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INTRODUCTION |
The proliferation of smooth muscle cells is closely related to the
pathogenesis of atherosclerosis and the restenosis after percutaneous
transluminal coronary angioplasty (1). However, the precise mechanism
of the proliferation is unknown. In this study, we purified a mitogen
for growth-arrested bovine aortic smooth muscle cells from the
supernatant of cultured
HUVEC1 and identified it as
TFPI-2, whose physiological function has been unknown.
TFPI-2 is structurally related to TFPI. TFPI is synthesized in
endothelial cells and exists on the endothelial surface and in plasma
(2-5). TFPI inhibits the initial steps of the extrinsic coagulation
pathway and regulates the hemostasis. Recently, an antiproliferative
action of TFPI has been reported. In the atherosclerotic rabbit
arterial injury model, treatment with recombinant TFPI reduced
angiographical restenosis and decreased neointimal hyperplasia (6).
Inhibition of TF-mediated coagulation by recombinant TFPI administration during the first 24 h after balloon-induced
arterial injury at the carotid artery of minipigs seems to be effective for attenuating subsequent neointimal formation and luminal stenosis (7). TFPI exhibits inhibitory activity toward cultured human neonatal
aortic smooth muscle cells (8). However, TFPI-2 has a mitogenic
activity despite its similarity of structure to TFPI. This new function
of TFPI-2 may play an important role in the pathogenesis of
atherosclerosis and neointimal hyperplasia after percutaneous
transluminal coronary angioplasty.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human umbilical vein endothelial cells were
cultured as described previously (9). Bovine aortic smooth muscle cells
were isolated from the medial layers of adult bovine aorta by a
modification of the explant technique of Ross as described previously
(10). In brief, they were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells were grown at 37°C
in a humidified atmosphere of 5% CO2 in air. Culture
medium (DMEM + 10% FCS) was changed every 3 days, and a confluent
smooth muscle cell monolayer was obtained after about 7 days. Cells
were used from the second to the sixth passage. Cells were harvested with 0.1% trypsin-0.02% EDTA solution and plated at a density of
2.0 × 105 cells in 10-cm dish (Nalge Nunc) for
48 h, after which their growth was arrested with DMEM containing
0.1% FCS. After 48 h, fresh medium (DMEM + 0.1% FCS) and
different concentrations of mitogen (TFPI-2) were added simultaneously
to the growth-arrested cells. After 48 h, cells were recovered
using the trypsin/EDTA solution, and cell counts were performed with a
hemocytometer. For the detection of DNA synthesis, cells were plated at
a density of 3,000 cells/well in 96-well plates. Biotrak Cell
proliferation enzyme-liked immunosorbent assay system (Amersham
Pharmacia Biotech) was used. 5-Bromo-2'-deoxyuridine (BrdUrd) was added
24 h after TFPI-2 addition. After 9 h, incorporated BrdUrd
was assayed.
Purification and Identification of TFPI-2--
A mitogen was
purified from the conditioned HUVEC medium. Medium (800 ml) was
concentrated to 20 ml using an ultrafiltration membrane (YM 10, Amicon)
at 4° C. This 20-ml solution was applied to HiTrap Heparin affinity
column (5 ml, Amersham Pharmacia Biotech) previously equilibrated with
50 mM Tris-HCl (pH 7.4). Following sample application, the
column was washed with 50 ml of 50 mM Tris-HCl (pH 7.4)
solution. Mitogenic activity was eluted with 20 ml of 50 mM
Tris-HCl (pH 7.4) containing 1 M NaCl. The eluent was
concentrated to 0.5 ml by Centriprep 10 (Amicon). This sample was
injected to ProRPC HR 5/10 column (Amersham Pharmacia Biotech) using
LC-6A high performance liquid chromatography system (Shimadzu Co.). The
flow rate was 1 ml/min. The peak was monitored at 280 nm. The column
was eluted with a gradient formed from 0.1% trifluoroacetic acid in
20% acetonitrile to 0.1% trifluoroacetic acid in 100% acetonitrile. The mitogenic fraction eluted from the column was freeze-dried. Amino acid sequence analysis was carried out by automated
Edman degradation using an Applied Biosystems 470 A gas-phase sequencer
(Perkin-Elmer).
Gel Electrophoresis--
SDS-polyacrylamide gel electrophoresis
was performed using Phast System (Amersham Pharmacia Biotech). The
protein was stained with Coomassie Brilliant Blue. The molecular size
marker used was obtained from Amersham Pharmacia Biotech.
Construction of TFPI-2 cDNA Expression Vector--
The
mammalian expression vector pK4K was used for the construction of the
expression vector designated as pK4KT2 (11). This vector contains
unique restriction sites for HindIII and BamHI. The TFPI-2 cDNA was made using polymerase chain reaction (PCR) with
a sense oligonucleotide 5'-ATGGACCCCGCTCGCC-3' and antisense oligonucleotide 5'-GCCATAAAGACAAACAAGAT-3', which corresponded to
nucleotides 39-54 and 761-782 of TFPI-2, respectively. The template
was pBluescript II SK(
) containing TFPI-2 cDNA (Stratagene). This
PCR product was blunted (DNA Blunting Kit, Takara Biochemicals) and
ligated into the above vector after blunting. The sequence of PCR
product was confirmed with automated DNA sequencer 373A (Applied
Biosystems, Perkin-Elmer).
Transfection and Cell Culture--
Nontransfected baby hamster
kidney (BHK) tk
ts13 cells were grown in DMEM
supplemented with 5% FCS, streptomycin (30 µg/ml), and penicillin
(30 units/ml). BHK cells (2 × 105 cells) were
transfected with 5 µg of pK4KT2 by a modified CaPO4 precipitation technique using the CellPhect transfection kit (Amersham Pharmacia Biotech). The transfected cells, BHKT2, were grown in DMEM
containing 5% FCS. After selection with 250 nM
methotraxate, cell culture supernatants were collected and used for the
isolation of recombinant TFPI-2 (rTFPI-2).
Purification of rTFPI-2--
rTFPI-2 was purified from
conditioned BHK medium by the method described under "Purification
and Identification of TFPI-2." rTFPI-2 was identified by
the retention time on the chromatogram, SDS-PAGE analysis, and the
peptide sequence analysis (data not shown).
MAPK Phosphorylation by MAPK Kinase--
Serum-starved cells in
24-well plates were exposed to 500 nM TFPI-2 in DMEM + 0.1% FCS. After incubation for the periods indicated in Fig. 6, the
supernatant was removed. 100 µl of 10 mM Tris-HCl (pH
8.0), 1 mM EDTA, 2.5% SDS, and 5% mercaptoethanol was
added to the well. The dissolved cell fractions were separated on
8-25% gels by SDS-polyacrylamide gel electrophoresis using Phast
System. The proteins were then blotted onto nitrocellulose (Hoefer
Scientific Instruments) by semi-dry electroblotting with Phast Transfer
(Amersham Pharmacia Biotech) for 30 min. The blots were blocked for
1 h with 10% bovine serum albumin in Tris-buffered saline (20 mM Tris-HCl (pH 7.6) and 137 mM NaCl). The
blots were then washed five times in the same buffer containing 0.1%
Tween-20. This washing was performed between each subsequent step. The
blots were incubated sequentially with the monoclonal antibody (Promega
Inc.) against phosphorylated MAPK (P44/ERK1 and P42/ERK2, which show no
cross-activity with nonphosphorylated MAPK) diluted in Tris-buffered
saline (25 ng/ml) for 1 h, with the biotinylated
F(ab')2 rabbit anti-mouse immunoglobulin G (Serotec Ltd.)
for 1 h, and with the streptavidin-alkaline phosphatase conjugate
for 1 h. Finally, nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt were added to detect specific proteins, and the reaction was stopped by
washing in distilled water.
MAPK Activity--
Bovine aortic smooth muscle cells were plated
in 60-mm dishes (Nalge Nunc) for 48 h, after which their growth
was arrested with DMEM containing 0.1% FCS. After 48 h, fresh
medium (DMEM + 0.1% FCS) and different concentrations of mitogen
(TFPI-2) were added simultaneously to the growth-arrested cells.
PD098059 was added 30 min before rTFPI-2 stimulation. After incubation,
MAPK activity was measured using a P44/42 MAP kinase assay kit (New England Biolabs, Beverly, MA) according to the manufacturer's instructions. The chemiluminescent intensity was determined using NIH image.
c-fos Promoter Luciferase Reporter Assay--
The human
c-fos promoter (12) from
442 to +42 was made by PCR. A
sense oligonucleotide was 5'-GGGGTACCATTCTGCGCCGTTCC-3', and an
antisense oligonucleotide was 5'-GAAGATCTGCTCAGTCTTGGCTTCTC-3'. The
template was human genomic DNA from whole blood (Promega). The sequence
of PCR product was confirmed with automated DNA sequencer 373A (Applied
Biosystems, Perkin-Elmer). Dual-Luciferase Reporter Assay System
(Promega) was used. The PCR product was ligated into KpnI-BglII site in pGL3-Basic Vector. This
c-fos reporter plasmid (5 µg) was transiently transfected
in bovine smooth muscle cells (106 cells) with pRL-SV40
Vector (0.5 µg) by the CellPhect transfection Kit (Amersham Pharmacia
Biotech). After 12 h, the precipitate was removed, and the cells
were washed with phosphate-buffered saline. Cells were starved for
48 h in DMEM with 0.1% FCS. After incubation with 500 nM rTFPI-2 with or without PD098059 for 30 min, cells were
collected and lysed in lysis buffer. Luciferase activity was assayed in
Aloka BLR-201 Luminesence Reader according to the manufacturer's
instructions. PD098059 was added 30 min before rTFPI-2 stimulation.
Northern Blot Analysis of c-fos mRNAs--
Cells were
prepared as described under "MAPK Activity." Isolation of total
cellular RNA from stimulated cells (108 cells) was obtained
using Ultraspec-II RNA isolation system (Biotecx Laboratories, Inc.).
Total RNA (20 µg/lane) was size-fractionated by electrophoresis on
1% agarose gels containing 18% formaldehyde. Capillary transfer to
N+ nylon (Amersham Pharmacia Biotech) was performed
overnight. Prehybridizations were carried out at 42° C for 4 h
in prehybridization buffer (4× SSC, 1% SDS, and 1× Denhardt's
solution). Hybridizations were carried out at 42° C for 18 h in
hybridization buffer (4× SSC, 1× Denhardt's solution, 1% SDS, and
100 µg/ml salmon sperm DNA) containing 2 × 106cpm/ml of 32P-labeled
([
-32P]dCTP, Amersham Pharmacia Biotech) cDNA
probes. The cDNA probes, c-fos (Takara Biomedicals) and
glyceraldehyde-3-phosphate dehydrogenase (CLONTECH)
were labeled using a Ready-To-Go DNA labeling kit (Amersham Pharmacia
Biotech). Washes were performed at 42° C in 2× SSC, 0.1% SDS,
55° C in 2× SSC, 0.1% SDS and finally at 55° C in 0.5× SSC,
0.1% SDS. Glyceraldehyde-3-phosphate dehydrogenase was used as a
control to check for equal loading in each lane and washed in the same
conditions as above. Autoradiograms were obtained using BAS 2000 system
(Fuji Film).
Protein Determination--
Protein concentration was determined
by Bio-Rad protein assay.
Reagents--
All reagents were of analytical grade. PD098059
was purchased from Calbiochem. Polyclonal anti-human PDGF-AB antibody
(neutralizing) was obtained from Upstate Biotechnology Inc.
Data Analysis--
The results are expressed as the means ± S.E. The Statistical Analysis System was used for the statistical
analysis for the experiment described in Table II. A two-tailed value
of p < 0.05 was considered to be significant.
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RESULTS |
Purification and Identification of TFPI-2--
Table
I shows the step for purification of
mitogen for growth-arrested bovine aortic smooth muscle cells. In Fig.
1, results of ProRPC HR5/10 column
chromatography are shown. The solid column shows the peak in
cell growth activity. On SDS-PAGE analysis, the molecular mass of this
peak was 32 kDa (Fig. 2B). The
peptide sequence analysis revealed the N-terminal sequence to be
DAAQEPTGNNAEI, which was identical to TFPI-2 or placental protein 5 (3,
5).
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Table I
Purification of TFPI-2 from the conditioned medium of the cultured
HUVEC
Specific activity was defined as optical density increase in
growth-arrested bovine smooth muscle cell/µg protein compared with
the control (0.1% FCS). BrdUrd incorporation was assayed as described
under "Experimental Procedures."
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Fig. 1.
Reverse-phase high performance liquid
chromatography on a ProRPC HR 5/10 column. Eluent from HiTrap
Heparin column (5 ml) was concentrated by Centriprep 10 to 0.5 ml. This
sample was injected into ProRPC HR5/10 column. The gradient is shown by
the dotted line from 0.1% trifluoroacetic acid in 20%
acetonitrile to 0.1% trifluoroacetic acid in 100% acetonitrile. The
solid peak shows the cell proliferating activity for the
growth-arrested bovine aortic smooth muscle cells. The flow rate was 1 ml/min, and the peak was monitored at 280 nm.
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Fig. 2.
SDS-PAGE of purified TFPI-2
(B) and rTFPI-2 (C). Samples
(0.1 µg) were subjected to SDS-PAGE in Phast System. After
electrophoresis, the gel was stained with Coomassie Brilliant Blue. The
molecular size markers used (A) were as follows:
phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin
(43,000), carbonic anhydrase b (30,000), soybean trypsin inhibitor
(20,100), and -lactalbumin (14,400).
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rTFPI-2--
We produced the rTFPI-2 by using the mammalian
expression vector in the BHK cells. The supernatant in the transfected
BHK was collected and purified by the same method employed for the cultured HUVEC. Results of SDS-PAGE of the purified rTFPI-2 are presented in Fig. 2C.
Mitogenic Effect of rTFPI-2 on the Growth-arrested Bovine Aortic
Smooth Muscle Cells--
Fig. 3 shows
the effect of rTFPI-2 on the cell proliferation of the growth-arrested
bovine aortic smooth muscle. In a dose-dependent manner
(1-500 nM), rTFPI-2 increased the cell growth. Fig.
4 shows the time course of the cell
proliferation. Cell counts increased from day 0 to day 6. To confirm
the mitogenic character of rTFPI-2, we assayed BrdUrd incorporation
into DNA. In Fig. 5, BrdUrd was found to
be incorporated into DNA by rTFPI-2 stimulation
dose-dependently. In the Fig.
6 experiment, instead of 0.1% FCS, 10 ng/ml PDGF was used. In the presence of PGDF, the increased
incorporation of BrdUrd was also observed.

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Fig. 3.
Mitogenic effect of rTFPI-2 on bovine aortic
smooth muscle cells. Bovine aortic smooth muscle cells were plated
(2.0 × 105 cells) in 10-cm dish in DMEM + 0.1% FCS.
After 48 h, growth-arrested cells were exposed to rTFPI-2 in the
presence of 0.1% FCS. After 48 h, cell numbers were evaluated
with a hemocytometer. Data are the means ± S.E.
(n = 4).
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Fig. 4.
Effect of rTFPI-2 on cell proliferation in
vascular smooth muscle cells for 6 days. Bovine aortic smooth
muscle cells were plated (2.0 × 105 cells) in a 10-cm
dish in DMEM + 0.1% FCS. After 48 h, growth-arrested cells were
exposed to rTFPI-2 (500 nM) in the presence of 0.1% FCS.
rTFPI-2 (500 nM) was newly changed every 48 h. After
2, 4, and 6 days, cell numbers were evaluated with a hemocytometer. The
control experiment was also shown. Data are the means ± S.E.
(n = 4).
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Fig. 5.
Mitogenic effect of rTFPI-2 on bovine aortic
smooth muscle cells. Bovine aortic smooth muscle cells were plated
(3,000 cells/well) in 96-well plates in DMEM + 0.1% FCS. After 48 h, growth-arrested cells were exposed to rTFPI-2 in the presence of
0.1% FCS. After 24 h, BrdUrd was added, and its incorporation
into DNA was determined after 12 h. Data are the means ± S.E. (n = 6).
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Fig. 6.
Mitogenic effect of rTFPI-2 on bovine aortic
smooth muscle cells. Bovine aortic smooth muscle cells were plated
(3,000 cells/well) in 96-well plates in DMEM + 0.1% FCS. After 48 h, growth-arrested cells were exposed to rTFPI-2 in the presence of 10 ng/ml PDGF. After 24 h, BrdUrd was added, and its incorporation
into DNA was determined after 12 h. Data are the means ± S.E. (n = 6).
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Effect of Anti-Human PDGF-AB Antibody on rTFPI-2 or PDGF-induced
BrdUrd Incorporation into Smooth Muscle Cell--
BrdUrd incorporation
by rTFPI-2 was not inhibited by anti-human PDGF-AB antibody.
PDGF-induced BrdUrd incorporation was inhibited by anti-human PDGF-AB
antibody (Table II).
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Table II
Effect of anti-human PDGF-AB antibody on rTFPI-2 or PDGF-induced BrdUrd
incorporation into smooth muscle cell
Bovine aortic smooth muscle cells were plated (3,000 cells/well) in
96-well plates in DMEM + 0.1% FCS. After 48 h,
growth-arrested cells were exposed to the above substances in the
presence of 0.1% FCS. After 24 h, BrdUrd was added, and its
incorporation into DNA was determined after 12 h. Data are the
means ± S.E. (n = 6).
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MAPK Activation and MAPK Activity--
To study the
phosphorylation of MAPK in rTFPI-2-induced smooth muscle cell growth,
the Fig. 7 experiment was done. Using an antibody specific for dually phosphorylated MAPK (P44/ERK1 and P42/ERK2), a rapid and transient phosphorylation was found to occur
after the stimulation by rTFPI-2. A specific inhibitor of MAPK kinase,
PD098059 (100 µM), inhibited these phosphorylations. Fig. 8 shows the time course of MAPK
activity stimulated by rTFPI-2 PD098059 inhibited MAPK activity and
BrdUrd incorporation in a dose-dependent manner (Fig.
9).

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Fig. 7.
Phosphorylation of MAPK by MAPK kinase.
Growth-arrested bovine aortic smooth muscle cells (5 × 106cells) were stimulated to 2 h with 500 nM rTFPI-2 in the presence of 0.1% FCS. Cells were
analyzed for phosphorylated MAPK by Western blot analysis as described
under "Experimental Procedures." PD098059 (100 µM)
was pretreated 30 min before rTFPI-2 administration.
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Fig. 8.
Time course of MAPK activity stimulated by
500 nM rTFPI-2. Densitometric analysis was
performed by NIH image. Data are the means ± S.E.
(n = 4).
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Fig. 9.
Inhibition of MAPK activity and BrdUrd
incorporation by PD098059. MAPK activity and BrdUrd incorporation
was assayed as described under "Experimental Procedures." The
concentration was 500 nM. Data are the means ± S.E.
(n = 4).
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Transcriptional Activation of c-fos by rTFPI-2 and Inhibition by
PD098059--
TFPI-2 increased the c-fos promoter activity
by luciferase assay. PD098059 was found to inhibit the promoter
activity (Fig. 10).

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Fig. 10.
Transcriptional activation of
c-fos by rTFPI-2 and inhibition by PD098059.
c-fos promoter activity was determined as described under
"Experimental Procedures." Luciferase-generated light activity was
shown as relative activation of rTFPI-2-treated cells with or without
PD098059 versus untreated cells transfected in parallel.
Normalization was done using the co-transfected pRL-SV40 Vector. Data
are the means ± S.E. (n = 4).
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Effect of rTFPI-2 on c-fos Expression and the Inhibition by
PD098059--
Northern blotting analysis was done to study the
induction of proto-oncogene c-fos (Fig.
11). After stimulation with rTFPI-2, there was a rapid increase in c-fos expression. At 30 min, a
rapid increase was found in c-fos. After 2 h,
c-fos expression disappeared. This c-fos
expression was inhibited dose-dependently by PD098059 (Fig.
12).

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Fig. 11.
Rapid induction of c-fos
mRNA by rTFPI-2. Growth-arrested smooth muscle cells
(108 cells) were incubated with 500 nM rTFPI-2
in the presence of 0.1% FCS for 30 min to 4 h, and Northern blot
analysis was performed. Expression of c-fos and the
housekeeping glucose-3-phosphate dehydrogenase (G3PD) was
determined as described under "Experimental Procedures." Depicted
is an autoradiogram that represents three independent
experiments.
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Fig. 12.
Inhibition of c-fos
expression by PD098059. Growth-arrested smooth muscle cells
(108 cells) were incubated with 500 nM rTFPI-2
and PD098059 in the presence of 0.1% FCS for 30 min, and Northern blot
analysis was performed. PD098059 was pretreated 30 min before the
experiment. Expression of c-fos and the housekeeping
glucose-3-phosphate dehydrogenase (G3PD) was determined as
described under "Experimental Procedures." Depicted is an
autoradiogram that represents three independent experiments.
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DISCUSSION |
To study the effect of the endothelial cells on the smooth muscle
cell growth, the following co-culture system was used; a culture insert
with a 1.0 µM membrane (3102, Becton Dickinson) along
with cultured HUVEC was placed over bovine aortic smooth muscle cells
grown in 6-well micro-test plates (3502, Becton Dickinson). When
endothelial cells existed in the insert, the smooth muscle cell growth
was found to be stimulated (data not shown). Based on this finding, we
purified the mitogenic substance from the conditioned medium of the
cultured HUVEC (Table I and Figs. 1 and 2), and this purified peptide
proved to be identical to TFPI-2. The mitogenic activity of TFPI-2 has
not been reported previously. To elucidate the mechanism of the
mitogenic activity of rTFPI-2, we studied the signal transduction
pathway for smooth muscle cell growth. TFPI-2 activated the 44- and
42-kDa MAPK rapidly and transiently (Fig. 7). MAPK/ERK is a key
intermediate in a signal transduction pathway that links many types of
cell surface receptors with nuclear events that initiate mitosis
(12-14). TFPI-2 induced the activation of c-fos promoter
(Fig. 10) and subsequent rapid and transient expression of
c-fos mRNA (Fig. 11). PD098059, a specific inhibitor of
MAPK kinase inhibited dose-dependently the activation of
MAPK (Figs. 7 and 9), c-fos promoter activation (Fig. 10),
and expression of c-fos mRNA (Fig. 12). These data
suggest that TFPI-2 stimulates cell proliferation through MAPK
activation and subsequent c-fos expression.
Vascular smooth muscle cell growth is a key event in atherogenesis and
the restenosis after percutaneous transluminal coronary angioplasty. In
animal experiments, inhibition of TF-mediated coagulation with rTFPI
has been reported to be effective in preventing neointimal formation
and stenosis (6, 7, 15). Thrombin is a potent mitogen for smooth muscle
cells. TFPI is an inhibitor of factor Xa alone or factor VIIa-TF
complex in the presence of factor Xa. Factor Xa and factor VIIa-TF
cause thrombin generation. Thus, the inhibitory action of cell
proliferation and restenosis by rTFPI is considered due to the reduced
thrombin generation secondary to inhibition of VIIa/TF and factor Xa.
After balloon angioplasty, TFs exposed on the luminal surface of the
vessel and factor X activation seem to play an important role in
thrombus formation and the generation of thrombin. As a mechanism of
prevention of restenosis by rTFPI, the direct inhibitory effect of
rTFPI on the proliferation was proposed using cultured human neonatal aortic smooth muscle cells (8). The following mechanisms are proposed
for mitogenic activity of rTFPI-2: (i) rTFPI-2 directly binds to its
receptor on smooth muscle cell and stimulates the cell proliferation
and (ii) similar to factor Xa (16), TFPI-2 functions via the
stimulation of PDGF. However, this second possibility seems to be
unlikely, because BrdUrd incorporation was not inhibited by anti-human
PDGF-AB neutralizing antibody (Table II).
In conclusion, TFPI has been reported to exhibit antiproliferative
action against vascular smooth muscle after arterial injury in addition
to the inhibition of the activation of the extrinsic coagulation
cascade. However, the structurally similar TFPI-2 has been found to be
mitogenic for smooth muscle cell. There may be a new mechanism by which
these two peptides regulate smooth muscle cell proliferation.