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(Circulation. 2004;109:2911-2916.)
© 2004 American Heart Association, Inc.

Basic Science Reports

Tissue Factor Binding of Activated Factor VII Triggers Smooth Muscle Cell Proliferation via Extracellular Signal–Regulated Kinase Activation

Plinio Cirillo, MD, PhD^*; Gaetano Calì, PhD^*; Paolo Golino, MD, PhD; Paolo Calabrò, MD; Lavinia Forte, MD; Salvatore De Rosa, MD; Mario Pacileo, MD; Massimo Ragni, MD, PhD; Francesco Scopacasa, MD; Lucio Nitsch, MD; Massimo Chiariello, MD

From the Division of Cardiology (P. Cirillo, P. Calabrò, S.D., M.P., M.R., M.C.), Department of Biology and Cellular and Molecular Pathology "L. Califano" (L.N.), and Department of Biochemistry and Biotechnology (F.S.), University of Naples "Federico II"; Division of Cardiology, Second University of Naples (P.G., L.F.); and IEOS "G. Salvatore"–National Council of Research (G.C.), Naples, Italy.

Correspondence to Paolo Golino, MD, PhD, Division of Cardiology, Second University of Naples, c/o Ospedale Monaldi, Via Leonardo Bianchi, 80131 Naples, Italy. E-mail paolo.golino{at}unina2.it

Received July 22, 2003; de novo received December 5, 2003; revision received March 9, 2004; accepted March 9, 2004.

	Abstract

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Background— Tissue factor (TF) is the main initiator of coagulation in vivo. Recently, however, a role for TF as a cell receptor involved in signal transduction has been suggested. The aim of the present study was to assess whether activated factor VII (FVIIa) binding to TF could induce smooth muscle cell (SMC) proliferation and to clarify the possible intracellular mechanism(s) responsible for this proliferation.

Methods and Results— Cell proliferation was induced by FVIIa in a dose-dependent manner, as assessed by [³H]thymidine incorporation and direct cell counting, whereas no response was observed with active site–inhibited FVIIa (FVIIai), which is identical to FVIIa but is devoid of enzymatic activity. Similarly, no proliferation was observed when binding of FVIIa to TF was prevented by the monoclonal anti-TF antibody AP-1. Activation of the p44/42 mitogen-activated protein (MAP) kinase (extracellular signal–regulated kinases 1 and 2 [ERK 1/2]) pathway on binding of FVIIa to TF was demonstrated by transient ERK phosphorylation in Western blots and by suppression of proliferation with the specific MEK (MAP kinase/ERK kinase) inhibitor UO126. ERK phosphorylation was not observed with FVIIai or when cells were pretreated with AP-1.

Conclusions— These data indicate a specific effect by which binding of FVIIa to TF on the surface of SMCs induces proliferation via a coagulation-independent mechanism and possibly indicate a new link between coagulation, inflammation, and atherosclerosis.

Key Words: coagulants • signal transduction • cell division

	Introduction

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Tissue factor (TF) is a cell surface–anchored glycoprotein that binds both the zymogen factor VII (FVII) and the active serine protease factor VIIa (FVIIa).¹ TF/FVIIa complex activates coagulation by cleaving its natural substrates, factors IX and X, ultimately leading to thrombin generation, fibrin deposition, and platelet activation.^1,2 On these grounds, TF has been considered to function as a major initiator of blood coagulation in vivo. Recently, however, some evidence has suggested that TF may mediate important coagulation-independent biological phenomena, including embryonic and oncogenic angiogenesis,^3,4 and inflammation.⁵ In addition, recent data indicate that TF/FVIIa interaction may activate different intracellular signals, culminating in a variety of cell responses, thus suggesting a role for TF as a "true" cell membrane receptor. For example, TF/FVIIa interaction has been shown to be a strong chemotactic stimulus for smooth muscle cells (SMCs), mediating their migration in vitro.⁶ FVIIa has been also shown to induce phosphorylation of the extracellular signal–regulated kinases 1 and 2 (ERK 1/2)⁷ and to upregulate poly(A) polymerase,⁸ urokinase-type plasminogen activator receptor,⁹ and a host of genes identified with the use of cDNA microarrays.¹⁰ However, despite evidence suggesting close links between TF, activation of coagulation, and intracellular signaling, no direct effects of TF/FVIIa on SMC proliferation have yet been demonstrated. Thus, in the present study, we investigated whether binding of FVIIa to TF may stimulate proliferation of a cell population widely represented in the arterial wall, such as SMCs. In addition, we investigated the possible molecular mechanisms involved in this phenomenon, evaluating the hypothesis that TF/FVIIa-induced SMC proliferation is the result of signal transduction via activation of the ERKs.

	Methods

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Human recombinant FVIIa and human recombinant active site–blocked factor VII (FVIIai) were a generous gift of Dr Mirella Ezban (Novo Nordisk A/S, Gentofte, Denmark). FVIIai is identical to FVIIa but, because the active site is blocked, is devoid of enzymatic activity.¹¹

Vascular SMC Isolation and Culture
SMCs were isolated from the thoracic aortas of New Zealand White rabbits by nonenzymatic dissociation as previously described.¹² At confluence, cells were identified by immunostaining of smooth muscle -actin with the use of a specific monoclonal antibody labeled with fluorescein (Sigma). Nearly 100% of the cells showed a positive fluorescence. All experiments were performed on cells synchronized in the G₀ phase of the cell cycle. SMCs were subcultured into 24-well culture plates in DMEM containing 10% FCS; at 70% to 80% confluence, cells were made quiescent by incubation in fresh DMEM containing 0.1% FCS for 48 hours.

Effects of FVIIa on SMC Proliferation
The growth effects of FVIIa were quantified by measuring the extent of [³H]thymidine incorporation into cell DNA and by direct cell counting. Thirty minutes after the addition of FVIIa or FVIIai, cells were washed, and fresh medium (DMEM containing 0.1% FCS) was added. Twenty-four hours later, cells were pulsed with 1 µCi [³H]thymidine, and, after 24 additional hours, the culture medium was discarded, and the cells were rinsed twice with PBS and lysed with 0.2% perchloric acid. The contents of the wells were aspirated, 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 unstimulated, serum-deprived SMCs, whereas positive control experiments included SMCs incubated with 10% FCS. In additional parallel experiments, cell counts were determined by a Coulter counter (GEN-S II, Coulter). Briefly, at the end of the experiments, performed as above, cells were washed with PBS and harvested with trypsin/EDTA diluted in PBS, collected, and resuspended in 500 µL of DMEM containing 10% FCS and counted. Six different experiments were performed for each experimental condition.

FVIIa-Induced SMC Proliferation: Molecular Mechanisms
ERK, c-Jun, and Jun N-Terminal Kinase Western Blotting
The following antibodies were used: a polyclonal anti–ERK 1/2 and a mouse monoclonal anti–phospho-ERK from Santa Cruz Biotechnology, Inc. SMCs were seeded onto 100-mm-diameter plastic dishes and starved in serum-free medium as described above. Cells were incubated with 100 nmol/L FVIIa for 1, 2, 5, 15, 20, 30, and 60 minutes. Cells were then washed twice and then lysed in 400 µL of lysis buffer (68.6% wash buffer, 1.5% NP-40, 10% glycerol, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L Na₃VO₄, 2 mmol/L EDTA, 1 mmol/L NaHPO₄, 10 µmol/L leupeptin, 10 mmol/L NaF, pH 7.6) on an ice bath. Protein concentration was determined with the use of Bio-Rad protein assay. Twenty micrograms of total proteins was separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Filters were washed extensively, blocked for 2 hours with 5% nonfat dry milk, and incubated for 1 hour at room temperature with the anti–p-ERK antibody (1:500). The filters were then washed extensively and incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibody (Amersham) (1:1000). Filters were then extensively washed and developed by an ECL detection method (Amersham).

Filters were rehydrated and stripped for 3 minutes in acid solution (0.5 mol/L CH₃COOH, 0.5 mol/L NaCl), neutralized with 0.2 mol/L Tris-HCl (pH 8), and washed extensively. Filters were reblocked, washed, reprobed with anti–ERK-1 polyclonal antibody (1:500), anti–phospho–c-Jun ser 63 diluted (1:5000), or anti–phospho–Jun N-terminal kinase (JNK) (1:3000), processed as above, and finally developed with the use of ECL.

In another set of experiments, SMCs were incubated, 15 minutes before FVIIa was added to the medium, with the monoclonal antibody against rabbit TF, AP-1 (100 µg/mL), or with 100 nmol/L of FVIIai. SMCs were processed as described above to evaluate activation of ERK.

Three different experiments were performed for each experimental condition.

ERK Immunofluorescence
Immunofluorescence studies were performed on cells seeded at low confluence on glass coverslips coated with poly-L-lysine (15 µg/mL), serum-starved for 48 hours, and then stimulated with 100 nmol/L FVIIa. At baseline or at 5 minutes after stimulation, SMCs were washed twice with PBS, fixed for 20 minutes with 4% paraformaldehyde in PBS at room temperature, washed twice in PBS containing 50 mmol/L NH₄Cl, incubated for 5 minutes in methanol at –20°C, and finally blocked for 30 minutes in PBS containing 1% BSA. Cells were then incubated for 1 hour with a polyclonal antibody against ERK-1 and a monoclonal antibody against p-ERK. In parallel experiments, cells were pretreated with UO126 (10 µmol/L). Cells were incubated for 30 minutes with rhodamine-tagged goat anti-mouse and fluorescein-tagged anti-rabbit secondary antibody (1:50) (Jackson ImmunoResearch). In control experiments, SMCs treated as described above were probed only with the secondary antibodies. Coverslips were mounted on a microscope slide and examined with a Zeiss Axiophot or Zeiss Laser Scanner 501 microscope. Three different experiments were performed for each experimental condition.

Statistical Analysis
Data are presented as mean±SD. Differences between groups were determined by a 1-way ANOVA followed by a Student t test with Bonferroni correction. A probability value <0.05 was considered statistically significant.

	Results

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FVIIa Induces SMC Proliferation
To investigate the effects of FVIIa on SMC proliferation, cells were stimulated with increasing concentrations of FVIIa. To determine whether FVIIa requires binding to TF to induce proliferation, SMCs were pretreated, 15 minutes before FVIIa was added to the medium, with AP-1 (100 µg/mL), a monoclonal antibody against rabbit TF.¹³ This concentration exceeds 10 times that necessary to double prothrombin time in rabbit plasma.¹³ In addition, to determine whether FVIIa enzymatic activity is also required to promote SMC proliferation, cells were incubated with 100 nmol/L of FVIIai. Results are expressed as percent increase in [³H]thymidine incorporation with respect to negative control experiments, represented by unstimulated, nonproliferating cells kept under conditions of serum deprivation. As per protocol design, proliferation of these cells was considered to be 0%.

FVIIa caused a dose-dependent increase in [³H]thymidine incorporation, reflecting cell proliferation, up to 2-fold of the control (Figure 1). Similarly, a dose-dependent increase in cell number in response to FVIIa was observed in parallel experiments employing direct cell counting (Figure 1). Binding of FVIIa to TF appeared to be a strict requirement to induce proliferation because pretreatment with AP-1 completely inhibited proliferation. Similarly, the enzymatic activity of FVIIa was also required to activate proliferation because incubation of SMCs with FVIIai did not result in any significant proliferation (Figure 1). The hypothesis that the proliferative effects of FVIIa could be mediated, at least in part, via formation of other downstream coagulation enzymes also known to trigger cell proliferation, such as FXa and thrombin, was tested in additional experiments performed in the presence of tick anticoagulant peptide (TAP) (1 µmol/L) or hirudin (0.1 mg/mL); neither TAP nor hirudin decreased significantly FVIIa-induced SMC proliferation (Figure 2). Similarly, the role of the LDL receptor–related protein (LRP), a multifunctional endocytic and signaling receptor capable of binding both TF/FVIIa and TF/FVIIa/FXa/TF pathway inhibitor (TFPI) complexes,¹⁴ was also excluded in other experiments performed after incubation of SMCs with recombinant receptor-associated protein (RAP) (2 µmol/L, Progene), a universal antagonist for ligand binding to LRP. Additionally, in these experiments FVIIa-induced proliferation remained unchanged after pretreatment with RAP (Figure 2). Finally, we also demonstrated that TFPI can inhibit FVIIa-induced SMC proliferation, but only after addition of small amounts of FXa, indicating that its inhibitory action was due solely to inhibition of the enzymatic activity of FVIIa in the TF/FVIIa complex, via binding of FXa (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 1. Effects of FVIIa on SMC proliferation, assessed by [3H]thymidine incorporation (A). Thirty minutes after the addition of FVIIa or FVIIai, cells were washed, and fresh medium containing 0.1% FCS was added. Twenty-four hours later, cells were pulsed with 1 µCi [3H]thymidine and, after 24 additional hours, they were lysed, and radioactivity was counted in the cell lysates. Negative control experiments included unstimulated SMCs incubated under conditions of serum deprivation, whereas positive control experiments included SMCs incubated with 10% FCS. In parallel experiments, direct cell counting was used instead of [3H]thymidine incorporation (B). Data are expressed as percent of quiescent, serum-deprived cells. FVIIa induced a dose-dependent increase in SMC proliferation, which was completely blocked by AP-1, a monoclonal antibody against TF, and FVIIai, which has the same affinity for TF as native FVIIa but is devoid of enzymatic activity. Each bar represents the mean±SD of 6 experiments; *P<0.01 vs FVIIa at each dose.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of different antagonists of the coagulation cascade and inhibitors of LRP on FVIIa-induced SMC proliferation. SMCs were stimulated with 100 nmol/L FVIIa either alone or in the presence of the following inhibitors: TAP (1 µmol/L), an inhibitor of FXa; hirudin (1 µmol/L), a direct thrombin inhibitor; RAP (2 µmol/L), a universal antagonist for ligand binding to LRP; and TFPI (100 nmol/L), either alone or in the presence of small amounts of FXa. The proliferative effects of FVIIa were not significantly affected by TAP and hirudin, ruling out the possibility of an indirect effect mediated by the formation of FXa or thrombin. Similarly, the contribution of LRP to FVIIa-induced proliferation could be excluded by pretreatment with RAP. TFPI alone also had no effect, whereas in the presence of small amounts of FXa it completely inhibited cell proliferation via inhibition of TF/FVIIa enzymatic activity. Each bar represents the mean±SD of 6 experiments; *P<0.01 vs FVIIa.

FVIIa-Induced SMC Proliferation: Molecular Mechanisms
To elucidate the molecular mechanisms involved in FVIIa-induced SMC proliferation, we examined the role of the ERK pathway and of Jun N-terminal kinase (JNK) in modulating this phenomenon. Moreover, we investigated whether mitogen-activated protein (MAP) kinase activation induced by FVIIa leads to SMC proliferation involving c-Jun, a component of the AP-1 transcription factor. Finally, we evaluated whether UO126, an inhibitor of ERK phosphorylation (Roche), can block FVIIa-induced SMC proliferation. Figure 3 shows activation of different MAP kinases in SMCs exposed to 100 nmol/L FVIIa for different time periods: ERK activation (phosphorylation) is evident at 2 minutes, peaks at 5 minutes, and starts declining at 30 minutes. Pretreatment with AP-1 or stimulation with FVIIai did not result in any appreciable activation of ERK. Similarly to ERK, phosphorylation of JNK and c-Jun was closely correlated with ERK phosphorylation. Interestingly, preincubation of cells with UO126, a selective inhibitor of ERK phosphorylation, completely inhibited SMC proliferation (Figure 4).

View larger version (24K): [in this window] [in a new window]   Figure 3. Time course of ERK, JNK, and c-Jun activation in SMCs stimulated with FVIIa. SMCs were starved in serum-free medium and incubated with 100 nmol/L FVIIa. Cells were then washed twice lysed on an ice bath. Twenty micrograms of total proteins was solubilized in Laemmli buffer, boiled for 5 minutes, and separated on 10% SDS-PAGE gels and then blotted onto nitrocellulose transfer membrane. Filters were probed, stripped, and reprobed with the following antibodies: anti–phospho-ERK, anti–phospho–c-Jun, anti–phospho-JNK, and anti–-tubulin. An appropriate secondary, horseradish peroxidase–conjugated antibody was then used. Control experiments included SMCs preincubated with the monoclonal antibody against rabbit TF, AP-1 (100 µg/mL), and SMCs incubated with 100 nmol/L of FVIIai. Three different experiments were performed for each experimental condition. A time-dependent activation of ERK and JNK was observed, starting at 2 minutes and peaking at approximately 5 minutes; a slight delay in the activation of c-Jun was also observed. Pretreatment with AP-1 or stimulation with FVIIai did not result in any appreciable activation of ERK.

View larger version (19K): [in this window] [in a new window]   Figure 4. Effects of UO126, a selective inhibitor of ERK phosphorylation, on FVIIa-induced SMC proliferation assessed by [3H]thymidine incorporation and expressed as percent of quiescent, serum-deprived cells. Each bar represents the mean±SD of 6 experiments; *P<0.01 vs FVIIa.

Immunofluorescence experiments performed in intact cells showed that 5 minutes after stimulation with FVIIa, an increase in p-ERK with a more intense perinuclear staining occurred compared with baseline (Figure 5). Interestingly, treatment of SMCs with the ERK inhibitor UO126 caused a significant reduction with a different localization of the phosphorylated (activated) form of ERK.

View larger version (65K): [in this window] [in a new window]   Figure 5. SMC immunostaining for total ERK and phospho-Erk (which represents the activated form) at baseline (A and B, respectively) and at 5 minutes after stimulation with FVIIa (C and D). Cells were seeded at low confluence on glass coverslips, serum-starved for 48 hours, and then stimulated with 100 nmol/L FVIIa. At baseline or at 5 minutes after stimulation, SMCs were fixed with 4% paraformaldehyde and incubated for 1 hour with a polyclonal antibody against ERK-1 and a monoclonal antibody against p-ERK. In parallel experiments, cells were pretreated with UO126 (10 µmol/L). Cells were then incubated with rhodamine-tagged goat anti-mouse and fluorescein-tagged anti-rabbit secondary antibody. FVIIa induced an intense activation of p-ERK (D) with respect to unstimulated cells (B). Pretreatment with UO126, a selective inhibitor of ERK activation, significantly reduced ERK phosphorylation both at baseline (F) and 5 minutes after FVIIa stimulation (H).

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
TF, a 47-kDa transmembrane glycoprotein, is the primary initiator of coagulation, which serves as a cell-associated, nonenzymatic cofactor for plasma FVII(a).^1,2 For many years, therefore, the primary function of TF was considered to be exclusively related to the initiation of coagulation.¹⁵ In recent years, however, it became evident that TF has additional biological functions apart from activating coagulation. One of the first indications for a nonhemostatic function of TF was its characterization as an immediate early gene that can be induced during cell division¹⁶ and in response to pathophysiologically relevant stimuli, such as cytokines,¹⁷ growth factors,¹⁸ oxidized LDL,¹⁹ and oxygen free radicals.²⁰

An exciting recent development in TF biology was the demonstration that this protein, a member of the cytokine receptor superfamily, can function as a true signaling receptor. Indeed, signaling via TF has now been documented in many different cell types, including keratinocytes, bladder carcinoma cells, COS-1, and BHK cells.^5–10 However, those studies, although useful for increasing our knowledge of TF biology, have been performed on cells that do not constitutively express TF but rather have been transfected with a human TF cDNA or on cells not related to the arterial wall. Data of the present study demonstrate, for the first time, that TF signaling may also occur in SMCs, a cell population largely present in the arterial wall and often involved in the formation of atherosclerotic plaques and constitutively expressing TF, thereby reproducing more physiological conditions.

Interaction between FVIIa and TF culminates in SMC proliferation via activation of the p44/42 MAP kinase (ERK 1/2) and JNK pathways. Interestingly, our data also indicate that 2 conditions are necessary to activate cell proliferation: (1) FVIIa must bind TF on the cell surface because blocking of FVIIa binding to TF by the monoclonal anti-TF antibody, AP-1, resulted in a complete inhibition of cell proliferation; and (2) the catalytic activity of FVIIa must be preserved because the inactivated form of FVIIa (FVIIai) did not result in any significant proliferation.

Another interesting finding of the present study is that the specific inhibitor of the ERK pathway, UO126, completely inhibited FVIIa/TF-induced SMC proliferation. This finding indicates that, despite the simultaneous activation of ERK and JNK pathways, ERK phosphorylation seems to be the probable main route to FVIIa-induced cell proliferation. This pivotal role of ERK activation in promoting SMC proliferation induced by FVIIa is also further supported by the time course of c-Jun activation observed in our study. It is known, in fact, that Fos and Jun proteins combine to form stable AP-1 heterodimers, which bind to the AP-1 consensus sequence and activate several genes associated with cell proliferative response.

Data of the present study do not allow one to indicate the mechanism(s) by which the TF/FVIIa complex transmits the signal to the cytoplasm. Some studies reported that part of the biological functions of TF require phosphorylation of its cytoplasmic tail,²¹ whereas others did not.²² Indeed, in the present study, because of the requirement that factor VIIa must be enzymatically active to support cell proliferation, one intriguing possibility is that the TF/VIIa complex might signal through other membrane-associated molecules that specifically recognize the TF/FVIIa complex. One possible candidate in this respect is represented by the LRP, a multiligand, endocytic, and signaling receptor that recognizes at least 30 different ligands, mostly proteinases and proteinase-inhibitor complexes, including FVIIa and TFPI, the natural inhibitor of the TF-dependent coagulation pathway.¹⁴ However, the experiments in which LRP-binding properties to its numerous ligands were inhibited by RAP have shown that FVIIa-induced cell proliferation was not affected, ruling out the contribution of LRP to the proliferative effect of FVIIa. These observations suggest that FVIIa-induced cell proliferation might be the consequence of proteolytic activation of another signaling receptor via activation of members of the protease-activated receptor (PAR) family.²³ Further support for this hypothesis is given by the observation that targeted deletion of the cytoplasmic tail of TF in mice does not affect the normal development of the animals,²⁴ thus leaving transactivation of PAR as the only possible mechanism responsible for TF/FVIIa-mediated cell proliferation.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
The data of the present study describe the course of events linking activation of coagulation, cell signaling, and SMC proliferation. All these events may occur in the vessel wall after plaque rupture. Although previous reports have demonstrated the activation of MAP kinases in arteries in animal models of balloon injury and neointimal formation,^25–28 our study provides evidence that TF can link activation of blood coagulation and SMC proliferation. In this respect, inhibition of these new functions of TF may represent a novel, attractive therapeutic target.

	Acknowledgments

 
The authors thank Dr Antonio Porcellini (Department of Experimental Medicine and Pathology "La Sapienza" University, Rome, Italy) for reviewing the manuscript. Francesco D’ Agnello and Mario Berardone are also gratefully acknowledged. Finally, the continuous support of BIOGEM is appreciated.

	Footnotes

 
*The first 2 authors contributed equally to this work.

Presented in part at the 52nd Annual Scientific Session of the American College of Cardiology, Chicago, Ill, March 29 to April 2, 2003.

	References

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