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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 579-586
Regulation of Tissue Factor Pathway Inhibitor Expression in Smooth
Muscle Cells
By
Usha R. Pendurthi,
L. Vijaya Mohan Rao,
J. Todd Williams, and
Steven Idell
From the Departments of Molecular Biology, Biochemistry and Medical
Specialties, The University of Texas Health Center at Tyler, Tyler, TX.
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ABSTRACT |
Tissue factor pathway inhibitor (TFPI) is the primary physiological
inhibitor that regulates tissue factor-induced blood coagulation. TFPI
is thought to be synthesized, in vivo, primarily by microvascular endothelial cells. Little is known about how TFPI is regulated under
pathophysiological conditions. In this study, we determined mechanisms
by which TFPI expression is regulated by human pulmonary artery smooth
muscle cells (PASMC), because these cells contribute to remodeling of
the pulmonary vasculature in disease. PASMC in culture constitutively
synthesize and secrete TFPI. Exposure of PASMC to phorbol myristate
acetate, lipopolysaccharide, tumor necrosis factor  , thrombin,
interleukin-1, and transforming growth factor- had no significant
effect on expression of TFPI by PASMC. By contrast, treatment of PASMC
with serum and basic fibroblast growth factor (bFGF)/heparin markedly
upregulated the expression of TFPI activity and antigen. On Western
blot analysis, a protein consistent with full-length TFPI (42 kD) was
identified in the conditioned media of PASMC, and the levels of the
protein were much higher in the conditioned media of serum and
bFGF/heparin-treated cells. Northern blot analysis showed that PASMC
constitutively express TFPI mRNA, and treatment of cells with serum and
bFGF/heparin had a minimal effect on the steady-state levels of TFPI
mRNA. Nuclear run-on analysis did not show a significant increase in the transcriptional rate of TFPI gene in PASMC treated with serum or
bFGF/heparin. Cycloheximide, but not actinomycin-D, treatment inhibited
the serum and bFGF/heparin-induced increase in TFPI activity in PASMC.
In conclusion, our data demonstrate that PASMC constitutively
synthesize and secrete TFPI and serum or bFGF upregulate its
expression, suggesting that growth factors that can stimulate the
vessel wall in vivo might locally regulate TFPI expression. Our study
also suggests that control of TFPI expression by serum or bFGF occurs
via translational rather than transcriptional regulation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PULMONARY ARTERIAL vasculature
responds to various insults with a characteristic pattern of
remodelling that includes hyperplasia of smooth muscle cells and an
increase in connective tissue in the medial layer of the vessel
wall.1 Although the pathogenetic mechanism(s) of medial
hypertrophy is not fully characterized, the process appears to involve
functional derangements of endothelial cells, fibroblasts, and
pulmonary artery smooth muscle cells (PASMC).2 Thrombosis
plays an important role in pulmonary vascular injury.3 The
tissue factor (TF) pathway of coagulation plays a primary role in hemostasis and the pathophysiology of many diseases, including coronary thrombosis, sepsis, and cancer.4-8 In particular,
TF-induced procoagulant activity has been implicated in fibrin
deposition and fibrosis associated with lung injury.9
Furthermore, factor Xa and thrombin, the intermediary products of the
TF-pathway of coagulation, promote vascular smooth cell
proliferation10,11 and may thus play a role in the
development of intimal hyperplasia.
Tissue factor pathway inhibitor (TFPI), a multivalent protease
inhibitor with three Kunitz-type domains, is the primary inhibitor of
TF-mediated coagulation.12,13 TFPI directly inhibits factor Xa and blocks the procoagulant activity of the TF/VIIa complex by
forming a quaternary TF/VIIa/Xa/TFPI complex.13 Under
physiological conditions, TFPI is primarily synthesized by the
microvascular endothelium and in relatively smaller amounts by
megakaryocytes and macrophages.14-17 Human umbilical vein
vascular smooth muscle cells and lung fibroblasts were found to
synthesize only small amounts of TFPI.14 Little is
currently known about how the expression of TFPI is regulated within
the pulmonary vasculature.
In the present study, we sought to determine how the expression of TFPI
is regulated in vascular smooth muscle cells in response to various
agents implicated in the pathogenesis of pulmonary arterial
hyperplasia. The data demonstrate that PASMC constitutively express
TFPI and that serum and basic fibroblast growth factor (bFGF)
upregulate the expression of TFPI activity. Our data also provide
evidence, for the first time, that the control of TFPI expression in
PASMC occurs via translational rather than transcriptional regulation.
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MATERIALS AND METHODS |
Materials.
The cytokines used in the present study (bFGF, transforming growth
factor- [TGF-], tumor necrosis factor  [TNF], and
interleukin-1 [IL-1]) were human recombinant proteins from R&D
Systems (Minneapolis, MN). Phorbol myristate acetate (PMA) and
lipopolysaccharide (LPS; Escherichia coli serotype O111:B4)
were obtained from Sigma Chemical and Co (St Louis, MO). Fetal bovine
serum (FBS) was from GIBCO-BRL (Grand Island, NY). TRI Reagent was
obtained from Molecular Research Center Inc (Cincinnati, OH).
[ 32P]ATP (3,000 Ci/mmol) and
[32P]dCTP (3,000 Ci/mmol) were from Dupont NEN
(Boston, MA). Molecular biology grade chemicals were obtained from
either Boehringer Mannheim (Indianapolis, IN) or US Biochemicals
(Cleveland, OH). Other chemicals were obtained from Sigma and Fisher
(Fair Lawn, NJ).
Purified proteins.
Recombinant factor VIIa and full-length TFPI were gifts from
Novo-Nordisk (Gentofte, Denmark). Human plasma factor X18
and factor Xa19 were purified as described earlier or
purchased from Enzyme Research Laboratories Inc (Southbend, IN).
Thrombin was purchased from Enzyme Research Laboratories Inc.
Cell culture.
Primary cultures of PASMC and coronary artery smooth muscle cells
(CASMC) were obtained from Clonetics and cultured to
confluency in SmGM-2 growth medium (Clonetics, San Diego, CA) at
37°C under 5% CO2. The cells were subcultured by first
detaching the cells with trypsin solution and replating them in 24-well
culture plates (for measurement of TFPI activity and antigen) or in
T-75 flasks (for isolation of nuclei and RNA). When cells reached 90%
confluency, the growth medium was replaced with basal medium, SmBM
(Clonetics), supplemented with 0.5% bovine serum albumin (BSA), and
cultured for 24 hours. Cells between 5 to 10 passages were used in the experiments.
Stimulation of PASMC.
Quiescent PASMC (serum and growth factor-starved for 24 hours) in
24-well culture plate were stimulated with 1 mL of fresh basal medium
containing 10% (vol/vol) FBS or bFGF (10 ng/mL) in the presence of
heparin (15 U/mL) or other agonists as described in Results. At
specific intervals, 50 to 100 µL of supernatant was removed from the
well and stored at  20°C to measure TFPI activity and antigen
levels. As controls, cells were treated with basal medium for identical
lengths of time. To induce TF activity, quiescent PASMC were treated
for 8 hours with agonists. At the end of 8 hours, cell supernatants
were removed and the monolayers were washed twice with phosphate
buffer. Cell extracts were made by lysing PASMC in 0.25 mL of 15 mmol/L
octyl glucopyranoside. All treatments were performed under sterile
conditions, and the monolayers were allowed to stay in a culture incubator.
Measurement TFPI activity.
TFPI activity was determined in a two-stage chromogenic assay as
described earlier using purified human coagulant
proteins.20 A TFPI standard curve was obtained using
dilutions of 0.125% to 4% pooled human plasma. TFPI activity present
in 1 mL of normal plasma was taken as 1,000 mU. To measure TFPI
activity in cell lysates, the above assay was modified to contain a
limited concentration of factor VIIa (1 ng/mL) and an excess of TF (10 ng/mL) to avoid the interference of cell lysate TF in the assay. In
this assay, recombinant TFPI (0.25 to 4 ng/mL) was used to generate the
standard curve. Normal plasma contains 100 ng/mL TFPI, which is
equivalent to 1,000 mU TFPI activity. TFPI activity measured in FBS was
used to correct TFPI activity measurements of the conditioned media containing serum.
Measurement of TFPI antigen.
TFPI antigen was measured in an enzyme-linked immunosorbent assay
(ELISA) as described earlier using rabbit antihuman TFPI IgG as a
capturing antibody and biotinylated rabbit antihuman TFPI IgG as a
reporter antibody.20 Monospecific rabbit antihuman TFPI
antiserum was obtained by immunizing a rabbit with recombinant human
TFPI protein. A TFPI antigen standard curve was obtained using
dilutions of 0.625% to 10% pooled human normal plasma.
Measurement of TF activity.
TF activity was measured based as the ability of cell lysates to
support the activation of factor X with the addition of VIIa. Briefly,
cell lysates (45 µL) were incubated with a reagent mixture (5 µL)
containing factor VIIa (0.5 µg/mL), factor X (10 µg/mL), and
CaCl2 (5 mmol/L) in a microplate (all concentrations were final concentrations). At the end of 15 minutes, 50 µL of Chromozym X
containing 25 mmol/L EDTA was added to each well and the initial rate
of color development in mOD per minute at 405 nm was
measured with a microplate reader (Molecular Devices, Menlo Park,
CA). Purified relipidated human TF (0.0625 to 1 ng/mL) was
used to construct a standard curve.
Western blot analysis.
Cell supernatant samples (100 µL) were subjected to nonreduced sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12%
acrylamide gels and blotted onto a PVDF membrane. The blot was probed with rabbit antihuman TFPI IgG (100 µg/mL) or rabbit antiserum against TFPI C-terminal peptide (100-fold dilution) (kindly
provided by Dr George Broze, Washington University School of Medicine,
St Louis, MO) and developed using an enhanced chemiluminescence detection system kit (Amersham Life Sciences, Arlington Heights, IL).
RNA isolation and Northern blot analysis.
Total RNA was prepared from cells cultured in T-75 flasks by the acid
phenol method using TRI reagent according to the manufacturer's technical bulletin (Molecular Research Center, Inc, Cincinnati, OH).
Ten micrograms of total RNA was size-fractionated by gel electrophoresis in 1% agarose/6% formaldehyde gels and transferred onto a nitrocellulose membrane by a capillary blot method. Northern blots were hybridized using a 32P-labeled TFPI cDNA probe
described earlier.21 For quantitative purposes, the blot
was also exposed to a phosphor screen and the exposed screen was
analyzed in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using
ImageQuant software.
Nuclear run-on assay.
Nuclei from 4 to 6 × 106 PASMC were harvested and
run-on assays were performed with [-32P]UTP-labeled
RNA as described previously.22
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RESULTS |
Effect of various agonists on TFPI and TF expression by PASMC.
Quiescent monolayers of PASMC in 24-well culture plates were treated
for 24 hours with basal medium alone (control) or the basal medium
supplemented with various agonists and TFPI activity in the conditioned
media and cell lysate was measured. The data obtained with the
conditioned media showed that quiescent PASMC expressed low levels of
TFPI (60 mU/106 cells/mL/24 h) constitutively and the
treatment of cells with LPS, TNF-, thrombin, TGF-, and IL-1
slightly increased (<2-fold) the expression of TFPI activity.
However, the levels of TFPI secretion obtained with these treatments
was not statistically significantly higher than the level of TFPI
secretion observed with the cells treated with the control basal medium
(P values are in the range of .4 to .6, n = 3). By contrast,
treatment of PASMC with either serum (10% vol/vol) or bFGF/heparin
markedly enhanced (by 12- to 15-fold) the expression of TFPI activity
(Fig 1). The treatment of PASMC with either
bFGF or heparin alone had no effect on the expression of TFPI activity.
Smooth muscle cells from another vascular site (CASMC) also responded
to bFGF/heparin and serum treatments. Treatment of CASMC with
bFGF/heparin or serum increased the secretion of TFPI activity by
fourfold and fivefold, respectively. The actual levels of TFPI secreted
from CASMC are as follows: control (no treatment), 32 ± 7.8 mU/106 cells/mL/24 h; bFGF/heparin treated, 121 ± 45 mU/106 cells/mL/24 h; and serum (10% vol/vol) treated, 159 ± 63 mU/106 cells/mL/24 hours (n = 3 to 6). Measurement
of TFPI antigen in the conditioned media showed a fivefold to sixfold
increase in TFPI antigen levels in PASMC treated with serum or
bFGF/heparin over the cells treated with the basal medium alone
(Fig 2). A small increase in TFPI antigen
levels observed in PASMC treated with other agonists was not
statistically significant over TFPI antigen levels secreted in
untreated cells (P values varied between .15 and .65, n = 3).
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| Fig 1.
Effect of various pathophysiological agents on the
expression of TFPI activity by PASMC. Quiescent PASMC monolayers were
treated for 24 hours with one of the following agents: PMA (10 ng/mL),
serum (10% vol/vol), LPS (1 µg/mL), TNF- (10 ng/mL), thrombin (5 U/mL; 1.5 µg/mL), TGF- (10 ng/mL), IL-1 (10 ng/mL), bFGF (10 ng/mL), and heparin (15 U/mL). In control, no agonist was added. The
conditioned media, removed at the end of 24 hours of treatment, was
assayed for TFPI activity. The data shown are the mean of three
experiments performed in duplicate ± SD.
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| Fig 2.
Secretion of TFPI antigen by PASMC treated with various
pathophysiological agents. PASMC monolayers were treated as described
in Fig 1 and TFPI antigen levels in the conditioned media were
determined as described in Materials and Methods (n = 3).
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The measurement of TFPI activity in cell lysates showed that a small
fraction of TFPI activity was associated with cells (~5 to 10 mU/106 cells/mL/24 h). Treatment of the cells with
agonists, including serum and bFGF/heparin, did not increase TFPI
activity in cell lysates.
In addition to TFPI, we also measured the effect of various agonists on
the expression of TF activity by PASMC. PASMC constitutively expressed
substantial amounts of TF and serum treatment enhanced the expression
of TF activity by 2.5-fold, whereas bFGF/heparin had no effect on the
expression of TF activity. Thrombin and TGF- treatments increased TF
activity minimally (P = .095 for thrombin and P = .13 for TGF-), and LPS, IL-1, and TNF- had no effect on the
expression of TF activity by these cells
(Fig 3).
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| Fig 3.
Effect of various agonists on expression of TF activity
by PASMC. Quiescent PASMC monolayers were treated for with various
agonists as described in Fig 1 for 8 hours and the cell lysates were
harvested to measure TF activity. In control, no agonist was added to
cells. The data shown are the mean of three experiments performed in
duplicate ± SD.
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The effect of serum and bFGF/heparin on TFPI expression was found to be
both time- and dose-dependent. TFPI secretion from PASMC in culture was
increased, although not linearly but steadily, over a 72-hour
experimental time period (Fig 4). Treatment
of PASMC with varying concentrations of bFGF in the presence of a fixed
concentration of heparin showed a dose-dependent increase in TFPI
secretion. A concentration of 10 ng/mL bFGF enhanced TFPI expression
maximally (Fig 5A). Similarly, serum
treatment of PASMC enhanced the expression of TFPI in a dose-dependent
manner, and the expression of TFPI was maximal in cells treated with
5% (vol/vol) serum (Fig 5B).
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| Fig 4.
Time course of serum- and bFGF-enhanced TFPI secretion in
PASMC. Quiescent PASMC monolayers were treated with basal medium alone
(control) or with the basal medium containing serum (10% vol/vol) or
bFGF/heparin (10 ng/15 U/mL). At various time points, 50 µL of
aliquots was removed from the supernatant conditioned media and stored
at 80°C until assayed for TFPI activity. The data shown are the
mean of three experiments performed in duplicate ± SD.
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| Fig 5.
Serum and bFGF dose-dependent secretion of TFPI by PASMC.
(A) Monolayers of quiescent PASMC were treated with basal medium alone
(0) or with the basal medium containing varying concentrations of bFGF
in the presence of a fixed concentration of heparin (15 U/mL). (B)
PASMC were treated with basal medium alone (0) or varying
concentrations (vol/vol) of serum. At the end of 48 hours, the
conditioned media was removed and assayed for TFPI activity (n =3).
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Western blot analysis of TFPI expressed by PASMC.
Western blot analysis of conditioned media from the cells with
anti-TFPI IgG showed a protein of similar size (42 kD) to that of a
full-length rTFPI isolated from transfected BHK cells
(Fig 6A), suggesting that the TFPI secreted
in PASMC was the full-length form. Furthermore, a mixture of rTFPI and
the media containing TFPI was migrated as a single band on SDS-PAGE.
Moreover, immunoblot analysis of the conditioned media from both bFGF
and serum-treated cells with a TFPI C-terminal domain specific antibody
showed that the PASMC-secreted TFPI contained C-terminal domain (Fig
6B). Consistent with the measurements of TFPI activity and antigen, Western blot analysis also showed that quiescent PASMC constitutively express low levels of TFPI, and bFGF/heparin or serum markedly enhanced
the expression of TFPI (Fig 6A).
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| Fig 6.
Western blot analysis of PASMC-secreted TFPI. (A)
Quiescent PASMC were treated with basal medium alone (lanes 3 through
5) or the basal medium containing bFGF/heparin (10 ng/15 U/mL; lanes 6 through 8) or serum (10% vol/vol; lanes 10 through 12) for 8 (lanes 3, 6, and 10), 24 (lanes 4, 7, and 11), and 48 hours (lanes 5, 7, and 12).
Other lanes are as follows: lane 1, full-length recombinant TFPI from
BHK cells; lane 2, molecular weight markers; and lane 9 (zero time),
aliquot from the serum-containing medium that was removed immediately
after its addition to the cells. The blot was probed with monospecific
polyclonal anti-TFPI IgG. (B) One hundred microliters of conditioned
media from cells treated for 48 hours with serum-free (control), bFGF
(10 ng/mL)/heparin (15 U/mL) (bFGF), and serum (10%
vol/vol)-containing media were subjected to SDS-PAGE followed by
Western blot analysis. Other lanes are as follows: full-length
recombinant TFPI from BHK cells, diluted either in a buffer (rTFPI) or
in control serum (10% vol/vol). The blot was probed with a 100-fold
diluted rabbit antiserum raised against TFPI C-terminal domain peptide.
Molecular weight markers are Bio-Rad prestained markers (low range).
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Effect of serum and bFGF/heparin on the expression of TFPI mRNA in
PASMC.
Because serum and bFGF/heparin treatments enhanced TFPI activity and
antigen levels in PASMC, we next investigated the effect of these
treatments on the expression of TFPI mRNA by Northern blot analysis. As
shown in Fig 7, quiescent PASMC
constitutively express TFPI mRNA. Treating PASMC with serum or
bFGF/heparin for 8 and 24 hours did not increase the level of TFPI mRNA
expression, although at these time points we observed a marked increase
in the expression of TFPI activity and antigen in PASMC (see Figs 1, 2,
and 4). It is interesting to note that TFPI mRNA levels were slightly
but consistently higher (2-fold, P = .046, n = 4) in PASMC
treated with bFGF/heparin for 48 hours. Although both 4- and 1.4-kb
messages were present in PASMC, the predominant TFPI mRNA in smooth
muscle cell was 4 kb (~80%).
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| Fig 7.
Effect serum and bFGF on TFPI mRNA accumulation in PASMC.
Quiescent PASMC were treated with basal medium alone or the basal
medium containing serum (10% vol/vol) or bFGF/heparin (10 ng/15 U/mL)
for 8, 24, and 48 hours. Total RNA was isolated from the cells, 10 µg
of each RNA sample was used for Northern blot analysis, and the blot
was hybridized with a radiolabeled TFPI cDNA probe and exposed to x-ray
film (A). Because the TFPI 1.4-kb message in these cells was about 20%
of the total TFPI message and migrated as a diffused band, it was not
visible clearly in the figure. Ethidium bromide staining of 28S and 18S
ribosomal RNA of the same samples indicates equal RNA loading. (B)
Quantitative analysis of hybridization signal obtained with
PhosphorImager (n = 3). The symbols are as follows: C, control
treated (basal medium alone); S, serum treated; F, bFGF treated.
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Because quiescent PASMC were found to express TFPI, we used these cells
to determine the half-life of TFPI mRNA. Quiescent PASMC were treated
with actinomycin-D (2.5 µg/mL) to inhibit transcription, and the
decay of TFPI mRNA with time was analyzed by Northern blot
analysis. The data showed that TFPI mRNA in PASMC appeared to decrease
very slowly, and the apparent half-life appears to be between 48 and 72 hours (data not shown). Because prolonged treatments of PASMC with
actinomycin-D resulted in significant cell death, we were unable to
determine the half-life of TFPI mRNA in a more quantitative manner.
Effect of serum or bFGF/heparin on the transcription of TFPI.
Although, as described above, Northern blot analysis of TFPI mRNA
accumulation failed to show a significant increase in the steady-state
levels of TFPI mRNA in PASMC treated with serum or bFGF/heparin, it is
possible that serum or bFGF/heparin could have exerted their effect on
TFPI gene transcription. We, therefore, sought to determine if
treatment with bFGF/heparin or serum enhanced the transcription rate of
TFPI gene. Nuclear run-on experiments were performed to compare the
rate of TFPI transcription in control quiescent PASMC and PASMC treated
with serum or bFGF/heparin. As shown in Fig
8, minimal transcription of TFPI was observed in control PASMC, and the
rate of transcription of TFPI gene was minimally changed after either
serum or bFGF/treatment. Quantitation of hybridization signal using a
PhosphorImager showed only a 10% to 20% increase in TFPI gene
transcription (corrected to transcription of house-keeping gene,
tubulin) in cells treated with bFGF/heparin or serum. In a positive
control, the transcription urokinase-type plasminogen activator
(uPA) gene was increased by 4.5- to 13-fold upon treatment
with bFGF/heparin and serum, respectively.
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| Fig 8.
Effect of serum and bFGF on nuclear run-on transcription
of TFPI gene. Quiescent monolayers of PASMC were treated with control
basal medium (Control), bFGF/heparin (10 ng/15 U/mL), or serum (10%
vol/vol) for 8 hours. Three identical blots containing TFPI and other
target DNAs were hybridized with equal amounts of labeled transcripts
of nuclear RNA. DNAs used were as follows: -tubulin (-Tub), TFPI,
urokinase-type plasminogen activator receptor (uPAR), urokinase-type
plasminogen activator (uPA), tissue-type plasminogen activator (tPA),
and TF.
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Effect of actinomycin-D and cycloheximide on upregulation of TFPI
expression.
To examine the contribution of transcriptional and translational
regulation to serum and bFGF-induced increase in TFPI activity, cells
were treated with either serum or bFGF/heparin in the presence and
absence of actinomycin-D and cycloheximide. As shown in
Fig 9A, actinomycin-D had no significant
effect on the serum and bFGF/heparin-induced TFPI activity, whereas
cycloheximide markedly inhibited the serum and bFGF/heparin-induced
TFPI expression. The data were further corroborated by Western blot
analysis (Fig 9B), which showed a minimal decrease in TFPI antigen in
the conditioned media from the cells treated with actinomycin-D before
stimulation with either bFGF/heparin or serum. In contrast, no
demonstrable TFPI antigen was secreted into the conditioned media if
the cells were treated with cycloheximide before their stimulation.
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| Fig 9.
Effect of actinomycin-D and cycloheximide on bFGF- and
serum-induced TFPI expression. Quiescent PASMC monolayers were
pretreated with actinomycin-D (5 µg/mL) or cycloheximide (10 µg/mL)
for 1 hour and then treated with serum (10% vol/vol) or bFGF/heparin
(10 ng/15 U/mL) for 24 hours. Conditioned media was assayed for TFPI
activity (A) or analyzed for TFPI antigen by Western blot analysis (B).
Control (in panel A) and none (in panel B) represent serum or
bFGF/heparin treatments in the absence of inhibitor.
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DISCUSSION |
Endothelial cells have been thought to be the primary source of TFPI
within the vasculature.14 Megakaryocytes and macrophages are shown to express small amounts of TFPI, whereas circulating monocytes and fibroblasts express none to negligible amounts of TFPI.17 In this study, we show that human PASMC in culture
constitutively synthesize and secrete TFPI. The levels of TFPI secreted
by PASMC (60 mU/106 cells/mL/24 h) is relatively similar to
the levels of TFPI secreted by endothelial cells (65 to 110 mU/106 cells/mL/24 h) but much higher than the level of
TFPI secreted by human umbilical vein SMC (5 mU/106
cells/mL/24 h).14,16 The difference in TFPI levels secreted by PASMC in the present study and the earlier study14 could be due to the difference in the vascular origin of SMC. Most of the
TFPI synthesized in PASMC was secreted, with less than 10% being
associated with cells. Although it had originally been thought that
TFPI synthesized in endothelial cells is primarily associated with
cells,23-25 recent data showed that virtually all TFPI
synthesized in endothelial cells26,27 and vascular smooth
muscle cells27 is secreted.
Little is known about how TFPI is regulated by the pulmonary
vasculature. Earlier studies with endothelial cells failed to provide
much information about the regulation of TFPI, because TFPI synthesis
in these cells is minimally affected by inflammatory agents, such as
TNF, IL-1, PMA, and LPS.16 In a preliminary report, Bajaj
et al28 reported that serum stimulation of human fetal lung
fibroblasts increased the expression of TFPI by sixfold. While the
present manuscript was under review, Caplice et al27 reported that serum stimulation of coronary vascular smooth cells resulted in a fivefold increase in TFPI antigen and activity levels in
conditioned medium at 48 hours. A number of cytokine and growth factors, including IL-1, TGF-, thrombin, and bFGF, have been implicated in the pathogenesis of hyperplasia of the arterial vascular
wall. Therefore, we tested a range of cytokines and growth factors to
define their ability to regulate the expression of TFPI in PASMC. Our
data show that serum and bFGF/heparin caused a marked increase in the
expression of TFPI activity, whereas other agents, such as thrombin,
TNF-, TGF-, IL-1, and LPS, minimally increased the expression of
TFPI. The increase in TFPI activity is associated with an increase in
TFPI antigen levels. However, we found some discrepancy between TFPI
activity and TFPI antigen levels. For example, we found a 12- to
15-fold increase in TFPI activity in cells treated with serum and bFGF,
whereas the measurement of TFPI antigen levels showed a fivefold to
sixfold increase. It is unlikely that the difference could be due to a
possible contribution TFPI-2 to our functional activity assay, because monospecific anti-TFPI antibodies completely neutralized the VIIa/TF inhibitory activity of the conditioned media. Thus, the difference could be due to a difference in the sensitivity of the activity and the
antigen assays. Although we cannot entirely rule out the possibility,
it is unlikely that the specific activity of TFPI secreted by PASMC
treated with serum or bFGF varies from the specific activity of
constitutively expressed TFPI. Western blot analysis showed that both
constitutively expressed TFPI and TFPI-induced by serum and bFGF were
full-length and contain C-terminal domain.
The presence of heparin is required for bFGF to stimulate the
expression of TFPI. One should note that heparin or bFGF treatment alone had no effect on the secretion of TFPI activity and total cell-associated TFPI levels. This rules out the possibility that bFGF/heparin-mediated increase in TFPI secretion stems from the release
of cell-associated TFPI. Heparin was shown to potentiate FGF activity
by either directly interacting with growth factor itself or with the
FGF-receptor. It has been shown that heparin and heparan sulfate
interact structurally with bFGF to induce conformational change in the
polypeptide that stabilizes or increases its biological
activity.29-31 Heparin has also been shown to decrease the
kd for FGF binding to its receptor.31
Serum and bFGF-induced increase in TFPI expression in PASMC appears to
involve transcriptional regulation to a very small degree. Northern
blot analysis showed that PASMC constitutively express TFPI mRNA. The
level of TFPI mRNA accumulation was not changed or only minimally
increased after treatments with serum or bFGF for 8 and 24 hours,
whereas the TFPI antigen and activity levels were increased by sixfold
and 12-fold, respectively, after 24 hours of treatment. These data were
further supported by nuclear run-on analysis that showed a minimal
increase in the rate of activation of TFPI gene transcription. In
addition, the inability of actinomycin-D to inhibit the increased
levels of TFPI activity and antigen in serum- and bFGF-treated cells
suggested that the regulation of TFPI could involve posttranscriptional
or translational regulation. However, we cannot completely rule out
some transcriptional regulation of TFPI, particularly in cells exposed
to bFGF for prolonged periods, because we consistently observe a small
increase in TFPI mRNA levels in these treatments.
Because TFPI mRNA appears to be relatively stable in the absence of any
treatment32 (and present data), we inferred that it would
be unlikely that an increase in TFPI mRNA stability could explain the
increased expression of TFPI activity and antigen in serum-or
bFGF-treated cells. Furthermore, we found no change in TFPI mRNA
stability in serum- or bFGF-treated cells over 48 hours after
transcriptional blockade. In addition, our data showed that
cycloheximide, but not actinomycin-D, treatment fully inhibited both
serum- and bFGF-induced increase in TFPI activity and TFPI antigen.
Overall, these data suggest that the regulation of TFPI gene expression
by serum and bFGF in PASMC is mainly regulated at the translational
level. Further experiments are needed to establish the mechanism(s) by
which serum and bFGF affects the translation of TFPI mRNA into a mature
functionally active protein.
The observed effect of serum and bFGF on the expression of TFPI could
be physiologically relevant in vascular remodelling associated with a
number of diseases, including pulmonary hypertension and
atherosclerosis. Several cytokines implicated in the pathogenesis of
these diseases stimulate expression of TF in endothelial cells, macrophages, and SMC, and the aberrant expression of TF in these cells
could lead to arterial thrombosis.3,5,7 Furthermore, thrombin10 and factor Xa,11 intermediary
products generated in TF-pathway of coagulation, are shown to promote
vascular smooth cell proliferation and thus may play a role in the
development of intimal hyperplasia.7 Serum- and
bFGF-stimulated expression of TFPI could inhibit the aberrant
expression of TF activity, which could restore hemostasis and temper
the factor Xa and thrombin-mediated proliferation of SMC. In this
regard, it may be important to note that our recent studies showed that
bFGF could suppress the aberrant expression of TF in endothelial
cells.33 Both endothelial cells and SMC were shown to
synthesize bFGF.34-36 Endothelial cell-derived bFGF is
associated with endothelial cells, subendothelial cell extracellular
matrix, and basement membrane.34 The denudation of
endothelium associated with vascular injury allows the circulating constituents of plasma to access the media and also leads to the release of stored bFGF, which would then be available for SMC in
neighboring intima.34,37,38 This bFGF could interact with heparin-like molecules produced by endothelial cells39 or
derived from degradation of extracellular matrix and basement membrane.
In summary, our data provide evidence that SMC could synthesize and
secrete functional TFPI, and the levels of TFPI synthesized by SMC are
similar to those of endothelial cells, the cells that were thought to
be the principal source of vascular TFPI. Our study also demonstrates,
for the first time, that the expression of TFPI in SMC is regulated by
serum and bFGF by a posttranscriptional mechanism. Serum- and
bFGF-mediated upregulation of TFPI in SMC could be physiologically
important for regulation of TF-coagulation pathway within the vessel
wall and thereby influence the course of hyperplasia associated with
pulmonary hypertension and atherosclerosis.
| |
ACKNOWLEDGMENT |
The authors thank Jason Voigt for his technical assistance.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted March 11, 1999.
Supported in parts by Grants No. HL 45018 and HL 58869 from the
National Heart, Lung and Blood Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Usha R. Pendurthi, PhD, Department of
Molecular Biology, The University of Texas Health Center at Tyler,
11937 US Highway 271, Tyler, TX 75708; e-mail: usha{at}uthct.edu.
| |
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ABSTRACT
INTRODUCTION