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(Circulation Research. 2004;94:340.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Thrombin- and Factor Xa–Induced DNA Synthesis Is Mediated by Transactivation of Fibroblast Growth Factor Receptor-1 in Human Vascular Smooth Muscle Cells

Bernhard H. Rauch, Esther Millette, Richard D. Kenagy, Guenter Daum, Alexander W. Clowes

From the Department of Surgery, University of Washington School of Medicine, Seattle, Wash.

Correspondence to Bernhard H. Rauch, MD, University of Washington School of Medicine, Department of Surgery, Box 356410, 1959 NE Pacific St, Seattle, WA 98195-6410. E-mail brauch{at}u.washington.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin and factor Xa (FXa) are agonists for G protein–coupled receptors (GPRCs) and may contribute to vascular lesion formation by stimulating proliferation of vascular smooth muscle cells (SMCs). Mitogenic signaling of GPCRs requires transactivation of receptor tyrosine kinases (RTKs). In rat SMCs, thrombin transactivates the epidermal growth factor receptor (EGFR) via a pathway that involves heparin-binding EGF-like growth factor (HB-EGF) as ligand for EGFR. The purpose of this study was to investigate in human SMCs the role of receptor transactivation in the mitogenic response to thrombin and FXa. Thrombin (10 nmol/L) and FXa (100 nmol/L) cause a 3.3- and 2.6-fold increase in DNA synthesis, respectively. In human SMCs, neither thrombin nor FXa causes EGFR phosphorylation, and blockade of EGFR kinase does not inhibit DNA synthesis. However, DNA synthesis and phosphorylation of fibroblast growth factor receptor-1 (FGFR-1) induced by thrombin or FXa are inhibited by antibodies neutralizing basic fibroblast growth factor (bFGF) or by heparin. Hirudin inhibits thrombin-, but not FXa-induced mitogenesis, indicating that FXa acts independently of thrombin. We further demonstrate by ELISA that upon thrombin and FXa stimulation, bFGF is released and binds to the extracellular matrix. Our data suggest that in human vascular SMCs, both thrombin and FXa rapidly release bFGF into the pericellular matrix. This is followed by transactivation of the FGFR-1 and increased proliferation. Heparin may inhibit the mitogenic effects of thrombin and FXa in human SMCs by preventing bFGF binding to FGFR-1.

Key Words: thrombin • factor Xa • basic fibroblast growth factor • fibroblast growth factor receptor-1 • epidermal growth factor receptor

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular smooth muscle cell (SMC) proliferation and migration are key events in atherosclerosis and restenosis after vascular injury.^1,2 Mitogenic signaling of G protein–coupled receptors (GPCRs) involves transactivation of receptor tyrosine kinases (RTKs).³ In several cell lines, it has been shown that the GPCR agonist thrombin mediates cell proliferation through transactivating the epidermal growth factor receptor (EGFR) via a metalloproteinase-mediated cleavage and release of pro-heparin binding EGF-like factor (HB-EGF), which then binds to EGFR.³ We have shown that this mechanism is present in rat SMCs and is required for thrombin-induced migration.⁴ Heparin, which inhibits SMC proliferation and migration in vivo and in vitro,^5–7 binds HB-EGF and interferes with this pathway.⁴ We have investigated the possible role of receptor transactivation in the proliferation of human SMCs mediated by thrombin and the activated coagulation factor X (FXa). FXa is a serine protease that in addition to cleaving prothrombin activates thrombin receptors (protease-activated receptors, PARs), which are members of the GPCR family.⁸ FXa acts as a thrombin-independent mitogen,^9,10 which is also sensitive to heparin inhibition.¹¹ In this study, we demonstrate that proliferation of human SMCs induced by thrombin and FXa does not involve EGFR transactivation, but the autocrine release of basic fibroblast growth factor and activation of the fibroblast growth factor receptor-1 (FGFR-1). This FGFR activation is inhibited by heparin and might in part account for the inhibitory effect of heparin on human SMC proliferation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Antibodies (Abs) against FGFR-1 and EGFR were from Santa Cruz Biotechnology (Santa Cruz, Calif). The phosphotyrosine Ab (clone 4G10) was from Upstate Biotechnology (Lake Placid, NY). EGF and the tyrphostin AG1478 were from Calbiochem (San Diego, Calif). Protein A–agarose was from Roche Diagnostics (Indianapolis, Ind). Neutralizing Ab against human basic fibroblast growth factor (bFGF) was a generous gift from Dr Michael A. Reidy (University of Washington, Seattle, Wash). Human -thrombin was from American Diagnostica (Greenwich, Conn), and human factor Xa was from Enzyme Research Laboratories (South Bend, Ind). The bFGF ELISA kit was from R&D Systems (Minneapolis, Minn). Recombinant bFGF and heparin (porcine intestinal mucosa) were from Sigma-Aldrich (St Louis, Mo). Batimastat (BB94) was from British Biotech (Oxford, UK). Activating peptides (APs) for PARs were from Anaspec (San Jose, Calif): AP-1, TFFLRN (PAR-1), AP-2, SLIGKV (PAR-2), AP-3, TFRGAP (PAR-3), and AP-4, AYPGKF (PAR-4).¹²

Cell Culture
Human aortic SMCs were prepared by the explant technique as described previously.⁴ Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 200 U/mL penicillin, and 200 µg/mL streptomycin. Cells at passages 5 to 12 were used for experiments.

Immunoprecipitation and Western Blotting
Immunoprecipitation (IP) of EGFR was carried out as previously described.⁴ For IP of FGFR-1, cells from 100-mm culture dishes were harvested in 1 mL ice-cold HEB buffer (25 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L NaF, 2 mmol/L sodium vanadate, 1 mmol/L benzamidine, 0.1% 2-mercaptoethanol, 1% Nonidet P-40, 1 mmol/L pepstatin A, 25 µg/mL leupeptin, and 20 kallikrein inhibitor units/mL aprotinin, and 1% saturated PMSF solution [in isopropanol]) and lysed for 30 minutes on ice. The lysates were cleared by centrifugation at 10 000g for 5 minutes in a microfuge. Three micrograms of Abs was added to the supernatants and incubated overnight at 4°C with constant agitation, then 15 µL protein A slurry was added for 1 hour at 4°C. Beads were washed once in HEB and boiled in 30 µL Laemmli sample buffer. Immunocomplexes were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunodetection was performed with the enhanced chemiluminescence kit (Amersham) according to the manufacturer’s protocol.

DNA Synthesis
SMCs at 60% to 80% confluence were incubated for 48 hours in media without fetal bovine serum followed by a change of serum-free media for another 24 hours. Cells were stimulated by the addition of thrombin, FXa, or PAR agonistic peptides. [³H]thymidine (1 µCi/mL) was added 16 to 18 hour after stimulation. After 26 to 28 hours, cells were washed 3 times with ice-cold PBS followed by incubation in 10% trichloroacetic acid (TCA) overnight at 4°C. Cells were washed in TCA, and DNA was solubilized in 0.1 N NaOH. Radioactivity was measured by liquid scintillation counting.

Enzyme-Linked Immunosorbant Assay (ELISA)
Levels of bFGF were determined using an ELISA according to the manufacturer’s instructions. SMCs in 6-well plates were incubated in serum-free media for 72 hours. After stimulation, media was removed and cells were incubated with 1 mL of 10 µg/mL heparin in PBS for 20 minutes at room temperature on a rocking shaker. The heparin solution was removed, cells were detached with trypsin/EDTA, washed with PBS, and cell pellets were lysed in 1 mL HEB. Media, heparin wash, and cell lysates were stored at -80°C.

Statistics
All experiments were performed at least three times in duplicate or triplicate. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s multiple comparison test or by a paired two-tailed t test as indicated. Values of P<0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin and FXa Do Not Transactivate EGFR in Human Vascular SMCs
In human aortic SMCs, we found no tyrosine phosphorylation of EGFR after stimulation with thrombin (10 nmol/L) or FXa (100 nmol/L) (Figure 1A). Treatment with EGF (10 ng/mL) caused a strong phosphorylation of EGFR, indicating the presence of the receptor in these cells. In addition, thrombin- and FXa-induced DNA synthesis was not affected by the specific EGFR receptor kinase inhibitor AG1478 (150 nmol/L). In comparison to thrombin and FXa, EGF was a weaker mitogen, and EGF-induced DNA synthesis was inhibited by AG1478 (Figure 1B). These findings suggest that EGFR is functional in human SMCs, but that thrombin and FXa do not transactivate EGFR.

View larger version (38K): [in this window] [in a new window]   Figure 1. Thrombin and FXa do not transactivate EGFR in human SMCs. A, EGFR was immunoprecipitated from nonstimulated human SMCs (Con) or cells treated with EGF (10 ng/mL), FXa (100 nmol/L), and thrombin (10 nmol/L) for 10 minutes. Immunocomplexes (IP) were analyzed by Western blotting (IB) for phosphotyrosine (p-tyr). B, DNA synthesis was determined by [3H]thymidine incorporation as described in Materials and Methods. Cells were pretreated with the EGFR kinase inhibitor AG1478 (150 nmol/L) 30 minutes before stimulation. Values are mean±SEM of 6 independent experiments. *P<0.05 (t test).

Thrombin- and FXa-Induced DNA Synthesis Is Mediated by bFGF in Human Vascular SMCs
Because proliferation mediated by thrombin or FXa is inhibited by heparin, and heparin blocks FGF binding to cell surface receptors, the contribution of bFGF to thrombin- and FXa-induced DNA synthesis was investigated. SMCs were preincubated with increasing concentrations of neutralizing bFGF antibody (3 to 30 µg/mL).¹³ We found a concentration-dependent inhibition of DNA synthesis stimulated by FXa or thrombin, whereas nonspecific IgG (30 µg/mL) had no effect (Figure 2). Preincubation of cells with heparin (100 µg/mL) showed an inhibitory effect comparable to bFGF neutralization (Figure 2). In contrast to thrombin-induced mitogenesis, FXa-induced DNA synthesis was not inhibited by hirudin, demonstrating that FXa acts as a thrombin-independent mitogen.

View larger version (45K): [in this window] [in a new window]   Figure 2. Thrombin- and FXa-induced DNA synthesis is mediated by bFGF. DNA synthesis in human SMCs induced by FXa (100 nmol/L) or thrombin (10 nmol/L) was determined by [3H]thymidine incorporation. Cells were preincubated either with bFGF-neutralizing antibodies (3, 10, and 30 µg/mL), heparin (100 µg/mL), nonspecific IgG (30 µg/mL), or hirudin (10 µmol/L) 30 minutes before stimulation. Values are mean±SEM of 4 to 6 independent experiments. *P<0.05 for controls (black bars) vs indicated experimental groups (ANOVA).

Thrombin and FXa Activate FGFR-1 in Human Vascular SMCs
FGFR-1 was immunoprecipitated from cell lysates as described in Materials and Methods. Western blotting with anti-phosphotyrosine antibodies revealed bands of approximately 120 and 140 kDa, corresponding to FGFR-1 and ß isoforms known to contain tyrosine phosphorylation sites.¹⁴ Tyrosine phosphorylation of FGFR-1 was detected 15 minutes after stimulation with either FXa or thrombin. FGFR-1 phosphorylation was inhibited by bFGF-neutralizing antibody and heparin, but not by nonspecific IgG (Figure 3A). Thrombin- but not FXa-induced FGFR-1 phosphorylation was inhibited by preincubation with hirudin (Figure 3B). The matrix metalloproteinase (MMP) inhibitor batimastat did not prevent FGFR-1 phosphorylation by thrombin or FXa (Figure 3B). Recombinant human bFGF was used as a positive control.

View larger version (54K): [in this window] [in a new window]   Figure 3. Thrombin and FXa transactivate FGFR-1 in human SMCs. FGFR-1 immunoprecipitates (IP) of cells stimulated with thrombin (10 nmol/L), FXa (100 nmol/L), or bFGF (20 ng/mL) for 15 minutes were analyzed by Western blotting (IB) for tyrosine phosphorylation (p-tyr). A, Cells were preincubated with bFGF-neutralizing antibodies (30 µg/mL), heparin (100 µg/mL), or nonspecific IgG (30 µg/mL) 30 minutes before stimulation. B, Cells were preincubated with hirudin (10 µmol/L) or batimastat (5 µmol/L) 30 minutes before stimulation. The double band of approximately 120 and 140 kDa presumably represents 2 different FGFR-1 isoforms.14 Blots were reprobed for total FGFR-1.

Thrombin and FXa Release bFGF Into the Extracellular Matrix
Heparan sulfate proteoglycans (HSPGs) function as an extracellular matrix or cell-surface reservoir for bFGF.¹⁵ To determine the localization of bFGF mediating the effect of thrombin and FXa, the cell layer was washed with 10 µg/mL heparin to remove any bFGF bound to the cell membrane or extracellular matrix as has been described.¹⁶ Levels of bFGF were measured by ELISA in the media, in cell lysates, and in the heparin wash solution. Both FXa and thrombin increased bFGF in the heparin wash solution at 15 and 30 minutes (Figure 4A), whereas levels of bFGF in the media and cell lysates were not significantly different between stimulated cells and nonstimulated controls (Figures 4B and 4C, respectively). Addition of heparin before stimulation decreased bFGF bound to the cell layer by 70% (Figure 5A). At the same time, a marked increase of bFGF was measured in the cell culture media (Figure 5B).

View larger version (35K): [in this window] [in a new window]   Figure 4. Thrombin and FXa release bFGF into the extracellular matrix. Basic FGF was removed from the cell layer by washing with heparin solution as described in Materials and Methods. Levels of bFGF in the heparin buffer (A), released into the media (B), and in the cells (C) after treatment with FXa or thrombin were determined by ELISA. Values are mean±SEM of 3 independent experiments. *P<0.05 for controls vs stimulation (ANOVA).

View larger version (24K): [in this window] [in a new window]   Figure 5. Pretreatment with heparin affects localization of bFGF. Cells were stimulated with FXa or thrombin for 15 minutes in the absence or presence of heparin (100 µg/mL). Basic FGF was removed from the cell layer by washing with heparin solution as described in Materials and Methods. Levels of bFGF in the heparin buffer (A) and released into the media (B) after treatment with FXa or thrombin were determined by ELISA. Values are mean±SEM of 4 independent experiments. *P<0.05 (t test).

PAR-1–Induced DNA Synthesis Is bFGF-Dependent
To determine which PARs may contribute to thrombin- and FXa-induced DNA synthesis, cells were stimulated with APs specific for PAR-1, -2, -3, and -4. Only PAR-1 agonistic peptide caused a significant increase in DNA synthesis, which was inhibited by preincubation of SMCs with either bFGF-neutralizing antibodies (30 µg/mL) or heparin (100 µg/mL) (Figure 6, data for PAR-2, -3, and -4 not shown).

View larger version (33K): [in this window] [in a new window]   Figure 6. PAR-1–induced DNA synthesis is bFGF-dependent. Cells were stimulated with activating peptide (AP1, 100 µmol/L) specific for PAR-1. DNA synthesis was determined by [3H]thymidine incorporation. Cells were preincubated either with bFGF-neutralizing antibodies (30 µg/mL), heparin (100 µg/mL), or nonspecific IgG (30 µg/mL) 30 minutes before stimulation with AP-1 (100 µmol/L). Values are mean±SEM of 3 independent experiments. *P<0.05 (ANOVA).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In various cell lines (HEK, COS), as well as in rat SMCs, thrombin transactivates EGFR by a metalloproteinase-dependent pathway, which involves cleavage of HB-EGF as a ligand for EGFR.^3,4,17,18 Whether EGFR transactivation in response to GPCR stimulation by thrombin plays an important role in human SMCs was the subject of this study. In contrast to rat SMCs,⁴ EGFR was not transactivated by thrombin in human SMCs (Figure 1). Because thrombin signaling is inhibited by heparin in SMCs from both species, we tested the possibility that FGF is involved in human SMCs. We found that proliferation induced by thrombin and FXa is inhibited by neutralizing bFGF antibodies (Figure 2). Consistent with a role for bFGF is that both GPCR ligands release bFGF in a time-dependent manner into the pericellular matrix (Figure 4) and stimulate phosphorylation of FGFR-1 (Figure 3). In contrast to EGFR transactivation,⁴ MMP inhibition by batimastat did not inhibit thrombin- or FXa-induced FGFR-1 phosphorylation (Figure 3B). The direct thrombin inhibitor hirudin reduced thrombin- but not FXa-induced DNA synthesis (Figure 2) and FGFR-1 phosphorylation (Figure 3B), which is consistent with previous reports that FXa is a thrombin-independent mitogen.^9,10 These observations suggest that FGFR-1 mediates the mitogenic activity of thrombin and FXa, although we cannot rule out that an additional member of the FGFR family¹⁹ is involved. Basic FGF is a mitogen for cultured SMCs and its contribution to intimal formation after arterial injury has been well described.^13,20–23 Thus, release of bFGF by GPCR ligands may be an important mechanism for the injury response. We found that bFGF is only transiently released with maximal release at 15 to 30 minutes after stimulation. It has been reported that FGFR-1 internalization peaks within 45 minutes after stimulation.²⁴ Thus, binding to its specific receptor and receptor internalization might contribute to the decrease of bFGF in the heparin-exchangeable bFGF fraction.

The mechanism by which bFGF is released from SMCs has not been described yet. Because bFGF lacks a typical amino acid sequence for externalization,²⁵ early studies speculated that bFGF is released on cell damage or by an exocytotic mechanism that is independent of the endoplasmic-reticulum-Golgi pathway.²⁶ Release of bFGF into the extracellular matrix is also induced by shear stress, which might cause a transient disruption of the cell membrane and thereby cause bFGF release.¹⁶ Induction of cell membrane permeability by thrombin and the thrombin receptor (PAR-1) results in increased release of von Willebrand factor,²⁷ which, like bFGF, lacks an amino acid sequence for secretion. Thus, increased cell membrane permeability induced by GPCR agonists appears to be an intriguing possibility for the release of bFGF. Whether von Willebrand factor and bFGF share a common release pathway remains to be determined.

To address which PARs are involved in the mitogenic response of human SMCs, we used PAR-specific agonist peptides. We found that only the PAR-1–specific peptide caused significant DNA synthesis, suggesting that the PAR-induced mitogenic response in human aortic SMCs is mainly PAR-1–dependent. This is consistent with the ability of both thrombin and FXa to activate PAR-1.⁸ Since PAR-1–induced DNA synthesis was inhibited by FGF antibodies and by heparin (Figure 6), we conclude that PAR-1 activation includes a bFGF-dependent pathway and that thrombin- and FXa-induced release of bFGF may be through PAR-1 activation. However, whether other PARs contribute to thrombin and FXa responses and to the release of bFGF and FGFR-1 transactivation requires further evaluation.

Heparan sulfate proteoglycans (HSPGs) bind bFGF with low affinity compared with FGF receptors and are required cofactors for the activation of FGF high-affinity receptors.^15,28 Further, binding to HSPGs protects bFGF from denaturation and proteolytic degradation and provides a matrix-bound or cell-surface reservoir of this factor for the cells.¹⁵ Heparin inhibits FGF signaling by competing with HSPGs for binding bFGF.^15,28 Our data suggest that bFGF is released from the cells after stimulation with thrombin and FXa and binds to HSPGs in the pericellular matrix (Figures 4A and 5 ). However, when high concentrations of heparin are present, binding of bFGF to the matrix is inhibited, indicated by the presence of bFGF in the heparin-containing media (Figure 5). One example of a bFGF binding HSPG is syndecan-4, which is a cofactor for FGFR-1 activation in fibroblasts.²⁹ Recently, it has been reported that bFGF and syndecan-4 may also be involved in the regulation of integrin functions,^30,31 which might be involved in a bFGF-dependent thrombin- and FXa-induced mitogenic response.

In summary, our data indicate that in human SMCs PAR-1 signaling involves activation of FGFR-1. This is different from recent observations in rat SMCs, where thrombin transactivates EGFR. Our study demonstrates that thrombin and FXa cause autocrine FGFR-1 phosphorylation via release of bFGF in human SMCs. This mechanism might contribute to thrombin- and FXa-induced mitogenesis. Our data suggest further that heparin inhibits mitogenesis induced by these stimuli by competing with HSPGs for released bFGF, which prevents FGFR-1 phosphorylation.

	Acknowledgments

 
This study was supported by the NIH (HL-18645) and a fellowship from the German Academy of Nature Scientists Leopoldina to B.H.R. (BMBF-LPD 9901/8-53). The authors are grateful to Dr Michael A. Reidy (University of Washington, Wash) for providing the bFGF-neutralizing antibody and for helpful discussion.

	Footnotes

 
Original received July 29, 2003; resubmission received November 12, 2003; accepted November 26, 2003.

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