial loss. Endothelium (1993) 1:5–22.[Medline]
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