Vascular endothelial growth factor production in polymyalgia rheumatica

Vascular Endothelial Growth Factor Production by Fibroblasts in Response to Factor VIIa Binding to Tissue Factor Involves Thrombin and Factor Xa V. Ollivier, J. Chabbat, J.M. Herbert, J. Hakim, D. de Prost Abstract—Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti– effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells. (Arterioscler Thromb Vasc Biol. 2000;20:1374-1381.) Key Words: vascular endothelial growth factor 䡲 tissue factor 䡲 activated factor VII 䡲 fibroblasts 䡲 proteases

T

issue factor (TF), the cell-surface receptor for activated factor VII (FVIIa), is the primary regulator of blood coagulation. The TF-FVIIa complex cleaves and activates factors IX and X into factors IXa and factor Xa (FXa), respectively, which lead to thrombin generation.1 Beyond its role as a procoagulant activator, TF participates in other cellular processes, including metastasis,2 tumor-associated angiogenesis,3 and embryogenesis.4 Accordingly, several cell functions are modified in response to FVIIa binding to TF, its receptor. These include intracellular signaling,5 activation of the p44/42 mitogen-activated protein (MAP) kinase pathway,6 induction of tyrosine phosphorylation in monocytes,7 upregulation of poly(A) polymerase,8 cell spreading and phosphorylation of focal adhesion kinase,9 enhanced expression of urokinase receptor by pancreatic cancer cell lines,10 and vascular endothelial growth factor (VEGF) production by human fibroblasts.11 The respective roles of the procoagulant activity of the TF-FVIIa complex and of intracellular signals

transmitted by the intracellular domain of TF are not fully understood. In most of the above-mentioned studies, the reported effect required catalytically active FVIIa to bind to TF. The TF-FVIIa complex may act by activating clotting factors downstream in the coagulation cascade; indeed, Fisher et al2 showed that TF-initiated thrombin generation activates signaling via the thrombin receptor on malignant melanoma cells. In other studies, an FVIIa-specific reaction independent of activated clotting factors appeared to be involved.6,10 VEGF is a key regulator of angiogenesis, stimulating endothelial cell proliferation and migration and increasing their permeability. We recently showed that TF was essential for the signaling events leading to VEGF synthesis by human lung fibroblasts11 in response to a plasma-derived human FVIIa concentrate (ACSET, LFB) and that this effect was mostly dependent on the proteolytic activity of the TF-FVIIa complex. It was unclear whether this was a direct effect of FVIIa protease or an indirect effect involving production of activated clotting

Received September 17, 1999; revision accepted November 11, 1999. From INSERM U479 (V.O., J.H., D.d.P.), Faculte´ Xavier Bichat, Paris; LFB Recherche et De´veloppement (J.C.), Les Ulis; Haemobiology Research Department (J.M.H.), Sanofi Recherche, Toulouse; and Service d’He´matologie et d’Immunologie Biologiques (D.d.P.), Hoˆpital Louis Mourier, AP-HP, Colombes, France. Correspondence to Dr D. de Prost, Hoˆpital Louis Mourier, Service d’He´matologie et d’Immunologie Biologiques, 178, rue des Renouillers, 92700 Colombes, France. E-mail [email protected] © 2000 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

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factors, such as thrombin or FXa. We now report that FXa and thrombin generation are involved in TF-FVIIa– dependent VEGF production by human fibroblasts.

method was used to count cells present in the well at the time of the assay, as previously described.11

Methods

Thrombin and FXa generated in the culture medium of ACSETtreated fibroblasts were quantified by hydrolysis of the thrombinand FXa-sensitive chromogenic substrates S-2238 and S-2765, respectively. Changes in absorbance at 405 nm (⌬A405/min) were measured for 30 minutes at 37°C on an ELISA plate reader (Molecular Devices). A calibration curve was constructed by using serial dilutions of thrombin (Sigma) or FXa (ERL).

Reagents Recombinant human FVIIa12 and Phe-Phe-Arg chloromethylketone– inactivated FVIIa (FVIIai)13 were kindly provided by Novo Nordisk (Gentofte, Denmark). Recombinant tick anticoagulant peptide (TAP) and the nematode anticoagulant peptides (NAPs) 5 and c2 were generously provided by Dr G.P. Vlasuk (Corvas Inc, San Diego, Calif). TAP and NAP5 are potent and specific inhibitors of FXa and prothrombinase activity; NAPc2 is a potent and specific inhibitor of the TF-FVIIa complex, which requires prior interaction with FXa.14 Recombinant hirudin was a gift from M. Lenoble (Hoechst, Paris, France). A pool of neutralizing anti-TF antibodies (TF8-5G9, TF86B4, and TF9-9C3) was generously provided by T.S. Edgington and N. Mackman (The Scripps Research Institute, La Jolla, Calif).15 Human FXa was purchased from Enzyme Research Laboratories. An antibody to the effector protease receptor-1 (EPR-1), the firstgeneration monoclonal antibody B6,16 was generously given by Dr D.C. Altieri (Yale University School of Medicine, New Haven, Conn). The interepidermal growth factor sequence L83GTRKL88(G) and its control scrambled variant K83FTGRLL88 were synthesized by Neosystem. Human thrombin was purchased from Sigma, and the thrombin receptor agonist peptide (TRAP, H-Ser-Phe-Leu-Leu-ArgAsn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-OH) was from CalbiochemNovabiochem. Chromogenic substrates S-2765 and S-2238 were purchased from Chromogenix. DX9065a was kindly given by Daichi Pharmaceuticals Co Ltd (Tokyo, Japan).

ACSET ACSET was prepared as previously described.11 The ACSET preparation contains 30% FVIIa and 70% factor VII (FVII); contaminating proteins include protein S, prothrombin, and factor X (FX).

Human FX Isolation The FX-containing fraction was obtained during the isolation of factor IX (FIX) and corresponded to the 0.28 mmol/L NaCl eluate,17 which was applied onto a ceramic hydroxyapatite column (Bio-Rad) equilibrated with 10 mmol/L K2HPO4 and 75 mmol/L NaCl (pH 8). After a wash with 30 mmol/L K2HPO4 and 0.25 mmol/L NaCl (pH 8), FX was eluted with 0.5 mmol/L K2HPO4 (pH 8) and then dialyzed overnight against 20 mmol/L Tris and 0.15 mmol/L NaCl (pH 7) at 4°C. To remove prothrombin, the FX-containing fraction was adsorbed to a chelating Sepharose Fast Flow column saturated with Cu2⫹ and equilibrated with 10 mmol/L K2HPO4, 10 mmol/L sodium citrate, and 0.5 mmol/L NaCl (pH 6.5). After a wash with the equilibration buffer and then with a solution containing 10 mmol/L sodium citrate, 10 mmol/L potassium phosphate, and 0.1 mmol/L sodium chloride (pH 7), FX was eluted with the same buffer containing 5 mmol/L glycine. FX exhibited a specific activity of 130 IU/mg protein and appeared as 1 protein band of 57 kDa after 12% SDS-PAGE. FVII:Ag and FIX:Ag, evaluated by ELISA (Diagnostica Stago), were ⬍1 ng/mL and ⬍0.3 ␮g/mL, respectively. Furthermore, this fraction had no amidolytic activity on S-2765 and S-2238 chromogenic substrates, demonstrating the absence of contaminating FXa and thrombin, respectively.

Cells and Culture Human lung fibroblasts (CCD-11Lu) were obtained from the American Type Culture Collection and grown as described.11 Subconfluent fibroblasts were starved for 24 hours in DMEM without FCS and subsequently incubated with human FVIIa in DMEM supplemented with 6 mmol/L CaCl2. Cell viability, monitored by lactate dehydrogenase release, was not altered by any of the experimental conditions (not shown).

FXa and Thrombin Generation Assays

Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

VEGF RT-PCR Total RNA was extracted from fibroblast cultures by using TRIzol Reagent (Life Technologies). A semiquantitative competitive reverse transcription (RT)–polymerase chain reaction (PCR) method was used as described elsewhere.11 A PCR MIMIC construction kit (Clontech Laboratories) was used to correct for variations in amplification efficiency in each reaction and to calculate relative changes in mRNA levels. The density of each band was normalized to the density of the mimic band and plotted in arbitrary units.

Measurement of Intracellular Calcium Increase Fibroblasts cultured as described above were detached with nonenzymatic cell dissociation solution (Sigma), scraped from the flasks, and centrifuged (400g for 10 minutes). Cells were then suspended in culture medium containing 5 ␮mol/L fura 2-AM and incubated for 10 minutes at 25°C. Thereafter, cells were first washed in culture medium and then in buffer (10 mmol/L HEPES/NaOH [pH 7.4], 137 mmol/L NaCl, 5.4 mmol/L KCl, 0.34 mmol/L Na2HPO4, 0.44 mmol/L KH2PO4, 0.8 mmol/L MgSO4, 5.5 mmol/L glucose, and 4.2 mmol/L NaHCO3) containing 0.1 mmol/L EGTA (to avoid cell adhesion) and kept in the dark at room temperature. Experiments were carried out with constant stirring in a Perkin-Elmer LS50 B spectrofluorometer with the use of ⬇300 000 cells in 3-mL fluorescence cuvettes at 37°C. Increasing concentrations of FXa were added to fura 2–loaded fibroblasts and incubated for 1 minute. [Ca2⫹]i was measured as described by Grynkiewicz et al.18

Western Blot Detection of Active MAP Kinases After 24-hour FCS starvation, confluent fibroblasts were stimulated with various ligands for the indicated periods of time (1 well of a 6-well plate per experimental point). Cells were then lysed in lysis buffer already described elsewhere,19 and lysates were loaded onto an 8% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The total amount of p44/42 MAP kinases was detected by using a cocktail of extracellular signal–regulated kinase (ERK)-1 and ERK-2 antibodies (Santa Cruz Biotechnology, Inc). Phosphorylated p44/42 MAP kinases were detected by using a phosphop44/42 MAP kinase monoclonal antibody (Biolabs).

Statistical Analysis Results are given as mean⫾SD. Data were compared by ANOVA. Where significant differences were inferred, the sample means were compared by the Fisher protected least significant difference test.

Results Effect of FVIIa on VEGF Secretion

Incubation of human lung fibroblasts constitutively expressing TF on their surface for 24 hours with 100 nmol/L FVIIa (a plasma-derived FVIIa concentrate provided by ACSET) resulted in a 4-fold induction of VEGF secretion (from Estimation of Cellularity 98⫾29 with control cells to 391⫾145 pg/mL, P⬍0.0001), To avoid changes in VEGF production due to differences in cell Downloaded from http://atvb.ahajournals.org/ by guest July 24,results 2015 11 (Figure 1A). proliferation according to the experimental conditions, a colorimetric confirming ouron previous

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Effect of Hirudin and FXa Inhibitors on ACSET-Induced VEGF Secretion To determine the role of thrombin and FXa in ACSETinduced VEGF production, we tested the effect of hirudin (the strongest known naturally occurring inhibitor of thrombin) and that of TAP and DX9065a (specific and direct FXa inhibitors). ACSET-induced VEGF secretion was inhibited by 55⫾25% by 10 U/mL hirudin, pointing to an important role of thrombin in this effect (Figure 1A). TAP and DX9065 caused 57⫾23% and 48⫾13% inhibition, respectively. Interestingly, the simultaneous addition of TAP and hirudin or of DX9065a and hirudin blocked 79⫾15% and 83⫾9% of VEGF secretion, respectively, in response to ACSET, suggesting that the bulk of the effect of ACSET was mediated by thrombin and FXa. DX9065a and TAP or hirudin had no effect on unstimulated cells; the baseline VEGF secretion is not modified in the presence of these FXa and thrombin inhibitors.

Thrombin Induces VEGF Secretion by Fibroblasts Figure 1. A, Effect of hirudin (Hir) and FXa inhibitors on ACSETinduced VEGF secretion. Confluent fibroblasts were incubated with 10 U/mL Hir, 10 ␮mol/L DX9065a (DX), 50 ␮g/mL TAP, or Hir in combination with either DX or TAP for 30 minutes at 37°C before a 24-hour incubation at 37°C with 100 nmol/L ACSET. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in treated fibroblasts compared with unstimulated fibroblasts (NS). Each point represents the mean⫾SD of at least 3 different determinations, each performed in triplicate. *P⬍0.0001 vs unstimulated fibroblasts. ⽧P⬍0.05 vs ACSET-stimulated fibroblasts. B, Kinetics of thrombin and FXa generation in the culture medium of confluent human fibroblasts incubated with 100 nmol/L ACSET. Thrombin and FXa generation were assessed by hydrolysis of the thrombin-sensitive chromogenic substrate S-2238 and by hydrolysis of the FXa-sensitive chromogenic substrate S-2765, respectively, in culture supernatants. Results of a typical experiment are shown.

As shown in Figure 2, incubation of cells with purified thrombin (0.5 to 10 U/mL) induced a dose-dependent increase in VEGF secretion (r⫽0.87, P⬍0.01), which was maximal after 12 hours. Stimulation of the cells with 10 U/mL thrombin resulted in a 3.3-fold increase in VEGF secretion relative to baseline (P⬍0.001). The thrombin receptor agonist TRAP also induced a dose-dependent increase in VEGF secretion, with a 2-fold rise at 100 ␮mol/L (P⬍0.001). Hirudin (100 U/mL) almost completely prevented VEGF secretion in response to 10 U/mL thrombin (⫺93⫾13%, data not shown). These results show that thrombin is an agonist of VEGF production and suggest that the effect occurs through protease-activated receptor-1, a specific thrombin receptor.

FXa-Induced VEGF Secretion by Fibroblasts

We then investigated whether FXa alone was able to induce VEGF production. FXa (22.8 to 228 nmol/L) incubated with FXa and Thrombin Generation in Culture fibroblasts for 24 hours induced dose-dependent VEGF seMedium of Fibroblasts Incubated With ACSET cretion, which was significant relative to baseline from 57 Because the ACSET preparation contained significant nmol/L FXa (P⬍0.05) and reached 2.5-fold at 228 nmol/L amounts of prothrombin and FX, we first assessed whether FXa (Figure 3A). The FXa inhibitors TAP and DX9065a thrombin and FXa were generated during its incubation with prevented VEGF secretion in response to 114 nmol/L FXa. fibroblasts. As shown in Figure 1B, thrombin and FXa TAP (50 ␮g/mL) and DX9065a (10 ␮mol/L) inhibited 100% (P⬍0.001) and 63⫾24% (P⬍0.01) of this effect, respecactivities were both detected as early as 1 minute after tively. VEGF secretion was not inhibited by hirudin or by incubation of the cells with 100 nmol/L ACSET. Thrombin anti-TF antibodies (Figure 3B). VEGF secretion induced by and FXa levels increased gradually, reaching 4.4⫾1.6 U/mL FXa was additive with VEGF secretion induced by thrombin and 49⫾22 nmol/L (n⫽3), respectively, after 24 hours of (insert, Figure 3A). incubation. In the presence of hirudin (10 U/mL), the To characterize the interaction between FXa and fibroACSET-induced thrombin activity was almost completely blasts, we first investigated whether FXa was able to induce eliminated (residual level 0.15⫾0.08 U/mL at 24 hours). The a cellular signal. As shown in Figure 4, treatment of fura FXa inhibitor TAP was also able to prevent 95% of the 2–loaded fibroblasts with increasing concentrations of FXa (1 thrombin generation. In the same way, ACSET-induced FXa to 1000 nmol/L) was associated with a concentrationactivity was largely inhibited in the presence of TAP at 50 2⫹ dependent [Ca ] i response. FXa (100 nmol/L) induced a 50% ␮g/mL (residual level 1⫾0.5 nmol/L at 24 hours) but not by [Ca2⫹]i increase over baseline, an effect that was inhibited in hirudin. Inhibition of thrombin and FXa activities by hirudin a concentration-dependent manner by DX9065a, with an IC50 and TAP, respectively, occurred after as little as 1 minute of of 120 nmol/L. Interestingly, preincubation of fibroblasts for incubation (not shown). Unstimulated cells generated insig30 minutes with an antibody (EPR-1 monoclonal antibody nificant levels of thrombin and FXa (0.04⫾0.02 U/mL and B6) directed against EPR-1, a reported receptor for FXa, at 0.8⫾0.5 nmol/L, respectively,Downloaded after 24 hours). from http://atvb.ahajournals.org/ by guest on 2015 concentrations ofJuly 50, 24, 100, and 300 ␮g/mL inhibited the

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Figure 2. A, Concentration effect of thrombin on VEGF production. Confluent fibroblasts were incubated for 24 hours with or without 0.5, 1, and 10 U/mL thrombin at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in treated fibroblasts compared with unstimulated fibroblasts (basal VEGF level 124⫾73 pg/mL). Each point represents the mean⫾SD of 4 different determinations, each performed in triplicate. *P⬍0.05 vs unstimulated fibroblasts. B, Kinetics of thrombin-induced VEGF secretion. Confluent fibroblasts were incubated with 10 U/mL thrombin for 2, 4, 6, 12, and 24 hours at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in thrombin-treated fibroblasts compared with unstimulated fibroblasts at the same time. Results of a typical experiment are shown. C, Concentration effect of TRAP on VEGF secretion. Confluent fibroblasts were incubated for 24 hours with or without 10, 50, and 100 ␮mol/L of TRAP at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction in VEGF secretion compared with unstimulated fibroblasts. Each point represents the mean⫾SD of 3 different determinations, each performed in triplicate. *P⬍0.05 vs unstimulated fibroblasts.

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Figure 3. A, Concentration of FXa on VEGF secretion. Confluent fibroblasts were incubated for 24 hours with or without 22.8, 57, 114, and 228 nmol/L of FXa at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with unstimulated fibroblasts. Each point represents the mean⫾SD of 3 different determinations, each performed in triplicate. *P⬍0.05 vs unstimulated fibroblasts. Insert, Additive effect of thrombin (IIa) and FXa on VEGF secretion. Confluent fibroblasts were incubated at 37°C for 24 hours with 1 U/mL of IIa or 100 nmol/L of FXa or a combination of both. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with unstimulated fibroblasts (NS). Each point represents the mean⫾SD of 3 different determinations, each performed in triplicate. ⽧P⬍0.01 vs NS. B, Effect of FXa inhibitors, Hir, and anti-TF antibodies on FXa-induced VEGF production. Confluent fibroblasts were incubated either with 10 ␮g/mL anti-TF antibodies (TFab) for 30 minutes at 4°C or with 10 ␮mol/L DX, 50 ␮g/mL TAP, or 10 U/mL Hir for 30 minutes at 37°C before 24-hour incubation at 37°C with 114 nmol/L FXa. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with NS. Each point represents the mean⫾SD of at least 3 different determinations, each performed in triplicate. *P⬍0.0001 vs NS. ⽧P⬍0.01 vs FXa-stimulated fibroblasts.

We also asked whether an increase in [Ca2⫹]i, as seen with the FXa binding to fibroblasts, was sufficient to induce VEGF secretion. Therefore, we used the calcium ionophore A 23187 (Sigma) to mimic an intracellular calcium increase. We were unable to induce VEGF secretion with this ionophore in

[Ca2⫹]i response induced by 100 nmol/L FXa by 18%, 47%, and 76%, respectively (2 experiments performed in triplicate, data not shown). These results strongly suggest that fibroblasts express EPR-1. Notably, preincubation of the cells for 1 hour with B6 at a concentration of 300 ␮g/mL did not influence the effect of thrombin (100 nmol/L) on [Ca2⫹]i, excluding a nonspecific effect of the antibody on [Ca2⫹]i. However, preincubation of the cells for 1 hour with B6 at concentrations up to 300 ␮g/mL (not shown) did not affect FXa-induced VEGF production even when the incubation Figure 4. Effect of FXa on [Ca2⫹]i in fibroblasts. Increasing contime with FXa was reduced to 6 hours (instead of 24 hours). centrations of FXa were added to fura 2–loaded fibroblasts and The use of B6 alone had no effect on basal VEGF secretion. incubated for 1 minute. FXa at the indicated concentrations was then added, and [Ca2⫹]i was measured as described in MethMoreover, the FX peptide Leu83-Leu88, representing the inods. Inset, Concentration-response curve obtained with DX. terepidermal growth factor sequence in FXa that mediates Data are the mean⫾SD of 2 experiments on 2 different batches ligand binding to EPR-1, neither induced VEGF secretion nor of cells. Results are expressed as percentage inhibition of the Downloaded from http://atvb.ahajournals.org/ by2⫹guest on Julyinduced 24, 2015 peak [Ca inhibited the FXa-induced VEGF secretion. ]i response by 1 ␮mol/L FXa.

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Figure 5. Effect of FXa inhibitors and TFab on FVIIa⫹FXinduced VEGF secretion. Confluent fibroblasts were incubated with FXa inhibitors (TAP, NAP5, and NAPc2 at 50 ␮g/mL and DX at 10 ␮mol/L) and with TFab for 30 minutes at either 37°C or 4°C, respectively, before 24-hour incubation with 100 nmol/L FVIIa and 90 nmol/L FX. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with NS. Each point represents the mean⫾SD of at least 3 different determinations, each performed in triplicate. *P⬍0.0001 vs NS. ⽧P⬍0.0001 vs FXa-stimulated fibroblasts.

concentrations up to 10 ␮mol/L. Moreover, an intracellular calcium chelation agent, BAPTA-AM (up to 60 ␮mol/L), did not prevent the FXa- or the thrombin-induced VEGF secretion (data not shown). From these data, we conclude that an increase in [Ca2⫹]i is not sufficient to induce VEGF secretion. Taken together, these results strongly suggest that EPR-1, an FXa receptor, does not appear to be involved in VEGF production in response to FXa.

Effect of FX Plus Recombinant FVIIa on VEGF Secretion We then determined the effect of FVIIa and FX in combination (Figure 5). Incubation of fibroblasts with 100 nmol/L FVIIa and 90 nmol/L FX increased VEGF secretion to 2.3-fold baseline (P⬍0.0001). By contrast, incubation of fibroblasts with 100 nmol/L recombinant FVIIa for 24 hours did not induce VEGF secretion (Figure 5). The FVIIa⫹FXinduced VEGF secretion was associated with FXa generation (shown by hydrolysis of the specific FXa chromogenic substrate S-2765) but not with thrombin generation (no hydrolysis of the specific thrombin chromogenic substrate S-2238 after 24-hour incubation of the cells with FVIIa-FX, data not shown). Furthermore, FVIIa⫹FX-induced VEGF production was abolished by inhibitors of FXa (TAP, NAP5, and NAPc2) used at a concentration of 50 ␮g/mL, by DX9065a at 10 ␮mol/L, and by anti-TF-antibodies (81⫾13% inhibition, Figure 5). Hirudin had no significant effect. The use of FVIIai in combination with FX did not induce VEGF secretion, indicating that the proteolytic activity of FVIIa was necessary, through the generation of FXa, to induce VEGF expression. The combination of FVIIa with purified FIX had no effect (data not shown).

FXa and Thrombin Induce VEGF mRNA Accumulation

Figure 6. Effect of activated clotting factors on VEGF mRNA induction. Confluent fibroblasts were incubated for 24 hours at 37°C with 100 nmol/L FVIIa in combination with 90 nmol/L FX, 114 nmol/L FXa, or 1 U/mL thrombin. Cells were preincubated for 30 minutes at 37°C either with 50 ␮g/mL TAP before incubation with FVIIa-FX and FXa or with 10 U/mL Hir before incubation with thrombin. Five micrograms of total RNA was analyzed by RT-PCR. A, RNA was quantified by scanning the Polaroid negative by laser densitometry. The density of the 180-bp band was normalized to the density of the mimic, and the fold induction of VEGF mRNA, induced in the different conditions of stimulation compared with NS, was plotted. Each point represents the mean⫾SD of 4 experiments. *P⬍0.05 vs NS. ⽧P⬍0.05 vs FVIIa-FX, FXa, or thrombin-stimulated fibroblasts. B, Photograph from a representative experiment is shown. Three VEGF mRNA transcripts are seen (180, 312, and 384 bp). The internal standard, mimic (M), is also visible. L indicates 100-bp DNA ladder.

312-bp and 384-bp transcripts increased similarly. A 1.9⫾0.3-fold increase (P⬍0.01) in the 180-bp transcript level was observed after FXa (114 nmol/L) stimulation, and a 1.6⫾0.27-fold increase (P⫽0.02) was seen after thrombin (1 U/mL) treatment. Preincubation of the cells with TAP (50 ␮g/mL) completely inhibited the VEGF mRNA accumulation induced by FVIIa-FX and by FXa (P⬍0.0001 and P⬍0.05, respectively). Hirudin (10 U/mL) prevented thrombininduced VEGF mRNA accumulation (P⬍0.05).

Activation of MAP Kinases ERK-1 and ERK-2 by Thrombin and FXa but Not by FVIIa Because binding of FVIIa to cell surface TF has been shown to activate signal transduction via p44/42 MAP kinases,6 we analyzed the contribution of this pathway by using a specific antibody against the phosphorylated Thr202/Tyr204 residues of these kinases. As shown in Figure 7A, exposure of the cells to 100 nmol/L of FVIIa or 100 nmol/L of FVIIai did not alter the phosphorylated p44/42 band. By contrast, thrombin and FXa transiently increased the phosphorylation of p44/42 MAP kinases, peaking at ⬇2⬘ and 10⬘, respectively, whereas the amount of total MAP kinases remained essentially constant (Figure 7B).

To identify the level of action of thrombin, FXa, and FVIIa in combination with FX, VEGF mRNA was studied after RT and amplification by PCR. Three VEGF transcripts of 180, Effect of MAP Kinase Inhibitors on FXa- and 312, and 384 bp coding for VEGF121, VEGF165, and VEGF189, Thrombin-Induced VEGF Production respectively, were detected. After 24 hours of treatment with To confirm the involvement of thrombin- and FXa-activated 100 nmol/L FVIIa in combination with 100 nmol/L FX, the MAP kinases in VEGF expression, we used PD 98059 to 180-bp transcript showed a 2⫾0.3-fold increase (P⬍0.001) selectively block the activation of p44/42 MAP kinases. PD Downloaded on July 24,preincubated 2015 98059 by (50guest ␮mol/L) was with fibroblasts for 30 over baseline (unstimulated cells, Figure 6).from Thehttp://atvb.ahajournals.org/ levels of the

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protease signaling mechanism. We confirmed that inactivated FVIIa did not enable VEGF production to occur, even in combination with factor X. These results show for the first time that the TF-FVIIa complex contributes to VEGF production by human fibroblasts through FXa and thrombin generation. Confirmation of the direct effect of thrombin and FXa on VEGF production was obtained by using purified enzymes. The effect of thrombin was significant from 0.5 U/mL, a concentration that may be reached locally in vivo, and was reproduced by the human selective peptide agonist of protease-activated receptor-1, a thrombin receptor expressed by fibroblasts.20 Mo¨hle et al21 recently reported thrombininduced release of VEGF by human megakaryocytes and platelets. We also found that FXa induced VEGF secretion. The FXa-induced increase in VEGF was blocked by TAP and DX9065a, clearly showing that its proteolytic activity was required. This effect was not mediated by contaminating or generated thrombin, because hirudin failed to block FXaFigure 7. A, Effect of FVIIa and FVIIai on p44/42 MAP kinase induced VEGF production while completely preventing the activation. Fibroblasts were incubated with either 100 nmol/L thrombin-induced increase in VEGF. In a previous study, we FVIIa or 100 nmol/L FVIIai for 5, 10, and 15 minutes. Phosphorylated p44/42 MAP kinases (p44ERK1 and p42ERK2) and total showed that ACSET induced VEGF production at the mRNA p44/42 MAP kinases (ERK1 and ERK2) were studied by Western level.11 In the present study, we found that FXa and thrombin blotting. B, Western blot analysis of thrombin and FXa-induced induced VEGF mRNA accumulation, an effect that was phosphorylation of p44/42 MAP kinases. Fibroblasts were incuprevented by TAP and hirudin, respectively. Similar results bated with 1 U/mL of thrombin for 2, 5, 7, and 10 minutes or with 100 nmol/L of FXa for 2, 5, 10, and 15 minutes. p44ERK1, were obtained by using FVIIa and factor X simultaneously. p42ERK2, ERK1, and ERK2 are shown. C, Effect of MAP kinase Recently, FXa has been shown to mediate a variety of inhibitors on FXa- and thrombin-induced VEGF secretion. Conbiological effects (in addition to its role in the activation of fluent fibroblasts were incubated with MAP kinase inhibitors coagulation), including mitogenesis, lymphocyte activation, (50 ␮mol/L PD 98059 [PD] and 10 ␮mol/L SB 203580 [SB]) for 30 minutes at 37°C before 24-hour incubation with either FXa cytokine secretion, and adherence molecule expression.22,23 (114 nmol/L) or thrombin (1 U/mL). Secreted VEGF was Our present results suggest that FXa not only plays a role in assessed by a specific ELISA. Results are expressed as fold inflammation but may also be involved in angiogenesis. We induction of VEGF secretion compared with unstimulated fibroblasts. Each point represents the mean⫾SD of 4 different detershow that EPR-1, a receptor that binds FXa and mediates minations, each performed in triplicate. *P⬍0.0005 vs NS. several of its cellular effects,24,25 is expressed on fibroblasts, ⽧P⬍0.05 vs FXa- or thrombin- stimulated fibroblasts. because FXa induced a calcium signal that was prevented by B6, a monoclonal antibody blocking the binding of FXa to minutes before 24 hours of cell stimulation with either 100 EPR-1. B6 antibody, however, was unable to block VEGF nmol/L FXa or 1 U/mL thrombin (Figure 7C). PD 98059 secretion even at very high concentrations or after a short inhibited 67⫾52% (P⬍0.05) of FXa-induced VEGF producincubation time (6 hours instead of 24 hours). We were also tion and 85⫾19% (P⬍0.0001) of thrombin-induced VEGF unsuccessful either in activating directly VEGF secretion or production, pointing to a role of the p44/42 MAP kinase in inhibiting FXa-induced VEGF secretion by the interepisignaling pathway in this production. SB 203580 (10 ␮mol/L), dermal growth factor peptide (Leu83-Leu88) that mediates a highly specific inhibitor of MAP kinase p38, had no EPR-1 recognition of FXa. Furthermore, the increase in significant effect on VEGF production induced either by FXa [Ca2⫹]i observed when FXa binds to fibroblasts was not or by thrombin. sufficient to induce VEGF secretion because the calcium ionophore A23187 failed to do it and because intracellular Discussion calcium chelation by BAPTA-AM was also without any We first confirmed our previous report11 that ACSET, a effect on FXa-induced VEGF secretion. This suggests that plasma-derived FVIIa concentrate, induces VEGF production EPR-1 is not involved directly in FXa-induced VEGF proby TF-expressing human fibroblasts. We further showed that duction and points to the involvement of another receptor, its use was associated with significant generation of FXa and alone or in association with EPR-1, that could be activated thrombin in the culture medium and that the increased VEGF by FXa. secretion was largely dependent on FXa and thrombin inasBecause binding of FVIIa to cell surface TF has been much as it was inhibited by 80% by the simultaneous addition shown to activate signal transduction via p44/42 MAP kiof hirudin and TAP or DX9065a. This small residual VEGF nases,6 we looked at direct FVIIa p44/42 MAP kinase secretion could result from an incomplete inhibition of activation with specific antibodies. In our cellular model, the thrombin and FXa generated in the presence of ACSET. p44/42 MAP kinase pathway was not significantly activated Indeed, the addition of recombinant FVIIa to TF-expressing by FVIIa. Thrombin and FXa, by contrast, induced p44/42 fibroblasts did not induce VEGF production unless FX was MAP kinase activation, which was maximal at 2 and 10 added simultaneously to allow FXa generation. The lack of minutes, respectively. PD 98059, a specific inhibitor of Downloaded froma http://atvb.ahajournals.org/ by 26 guest on July 24, 2015 effect of recombinant FVIIa alone excluded direct FVIIa MEK1/2, which blocks the p44/42 MAP kinase pathway,

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expression in both macrophages and poorly differentiated prevented 85% of the effect of thrombin and 50% of the effect of FXa on VEGF production, whereas SB 203580, an smooth muscle cells of human coronary atherosclerotic inhibitor of p38 MAP kinase, had no effect. These results plaques but not in normal coronary arteries.35 Moreover, in a show for the first time that FXa induces the p44/42 MAP porcine animal model, the response of the coronary artery to kinase activation that leads to VEGF production. Investigaballoon angioplasty has been associated with activation of tions are in progress to clarify the complete pathway from the adventitial fibroblasts, which undergo differentiation to myocellular system receptor to the VEGF gene in human fibrofibroblasts and migrate into the intima.36 This observation blasts. They are in keeping with previous work showing that raises the possibility that adventitial myofibroblasts contribthrombin induces strong MAP kinase activation in mouse ute to local VEGF production in response to coagulation lung fibroblasts20 and that the p44/42 MAP kinase module proteases. VEGF may contribute to atheromatous lesion plays a key role in transcriptional regulation of the VEGF progression by enhancing neovascularization within the legene.27 The present results suggest that in human lung sion. Alternatively, and as suggested by initial clinical results fibroblasts, direct activation of the p44/42 MAP kinase of gene therapy,37 VEGF may have a beneficial role in pathway triggered by the binding of FVIIa to TF does not reendothelialization and in improving collateral blood occur, unlike the results obtained by Poulsen et al6 in flow.38,39 The overall effect of VEGF production mediated by transfected BHK cells. In the present study, although the TF, FXa, and thrombin in atherosclerotic plaque remains to proteolytic activity of FVIIa was absolutely required, be established. FVIIa-TF signaling was not quenched by TAP or hirudin and was independent of the cytoplasmic domain of TF because Acknowledgments cells transfected with a cytoplasmic domain– deleted version We thank Dr Martine Jandrot-Perrus (Faculte´ Xavier Bichat, Paris, France) and Dr Nigel Mackman (The Scripps Research Institute, La of TF also supported FVIIa-induced MAP kinase activation.28 Jolla, Calif) for fruitful scientific discussions and critical reading of VEGF is a direct angiogenic agent in normal and abnormal the manuscript. We also thank Dr Jamel El Benna and Cedric Dewas physiological conditions. Angiogenesis is a crucial compofor technical advice concerning the MAP kinase Western blotting. nent of tumor growth and metastasis, and VEGF mRNA is markedly upregulated in the majority of human tumors.29 References Interestingly, in a recent study, Fukumura et al30 suggested an 1. Bach RR. Initiation of coagulation by tissue factor. CRC Crit Rev Biochem. 1988;23:339 –368. important contribution of stromal cells of the tumor micro2. Fischer EG, Ruf W, Mueller BM. Tissue factor-initiated thrombin genenvironment to tumor angiogenesis, demonstrating the actieration activates the signaling thrombin receptor on malignant melanoma vation of the VEGF promoter in fibroblasts of the fibrotic cells. Cancer Res. 1995;55:1629 –1632. tumor matrix. Our results contribute to clarifying the role of 3. Zhang Y, Deng Y, Luther T, Muller M, Ziegler R, Waldherr R, Stern DM, Nawroth PP. 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Vascular Endothelial Growth Factor Production by Fibroblasts in Response to Factor VIIa Binding to Tissue Factor Involves Thrombin and Factor Xa V. Ollivier, J. Chabbat, J. M. Herbert, J. Hakim and D. de Prost Arterioscler Thromb Vasc Biol. 2000;20:1374-1381 doi: 10.1161/01.ATV.20.5.1374 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2000 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

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