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Factor VII-activating protease (FSAP) inhibits growth factor-mediated cell proliferation and migration of vascular smooth muscle cells ¹

CHRISTIAN KANNEMEIER^*, NADIA AL-FAKHRI, KLAUS T. PREISSNER^* and SANDIP M. KANSE^*,2

^* Institute for Biochemistry and
Institute for Clinical Chemistry, Justus-Liebig-University Giessen, Germany

²Correspondence: Institute for Biochemistry, Justus-Liebig-University Giessen, Friedrichstr. 24, 35392 Giessen, Germany. E-mail: sandip.kanse{at}biochemie.med.uni-giessen.de

SPECIFIC AIMS

1. The homology of factor VII activating protease (FSAP) to proteases with known effects on vascular cellular functions such as hepatocyte growth factor activator, tissue plasminogen activator or urokinase, and the association of a genetic polymorphism in FSAP, Marburg I, with carotid stenosis prompted us to investigate the influence of FSAP on vascular smooth muscle cell (VSMC) proliferation and migration.

2. The effect of FSAP on rapid phosphorylation events in cells was analyzed.

3. Since heparin binds to and activates FSAP, its influence on the cellular effects of FSAP was investigated as was the role of serine protease inhibitors.

4. Proteolysis of platelet-derived growth factor-BB (PDGF-BB) or its receptor by FSAP was analyzed.

5. Interaction of FSAP with PDGF-BB and the influence of FSAP on PDGF-BB binding to cells were investigated.

6. The distribution of FSAP in normal and atherosclerotic vessels was determined.

PRINCIPAL FINDINGS

1. FSAP inhibits DNA synthesis and cell proliferation in VSMC
Plasma concentrations of single-chain FSAP (12 µg/mL) inhibited PDGF-BB-induced human VSMC proliferation by 37%. At neutral pH there is a spontaneous and rapid conversion of the enzymatically inactive single-chain FSAP into the enzymatically active two-chain form. PDGF-BB-induced DNA synthesis of human VSMC was reduced to 50% by concentrations of two-chain FSAP as low as 1–2 µg/mL whereas 5–6 µg/mL single-chain FSAP was required to achieve a similar inhibition. When FSAP was added together with heparin (10 µg/mL), a more pronounced inhibition in DNA synthesis was observed. Heparin alone did not have a significant inhibitory effect on the mitogenic response of human VSMC to PDGF-BB. Only unfractionated, high molecular weight heparin had an augmenting effect on FSAP-mediated inhibition of human VSMC DNA synthesis induced by PDGF-BB whereas fractionated, low molecular weight heparin was not effective. To assess the contribution of cell surface proteoglycans in mediating the cellular effects of FSAP, cells were pretreated with heparitinase III or chondroitinase ABC, then tested in DNA synthesis assays. No modulation of the inhibitory effect of FSAP was observed with these treatments, indicating that cell surface proteoglycans were not involved.

IGF-1- but not FCS-stimulated proliferation of human VSMC was inhibited by FSAP, indicating that not all growth stimulatory factors are inhibited by FSAP. Proliferation of MG63 osteosarcoma cells was inhibited by FSAP when they were stimulated with PDGF-BB or IGF-1 but not with FCS. The inhibitory effect of FSAP on PDGF-BB-induced DNA synthesis was abolished by preincubation of two-chain FSAP with PPACK, aprotinin, or the FSAP enzymatic activity blocking monoclonal antibody (mAb)570, but not by a nonblocking mAb677. Hence, the enzymatic activity of two-chain FSAP was essential for inhibition of PDGF-BB-induced DNA synthesis in human VSMC.

Preincubation of PDGF-BB with FSAP and heparin before its addition to the cells leads to a maximal inhibition of DNA synthesis. The addition of aprotinin at the start of the preincubation period led to a reversal of the inhibitory effect of FSAP; subsequent addition of aprotinin did not influence the inhibition of DNA synthesis by FSAP.

2. FSAP inhibits the migration of VSMC
The characteristics of inhibition of PDGF-BB-induced cell migration by FSAP were similar to that observed for DNA synthesis. In normal cell migration assays, the chemotactic agent was added to the lower well and cells were added to the upper wells, separated by a membrane across which the chemotactic gradient was established. Addition of FSAP to the cells did not elicit an inhibitory response whereas coaddition of FSAP and PDGF-BB or the addition of FSAP to the PDGF-BB and the cells led to a pronounced inhibition of cell migration due to PDGF-BB.

3. Effect of FSAP on PDGF-BB-stimulated phosphorylation in human VSMC
PDGF-BB induced a robust phosphorylation of MAPK-p42/p44 or protein tyrosine phosphorylation. FSAP stimulated the phosphorylation of MAPK-p42/p44 to a small extent but not protein tyrosine phosphorylation. Coaddition of the substances did not lead to modulation of PDGF-BB-induced cellular signaling; but after preincubation of FSAP and PDGF-BB in the presence of heparin, the phosphorylation of MAPK-p42/p44 and proteins with an apparent molecular weight of 170 and 180 kDa was abolished.

Since FSAP can activate factor VII, it may indirectly activate serum-derived proteases in the presence of cellular tissue factor eventually leading to activation of the general protease-activated receptor-2 (PAR-2). Polyclonal blocking antibodies to tissue factor or PAR-2 did not diminish the inhibitory effect of FSAP on PDGF-BB-induced phosphorylation of MAPK-p42/p44.

4. Proteolysis of FSAP and PDGF-BB
There was a rapid autoactivation (within minutes) of single-chain FSAP and a relatively slower (within hours) autocatalytic degradation in the presence of cells. Rapid autoactivation and proteolysis of single-chain FSAP was inhibited by aprotinin and slightly stimulated by heparin. A small but significant degradation of PDGF-BB by FSAP was observed both in the absence or presence of heparin. However, even after a long incubation (4 h) a significant amount of intact PDGF-BB was still available for activation of cells, hence proteolysis alone did not account for the inhibitory effect of FSAP on cells.

5. Influence of FSAP on the binding of ¹²⁵I-PDGF-BB to human VSMC
Binding of ¹²⁵I-PDGF-BB to human VSMC was specific and saturable and it could be competed for by a 100-fold excess of unlabeled PDGF-BB. The simultaneous addition of FSAP and heparin led to complete inhibition of ¹²⁵I-PDGF-BB binding to human VSMC whereas in the absence of heparin, no such effect was observed. When FSAP and ¹²⁵I-PDGF-BB were preincubated, there was reduced binding of the mitogen in the absence of heparin and complete inhibition of binding in its presence. These data indicated that FSAP inhibited ¹²⁵I-PDGF-BB binding to the cell surface of VSMC; this inhibition was accentuated by heparin.

6. PDGF-BB binding to FSAP
PDGF-BB bound to immobilized two-chain FSAP in a dose-dependent manner and this interaction exhibited saturation. The presence of heparin did not significantly alter these binding characteristics.

7. Immunostaining of FSAP in the vessel wall
Immunostaining of serial sections of atherosclerotic carotid arteries revealed a strong expression of FSAP in the neointima, as well as in the media. No notable FSAP immunoreactivity was observed in smooth muscle cells of normal, undiseased arteries. In the neointima, FSAP expression was predominantly found in the vicinity of foam cell accumulations of smooth muscle cell and macrophage origin (Fig. 1 ).

View larger version (156K): [in this window] [in a new window]   Figure 1. FSAP immunostaining in human carotid artery. Immunohistochemistry of arteriosclerotic carotid artery, visualized through streptavidin-alkaline phosphatase-conjugate, Fast Red stain and Mayer’s hemalum counterstain. General view (50-fold magnification) of the arterial wall showing FSAP heavy chain immunostaining (A). Extension of the neointima is indicated by a dotted line. The luminal border is marked by light arrowheads. The border of the neointima to the media, (i.e. the remainders of the lamina elastica interna) is marked by dark arrowheads. The foam cell accumulation marked by a rectangle is presented in parts (B–F) in higher power views. Serial sections (400-fold magnification) showing FSAP light chain (B), smooth muscle -actin (C), CD68 macrophage antigen (D), PCNA (E), and the negative control (F). Note that FSAP is expressed in smooth muscle -actin positive areas (red stain). Foam cell accumulation is composed of smooth muscle cells and macrophages. Proliferation was demonstrated in these regions; red arrowheads (E) point to individual PCNA-positive proliferating cells. Two separate FSAP mAbs, one to the light chain and one to the heavy chain, gave a similar staining pattern and there was no immunostaining reaction with the negative control antibody.

CONCLUSIONS AND SIGNIFICANCE

The development of vascular stenosis is a multifactorial process with a prominent role for atherothrombotic events. We demonstrate here that the proteolytic activity of FSAP is involved in the inhibition of VSMC proliferation and migration. Evidence is provided that FSAP immunoreactive material is present in high concentrations in the atherosclerotic vessel wall but not in normal arteries. The FSAP present in the atherosclerotic vessels may be synthesized locally or imported from the bloodstream due to increased vascular permeability.

FSAP is not a nonspecific inhibitor of cells since PDGF-BB- and IGF-1-mediated DNA synthesis was inhibited but the effect of FCS was not influenced. This could be due to the inhibition of the proteolytic activity of FSAP in the presence of serum protease inhibitors. A similar pattern of DNA synthesis was seen on MG63 osteosarcoma cells, suggesting the effect of FSAP is not cell specific.

A strong case can be made for the active enzyme as an inhibitor of cell migration and proliferation, since aprotinin or an FSAP mAb was found to neutralize this activity of FSAP. That the effect of FSAP is inhibitable by serine protease inhibitors indicates a certain specificity of this effect and suggests that in the in vivo situation a balance between FSAP and its inhibitors determines the extent of cellular inhibition.

Not only did FSAP inhibit the PDGF-BB-stimulated proliferation, DNA synthesis, and migration of mouse and human VSMC, but also the extent of protein tyrosine phosphorylation and activation of the p42/p44 MAPK pathway. In each case the FSAP-mediated inhibition could be neutralized by the serine protease inhibitor aprotinin. Inhibition of signal transduction and cell migration was maximal in the presence of exogenous heparin and dependent on the preincubation of FSAP and PDGF-BB. Moreover, if aprotinin was present from the beginning in the preincubation mixture, the effect of FSAP was neutralized, but if it was added at the end of the preincubation phase, aprotinin was unable to prevent the inhibitory effect of FSAP. In the time course of these experiments there was no degradation of PDGF-BB or its receptor.

These findings can be best reconciled with a model where intact two-chain FSAP or a fragment thereof forms a complex with PDGF-BB in the presence of heparin and prevents its binding to its cellular receptor (Fig. 2 ). In a purified system, heparin augmented the autoactivation and degradation of FSAP with loss of function within minutes; this explains the lack of a MAPK p42/p44 phosphorylation induced by preincubated FSAP. Endogenous cell surface proteoglycans were not involved in the FSAP-mediated inhibition of p42/p44 MAPK phosphorylation or DNA synthesis.

View larger version (43K): [in this window] [in a new window]   Figure 2. FSAP is a plasma serine protease also localized in areas of the vessel wall undergoing atherosclerotic remodeling. The single-chain form is readily converted to a 2-chain form by autoactivation and further degraded by autoproteolysis or by other proteases. The 2-chain form of FSAP can activate prourokinase and FVII. In the presence of heparin-like polymers, FSAP or its fragments may bind to PDGF-BB and prevent its interaction with the cell surface PDGF-ß receptor. This leads to inhibition of signal transduction and a concomitant reduction in cell migration and proliferation. Serine protease inhibitors can inhibit the effect of FSAP by preventing the autoactivation of single-chain FSAP into 2-chain FSAP and its degradation.

Different types of binding studies were performed to support the proposed hypothesis. Binding of radiolabeled PDGF-BB to VSMC was measured, and in the absence of heparin there was no modulation of specific PDGF-BB binding by FSAP. However, in the presence of heparin specific PDGF-BB binding was abolished by FSAP. PDGF bound to immobilized two-chain FSAP in a saturable manner, and this binding was not modified by the presence of heparin. Hence, the characteristics of PDGF-BB-FSAP binding are strongly influenced by the nature of the binding assay; these results need to be extended.

FSAP is not only inhibitory but can directly stimulate cells. This is illustrated by the fact that FSAP by itself promoted the phosphorylation of p42/p44 MAPK. FSAP also stimulated PDGF-BB-induced migration in the absence of heparin. A single nucleotide polymorphism in FSAP (Marburg I) that reduces its ability to activate prourokinase has been linked to the development of carotid stenosis. Whether the variant FSAP protein also demonstrates reduced inhibition of VSMC functions needs to be evaluated. Hence, FSAP, a plasma serine protease with interactions in the coagulation and fibrinolysis pathways, is an endogenous inhibitor of proatherogenic phenotype of vascular smooth muscle cells. FSAP, or variants thereof, could be used as pharmacological inhibitors in fibroproliferative disorders where PDGF-BB is involved.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0898fje; doi: 10.1096/fj.03-0898fje

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