(Circulation. 2000;101:2651.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Tissue Factor Overexpression in Rat Arterial Neointima Models Thrombosis and Progression of Advanced Atherosclerosis
From the Department of Surgery, University of Washington (D.H., H.L., A.W.C.), and Zymogenetics (C.E.H., S.L.), Seattle, Wash.
Correspondence to Dr Alexander W. Clowes, University of Washington, Department of Surgery, Box 356410, 1959 NE Pacific St, Seattle, WA 98195.
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Abstract |
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Background—Tissue factor located in the atherosclerotic plaque might cause the clinically significant thrombotic events associated with end-stage disease. It might also affect intimal area by increasing matrix accumulation and stimulating smooth muscle cell (SMC) migration and proliferation. To test this hypothesis, we overexpressed tissue factor in a rat model of the human fibrous plaque.
Methods and Results—A neointima was generated by seeding tissue factor–overexpressing rat SMCs onto the luminal surface of a balloon-injured syngeneic rat carotid artery. The cells attached and expressed tissue factor over the long term. Mural thrombus accumulation was present at 4 and 7 days and increased neointimal SMC numbers and area by 2-fold at 2 and 4 weeks. Tissue factor overexpression accelerated reendothelialization compared with controls at 2 weeks and 1 month. Tissue factor–overexpressing SMCs exhibited increased migration both in vitro and in vivo. The increased migration by tissue factor–overexpressing SMCs in vitro was not dependent on activation of the coagulation cascade and could be blocked by an inhibitor of tissue factor.
Conclusions—These results suggest that tissue factor plays a direct role in neointimal development by coagulation-dependent and -independent pathways.
Key Words: tissue factor • thrombosis • atherosclerosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Tissue factor is a 45-kDa membrane-bound glycoprotein that is the primary initiator of coagulation.1 Tissue factor is found on the surface of a variety of cell types normally located outside the vasculature.2 Tissue factor binds factor VII/VIIa, and the TF:VIIa complex can proteolytically activate factor X, which in turn activates thrombin and generates fibrin. Recently, 3 groups3 4 5 have reported that tissue factor deficiency is lethal during murine embryogenesis. Because these embryos exhibit abnormal vasculature, tissue factor may play an essential role in vascular development and integrity. Other groups have demonstrated that inhibition of tissue factor can inhibit intimal hyperplasia, supporting a role for tissue factor or downstream factors in intimal biology.6 7
Tissue factor is present only in the adventitia of the normal vessel and can be transiently induced by injury.8 9 In advanced atherosclerosis, tissue factor is expressed in the plaque and might play a role in the thrombotic response associated with disruption of the luminal surface and the fibrous cap.10 Although advanced atherosclerotic lesions can be generated in animals by cholesterol feeding or genetic manipulation, they do not model the critical terminal events of wall disruption and thrombosis found in humans.
To model the thrombotic aspect of advanced atherosclerosis, we constructed a synthetic neointima containing smooth muscle cells (SMCs) that constitutively expressed rat tissue factor. Rat SMCs were transduced in vitro with a retroviral vector containing tissue factor, and then the transduced SMCs were seeded back onto the vessel wall, where they permanently attached on the luminal surface of the carotid artery.11 In this study, we report that tissue factor plays an important role in neointimal development by increasing mural thrombus, increasing intimal lesion size, and accelerating endothelial regrowth. This process may depend on coagulation-dependent and -independent mechanisms.
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Methods |
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Construction of Rat Tissue Factor Retroviral Vector and SMC Transduction
The rat tissue factor coding sequence was introduced into the retroviral vector (LXSN) to construct the recombinant tissue factor vector (LTFSN) (Figure 1A
).12 The LXSN
vector alone was used as a control. DNA sequencing was performed to
verify the orientation and integrity of the retroviral construct.
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Viral packaging and cell transduction were performed according to
Miller and Rosman.12 Viral titers of
1x106 pfu/mL from PA317–tissue factor and
vector alone were used to transduce rat SMCs enzymatically isolated
from male Fischer 344 rat aortas.
Northern Analysis
Total cellular RNA was extracted13 and processed
for Northern analysis.14 Blots were probed with a
700-bp EcoRI fragment of the rat tissue factor cDNA and then
reprobed with 28s cDNA to document equal loading. Quantification
was performed with a PhosphorImager and ImageQuant (Molecular
Dynamics).
Tissue Factor and Factor VIIa Assays In Vitro
Tissue factor activity was determined by the ability of tissue
factor and exogenously added factor VIIa to convert factor X to Xa in a
chromogenic assay.15
The assay for tissue factor activity described above was modified as follows to measure factor VIIa. Purified factor X (American Diagnostica) was added to cells cultured for 5 days in serum-free growth medium (SmBM 3 supplemented with human epidermal growth factor 10 ng/mL, human fibroblast growth factor 2 ng/mL, transforming growth factor-ß1 0.5 ng/mL, and insulin 5 µg/mL, Clonetics Co). Samples were compared with identical wells given purified factor VIIa (0.1 to 1000 ng/mL, Zymogenetics).
Migration Assay for SMCs In Vitro
SMC migration in vitro was assayed with a 48-well modified
Boyden microchemotaxis chamber (Neuro Probe) and polycarbonate filters
(Nucleopore Corp) with 10-µm pores. The filters were precoated with
2.7 µg/well of basement membrane matrix (Matrigel, Collaborative
Research) in 0.5x PBS and dried overnight. Thirty minutes before use,
the matrix was reconstituted in 0.5x PBS and placed on top of the
lower chamber containing 20 ng/well of platelet-derived growth
factor (PDGF)-BB (Zymogenetics). SMCs were cultured in serum-free
growth medium (SmBM 3 with growth factors described above) for 5 days,
trypsinized, washed 3 times in serum-free medium, suspended at a
concentration of 5x105/mL in serum-free medium,
and added to the upper chamber. Recombinant hirudin (50 nmol/L,
CIBA-Geigy) and FFR-VIIai (a catalytically inactive form of factor VIIa
that retains full binding capacity to tissue factor, 4.8 µg/mL,
Zymogenetics) were added to the cells 10 minutes before the chamber was
loaded. The chemotaxis chamber was incubated for 5 hours at 37°C with
5% CO2. At the end of the assay, the cells that
had migrated were stained with Diff-Quick (Baxter) and reported as mean
number of cells per x400 field.
Long-Term Overexpression of Tissue Factor in the Carotid
Male Fischer 344 rats (250 to 300 g) were seeded with the
retrovirally transduced cells as previously described.11
To maintain vessel patency, both tissue factor–overexpressing and
control cells were pretreated with a tissue factor
inhibitor (10 µg FFR-VIIai/2.5x106
SMCs) for 10 minutes before seeding.
At various times, rats were killed, and the carotids were either removed or surgically exposed for further analysis. The rats were cared for according to the "Principles of Laboratory Animal Care" (formulated by the National Society of Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985).
Tissue Factor Activity Assay Ex Vivo
Tissue factor activity was assayed in isolated carotid segments.
Rats were killed with an overdose of pentobarbital and exsanguinated
through the abdominal aorta. The carotid artery was cannulated with
Silastic tubing (0.012-in ID, Technical Products) both proximal and
distal to the seeded area. Immediately after placement of the cannulas,
the artery was flushed with 3 mL M199 (Sigma), and then 2 U/mL
Proplex-T (Baxter) in M199 was infused and allowed to incubate in the
vessel for 10 minutes. The luminal volume was collected by flushing the
carotid artery with 200 µL M199, and the entire collected luminal
volume was placed in a 96-well plate. S-2765 (10 µL) was added, and
the OD was measured at 405 nm after a 20-minute incubation at 37°C.
Contralateral carotids and LXSN control seeded carotid arteries were
used as controls.
Migration of SMCs In Vivo
SMCs were genomically marked with bromodeoxyuridine
(BrdU) in vitro before seeding to allow tracking. This strategy allows
us to distinguish the seeded cells from endogenous SMCs by
immunohistochemistry. Subconfluent SMCs were cultured with 0.06 µg/mL
BrdU (Boehringer Mannheim) in DMEM with 10% FBS (Gibco-BRL)
for 48 hours and seeded as previously described. This concentration of
BrdU does not affect cell proliferation and labels >99% of SMCs. At
various time points, carotids were perfusion-fixed with formalin and
embedded in paraffin. Anti-BrdU antibody (Boehringer Mannheim)
and alkaline phosphatase with DAB (Vectastain) or True Blue (Kirkegaard
and Perry Laboratories), as described by the manufacturers, were used
for visualization. The number of BrdU-positive cells were expressed as
a percentage of total cells in each intimal quartile, starting with the
region closest to the internal elastic lamina and progressing toward
the lumen.
External Seeding Method for Measuring SMC Migration In
Vivo
A second method was developed to measure migration in vivo from
the adventitia into the carotid wall. Carotid arteries were surgically
exposed and stripped of loose connective tissue surrounding the
adventitia, and a Teflon sheet (1.5x3 cm) was placed under the
carotid to protect the surrounding tissue. The carotid artery was
decellularized by gentle touching of liquid nitrogen–cooled forceps to
the carotid artery until frozen. Freeze-thaw cycles were repeated 3
times. The Teflon sheet was removed, BrdU-labeled SMCs
(1.25x106 SMCs in 400 µL) were seeded onto the
adventitia of the carotid artery and incubated for 10 minutes, and then
the neck wound was closed. An Alzet osmotic minipump (model 2 ML1, Alza
Co) containing either the tissue factor inhibitor FFR-VIIai
(2.2 mg/mL) or saline was placed subcutaneously in the rat. Silicone
medical grade tubing (0.012-in ID, Technical Products) was used to
deliver FFR-VIIai (10 µL/h for 1 week) to the periadventitial region.
The total number of BrdU-positive cells migrating into the media and
intima per cross section was quantified in histochemical cross
sections.
Tissue Preparation, Morphometry, and Endothelial Staining
At various time points, tissue factor–overexpressing (LTFSN)
and control (LXSN) seeded carotids were flushed with lactated Ringer’s
solution (Baxter) and perfusion-fixed with 4% formalin, pH 7.4 (Fisher
Scientific) at 120 mm Hg pressure. Vessels were excised and
embedded in paraffin for histology and immunohistochemistry.
Cross-sectional areas were analyzed at 2 sites with a
digitizing pad (Opelco) and camera lucida. Endothelial
regeneration was visualized by Evans blue staining. Evans blue dye (60
mg/kg) (E-2129, Sigma) was injected into the tail vein 60 minutes
before euthanasia.16
Electron Microscopy
Carotid arteries were flushed and perfusion-fixed at 120
mm Hg with 4% paraformaldehyde for 3 minutes. Tissue
intended for scanning electron microscopy was pinned out to expose the
luminal surface and fixed in 2% osmium tetroxide before
sputter-coating.
Statistics
All values are expressed as mean±SD. Comparisons between tissue
factor and control groups and corrections for multiple comparisons were
made with combined Wilcoxon tests for blocked
data.17 Comparisons between tissue factor and control
groups at individual time points were made with Mann-Whitney
nonparametric tests. Statistical significance was set
at P<0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Construction and Overexpression of Rat Tissue Factor in SMCs In Vitro
Tissue factor–overexpressing cells had growth curves similar to those of controls (Figure 1B
). RNA from these cells was
analyzed by Northern blotting to verify expression of tissue
factor (Figure 1C). A 4.0-kB tissue factor transcript composed
of 3.1 kB of the retroviral vector sequence and 0.9 kB coding sequence
of rat tissue factor was easily identified in tissue
factor–overexpressing cells.
There was a 7-fold increase in tissue factor activity in vitro in
tissue factor–overexpressing cells compared with controls (Figure 2). Although uninjured SMCs in
vivo do not normally express tissue factor, SMCs in vitro do have
low-level expression.
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Migration In Vitro
The migration of tissue factor–overexpressing SMCs in vitro
through a basement membrane matrix toward the potent chemoattractant
PDGF-BB was increased compared with control SMCs (Figure 3). SMCs used in this assay were cultured
in the absence of serum and coagulation factors for 5 days; no residual
factor VIIa (<<1 pmol/L) was detected. Tissue factor–overexpressing
SMCs had an 2-fold increase in directed chemotaxis, with no increase
in chemokinesis. Addition of recombinant hirudin did not reduce
migration of tissue factor–overexpressing or control SMCs. Addition of
catalytically inactive factor VIIa reduced migration of tissue
factor–overexpressing SMCs but not control SMCs (Figure 3).
Factor VIIa or Proplex-T, a preparation of factor VIIa and X, also
reduced migration of tissue factor–overexpressing SMCs but not control
SMCs (data not shown). These experiments suggest that tissue factor can
increase migration independently of coagulation in response to
chemotactic factors.
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Tissue Factor Overexpression in the Carotid Artery
Tissue factor overexpression in the carotid artery resulted in
immediate thrombosis of >90% of the seeded vessels (data not shown).
To study the long-term effects of tissue factor overexpression, it was
necessary to temporarily attenuate tissue factor activity during the
seeding process. This was done by treating the tissue
factor–overexpressing cells with a catalytically inactive form of
factor VIIa that binds tissue factor but does not allow conversion of
factor X to Xa. Treatment with the inactive factor VIIa completely
blocked tissue factor activity for 
4 hours after seeding (Figure 2). Tissue factor activity remained elevated in the vessels
seeded with tissue factor–overexpressing SMCs at all time points
tested after 4 hours and was 5-fold greater than in vessels seeded
with control SMCs (Figure 2). This increased activity was
maintained even as the neointima became progressively
filled with a mix of endogenous and seeded SMCs. The level
of tissue factor expression after seeding of tissue
factor–overexpressing SMCs is higher than the increase seen after
injury alone.
Neointimal Area, Mural Thrombus, Endothelial
Regeneration
Tissue factor overexpression in the carotid artery resulted
in increased neointimal areas (Figures 4 and 5).
At early time points, this was due to a large increase in mural
thrombus (Figure 4). At 4 days, there was a clear boundary
between the seeded SMCs and an acellular area of fibrin accumulation,
which accounted for 50% of the neointimal area. At 1
week, the fibrin-rich mural thrombus was invaded by SMCs (Figure 4).
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) have larger
neointimas and smaller lumens than control carotids (•)
at 1, 2, and 4 weeks after seeding. Overall difference between
treatment groups, P>0.001 for both intimal area and
luminal area, corrected for multiple comparisons by combined
Wilcoxon test for blocked data. Difference at each time point
determined by Mann-Whitney test. At 1 week, P=0.014
intimal area, P=0.037 luminal area; n=11 control, n=9
tissue factor; 2 weeks, P=0.001 intimal area,
P=0.014 luminal area; n=10 control, n=9 tissue factor; 4
weeks, P=0.004 intimal area, P=0.029
luminal area; n=4 control, n=4 tissue factor. Data are mean±SD.
One week after seeding, the entire region of the tissue
factor–overexpressing but not control carotid artery was covered in
platelets (Figure 6). By 2 and 4
weeks, the surface was free of platelets and partly covered by
regenerating endothelium derived from the proximal and
distal uninjured regions adjoining the denuded area. Tissue
factor–overexpressing carotids had a 3-fold increase in
endothelial coverage of the seeded area at 2 weeks and
a 2-fold increase at 4 weeks compared with control seeded vessels
(Figure 7). The control LXSN
cell–seeded vessels were reendothelialized at the same
rate as the balloon-injured vessels not seeded with cells (data not
shown).
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At later time points, there was a significant increase in the number of
neointimal SMCs in the tissue factor–overexpressing
carotids and no significant differences in cell-to-matrix ratio (2-week
cell-to-matrix ratio: tissue factor 0.59±0.33, control 0.63±0.30,
P=NS, n5). SMC proliferation measured by BrdU labeling
index did not show any significant differences between experimental
control groups at 4 days, 1 week, 2 weeks, and 1 month (data not
shown). The absence of differences at late times suggests that
increased migration into the layer of thrombus might contribute in part
to this increased neointimal area.
Migration In Vivo
To confirm that tissue factor–overexpressing cells have increased
migration compared with control seeded cells in vivo, a labeling
strategy was used. Tissue factor–overexpressing cells or LXSN controls
were cultured in vitro with BrdU before cell seeding. The BrdU serves
as a genomic tag for subsequent identification of the seeded cells in
the carotid. The concentration of BrdU was not toxic, and the labeled
and unlabeled cells grew at the same rate (data not shown). By 2 weeks,
tissue factor–overexpressing SMCs tended to be located closer to the
lumen than control seeded SMCs (Figure 8). The LXSN control SMCs do not migrate
as rapidly as the tissue factor–overexpressing cells and tend to be
located closer to the internal elastic lamina.
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Seeding tissue factor–overexpressing SMCs in the lumen creates a large
mural thrombus that hinders comparisons of their migration with that of
controls. We have developed a novel migration assay to overcome this
problem. SMCs are seeded onto the adventitial side of a decellularized
carotid artery. This method does not require pretreatment with tissue
factor inhibitor and does not result in mural thrombus.
Using this method, we found that tissue factor–overexpressing SMCs
migrate more rapidly toward the lumen of the carotid (Figure 9). Increased migration of tissue
factor–overexpressing SMCs was reduced by continuous infusion of
catalytically inactive factor VIIai. Carotid arteries externally seeded
with tissue factor–overexpressing SMCs had an average of 21±15 tissue
factor–overexpressing cells per field in the media and intima at 1
week, compared with 4.5±3.5 cells when tissue factor
inhibitor was infused (n=5, P
0.05).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Increased expression of tissue factor in the intima might account for the thrombotic properties of advanced atherosclerosis. In this study, we demonstrate that tissue factor may influence lesion development through coagulation-dependent and -independent mechanisms. Tissue factor overexpression increased neointimal area by increasing mural thrombus and SMC migration as well as increasing endothelial regeneration. Tissue factor–overexpressing SMCs migrated more rapidly than control SMCs both in vitro and in vivo. Taken together, we conclude that tissue factor expression can play an important role in neointimal formation.
Tissue Factor Increases Neointimal Formation
The increase in neointimal area could be due to 3
mechanisms: increased matrix accumulation, increased proliferation or
decreased cell death, or increased migration of SMCs. Because SMC
density and proliferation were not altered in tissue
factor–overexpressing neointimas, it is likely that
enhanced SMC migration contributed to increased intimal thickening.
Sato et al18 19 showed that the catalytically active
tissue factor complex can induce chemotaxis in SMCs. Recent work by Ott
et al20 defined coagulation-independent and ligand (factor
VIIa)–dependent roles for tissue factor in cell adhesion and
migration. We have shown that tissue factor–overexpressing cells
themselves exhibit increased ability to migrate in vitro and in vivo.
Although we cannot exclude the possibility that tissue
factor–overexpressing SMCs also induce endogenous SMCs to
migrate, we conclude that tissue factor expression is able to directly
facilitate migration in an autocrine manner.
Tissue Factor Increases Migration In Vitro and In Vivo
Tissue factor–overexpressing SMCs have an increased rate of
migration in response to the chemoattractant PDGF-BB in vitro. This
assay was conducted in the absence of all coagulation cascade
components and thus rules out the possibility that factors such as
factor Xa or thrombin are responsible for tissue factor–induced
migration in vitro. Determining the cause of migration in vivo is
complicated by the massive platelet-rich mural thrombus that forms
as a result of tissue factor overexpression. The increased migration in
vivo could be due to a combination of factors derived from the
thrombus. Therefore, we propose that tissue factor in vivo facilitates
increased migration by coagulation-dependent and
coagulation-independent mechanisms. The coagulation-independent
mechanisms have not been defined but might include intracellular
signaling pathways or association with cytoskeletal
components.20 21
Tissue Factor Accelerates Endothelial Regeneration
Tissue factor overexpression is associated with accelerated
endothelial regeneration. This finding is
consistent with observations made by Lindner et
al22 showing that increased mural thrombus is associated
with accelerated endothelial regeneration. In other
experiments in which we have limited fibrinolytic activity by
overexpressing plasminogen activator
inhibitor-1, we have found a similar increase in
endothelial regeneration.23 From these
experiments, we cannot determine whether the increased
endothelial regeneration is a direct result of the
fibrin or of components derived from or attached to fibrin.
Relevance of the Rat Model to Advanced Atherosclerosis
Overexpression of tissue factor in the intima models several
aspects of the human lesion, including prolonged elevation of tissue
factor localized to the intima and increased mural thrombus. Tissue
factor might perform the same functions in the atherosclerotic plaque
as in the intima generated by cell seeding. It might also contribute to
intimal hyperplasia by increasing SMC migration into the intima. After
the fibrous cap ruptures, tissue factor would be expected to increase
thrombus formation at the site of plaque rupture. In addition, it might
encourage endothelial regeneration over the disrupted
lesion. In summary, tissue factor expression might encourage rapid
repair at the risk of increasing thrombosis at sites of vascular
injury.
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Acknowledgments |
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This work was supported by NIH grant HL-52459. Dr Hasenstab was supported by NIH training grant T32-HL-07312.
Received July 6, 1999; revision received December 16, 1999; accepted January 11, 2000.
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References |
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