© 2000 American Heart Association, Inc.
Thrombosis |
Local Plasminogen Activator Inhibitor Type 1 Overexpression in Rat Carotid Artery Enhances Thrombosis and Endothelial Regeneration While Inhibiting Intimal Thickening
From the Department of Surgery, University of Washington, School of Medicine, Seattle.
Correspondence to Dr Alexander W. Clowes, University of Washington, Department of Surgery, Box 356410, Seattle, WA 98195-6410. E-mail clowes{at}u.washington.edu
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Abstract |
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Abstract—Elevated levels of plasminogen activator inhibitor type 1 (PAI-1) are found in advanced atherosclerotic plaque compared with normal vessel and may contribute to plaque progression and complications associated with plaque rupture. Increased expression of PAI-1 probably contributes to the thrombotic properties of advanced atherosclerotic plaque by impeding plasmin generation and degradation of fibrin. To test this hypothesis, we have deliberately created synthetic neointimas by seeding onto the denuded luminal surface of rat carotid arteries smooth muscle cells transduced with replication-defective retrovirus encoding rat PAI-1. This cell-based gene transfer method results in stable, long-term, and localized gene expression. PAI-1 overexpression increases mural thrombus accumulation at 4 days but decreases neointimal area by 30% and 25% at 1 week and 2 weeks, respectively. PAI-1 overexpression accelerates reendothelialization of injured arteries compared with control arteries at 1 week, 2 weeks, and 1 month. PAI-1 overexpression does not alter matrix accumulation at 1 week. Increased PAI-1 expression in the rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening. These results suggest that PAI-1 could play a direct role in the development of advanced atherosclerotic plaque and in the repair of the diseased vessel after fibrous cap disruption.
Key Words: plasminogen activator inhibitor type 1 • carotid arteries • thrombosis • fibrinolysis • endothelium
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Introduction |
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Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiological inhibitor of the plasminogen activator system and thereby blocks the conversion of plasminogen to plasmin. The plasminogen activator (PA) system is composed of urokinase PA (uPA) and tissue PA that proteolytically convert plasminogen to the serine protease plasmin. Components of the PA system are localized to the cell surface by receptors or extracellular matrix binding sites.
Increased expression of PAI-1 has been demonstrated in atherosclerotic arteries.1 2 PAI-1 is elevated in mesenchymal-appearing intimal cells at the base of the plaque and in the necrotic core. Its function in the advanced atherosclerotic lesion is not known. PAI-1 may limit the fibrinolytic capacity of the plaque.3 It also might modulate cellular proliferation or migration in the lesion through changes in matrix composition and growth factor release. The effects of PAI-1 are likely to be exerted locally in view of the fact that a majority of patients with generalized atherosclerosis have normal plasma fibrinolytic profiles.4 5
The various biological effects of PAI-1 generate a dilemma.6 On the one hand, local PAI-1 overexpression should enhance fibrin accumulation and thereby contribute to the growth of atherosclerotic lesions.3 7 On the other hand, increased PAI-1 inhibits smooth muscle cell (SMC) migration and the formation of neointima in injured mouse vessels; this result supports the hypothesis that PAI-1 overexpression retards the growth of atherosclerotic lesions.8 9
In the following experiments, we have attempted to define the role of PAI-1 in the intima to resolve this dilemma and to understand how PAI-1 may influence the biology of the advanced atherosclerotic lesion. We have developed a rat model for this purpose. This model makes use of a novel approach that involves localized gene overexpression and is well suited for the study of genes that may be involved in atherosclerotic progression. Localized gene overexpression is achieved by constructing a synthetic neointima in the carotid artery by seeding rat SMCs transduced in vitro with a retroviral vector containing the gene of interest. We have previously demonstrated that this cell-seeding technique gives long-term and biologically significant gene expression in the intima.10
In the present study, we demonstrate that increased PAI-1 expression in the rat carotid artery inhibits neointimal formation, increases platelet accumulation, and accelerates endothelialization of the injured carotid artery. Our findings demonstrate that PAI-1 plays an important role in neointimal formation in the rat and suggests that PAI-1 may influence the biology of the atherosclerotic lesion in humans.
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Methods |
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Construction of Rat PAI-1 Retroviral Vector
Rat PAI-1 cDNA, supplied by T. Gelherter (University of Michigan, Ann Arbor),11 was modified to have a Kozak12 consensus sequence (ACCATGG) by polymerase chain reaction with appropriate primers. The PAI-1 coding sequence was introduced into the unique HpaI site of the retroviral vector (LXSN)13 to construct the recombinant PAI-1 vector (LPAISN, Figure 1
). The
LXSN vector without the PAI-1 coding region was used as a control. DNA
sequencing was performed to verify the orientation and integrity of the
retroviral construct.
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Rat SMC Transduction
Viral packaging was performed according to Miller and
Rosman.13 Two packaging cell lines were used. PE501 cells
were transfected with the retroviral constructs and used to produce
replication-defective retrovirus to infect the amphotropic packaging
cell line PA317. Viral titers were 
1x106
plaque-forming units per milliliter for LPAISN and LXSN. Rat SMCs were
enzymatically isolated from male Fischer 344 rat aortas and then
cultured in DMEM with 10% FBS. Supernatant harvested at 16 hours from
the packaging cell lines was used to infect rat SMCs. Colonies of G418
(1 mg/mL, GIBCO/BRL)–resistant cells were isolated.
RNA Isolation and Northern Analysis
Total cellular RNA was isolated from SMCs grown in DMEM
containing 10% FBS by using guanidinium
isothiocyanate–phenol–chloroform extraction as previously
described.14 Isolated RNA was resuspended in 0.5% SDS and
quantified spectrophotometrically. RNA samples were separated in a 1%
agarose/formaldehyde gel.15 RNA was transferred to a
Zeta-probe nylon membrane (Bio-Rad) as described by the manufacturer
and cross-linked to the membrane by using UV light (Stratagene). Filter
hybridizations were carried out as described by Church and
Gilbert.16 rRNA bands (18S) were visualized with UV light
to document equal loading.
PAI-1 Activity Assay In Vitro and Carotid Extracts
PAI-1 activity was determined by reverse zymography as
previously described.17 Equal amounts of protein from
cells grown in culture or from homogenized carotid arteries
were separated on an SDS-polyacrylamide gel, and the gel was
overlaid onto an agar gel containing plasminogen, uPA, and
casein. PAI-1 inhibits the caseinolytic activity of
plasminogen activated by urokinase. The reverse
zymogram was developed at 4°C overnight and then at 37°C for 3 to 6
hours. Dark-field illumination makes regions of undigested casein
appear white when viewed on a black background; quantification of bands
was performed by densitometry.
Zymography was also performed on carotid extracts by using the methods described above, except that uPA was omitted in the agar gel. Amiloride (1 mmol/L final concentration, Sigma Chemical Co) was added to some agar underlays to inhibit uPA activity. Rat urine was collected from euthanized rats by use of a hypodermic needle and syringe.
Migration, Attachment, and Proliferation In Vitro
The ability of rat SMCs to invade the matrix and migrate toward
a chemoattractant was assayed by using 2 experimental approaches. The
first approach made use of a modified Boyden chamber method that
involved a 48-well microchemotaxis chamber (Neuro Probe) and
polycarbonate filters (Nucleopore Corp) with 10-µm pores. The filters
were precoated with 2.7 µg per 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 the filter was assembled on top of the lower chamber containing
0.67 nmol/L platelet-derived growth factor (PDGF)-BB
(Zymogenetics). Cultured SMCs were trypsinized, washed 3 times in
serum-free medium, and resuspended at a concentration of
5x105 cells per milliliter in serum-free DMEM,
and then 40 µL of the cell suspension was added to the upper chamber.
The chemotaxis chamber was incubated for 5 hours at 37°C under 5%
CO2. At the end of the assay, the upper side of
the filter was scraped clean, and the cells that had migrated to the
bottom of the filter were stained with Diff-Quick (Baxter) and
quantified.
Attachment assays were conducted as described for the Boyden chamber above, but no chemoattractant was added to the lower wells. After 60 minutes of incubation, the filters were gently washed 3 times in 1x PBS, and the cells were counted on the upper side of the filter.
For longer term migration studies, a thick layer of collagen over pluronic gel containing PDGF-BB was used. Two milliliters of Pluronic gel (F-127, Sigma) per well in a 24-well plate containing 0.67 nmol/L PDGF-BB was allowed to gel at 37°C for 2 hours before adding 1 mL of chilled Vitrogen (Cohesion Technologies), which was allowed to polymerize for 2 hours at 37°C. SMCs were trypsinized (0.05% trypsin), counted, and suspended in DMEM containing 10% FBS (GIBCO/BRL) with 5 mmol/L hydroxyurea (Sigma) to inhibit proliferation. Rabbit anti-rat PAI-1 IgG (5 µg/mL, No. 1062, American Diagnostica) or control rabbit IgG (5 µg/mL, Santa Cruz Biotechnology) was added to the cells before they were plated on Vitrogen. Cells were plated on the surface of the polymerized Vitrogen at a density of 1.0x103 cells per square millimeter. After 48 hours, the medium was removed, and each well was fixed with 100% methanol and stained with Diff-Quick stain. The number of cells that had migrated into the collagen was determined by phase microscopy in 4 fields per well. The experiments were performed in duplicate or triplicate 4 times.
To measure growth rates in vitro, 103 cells per square centimeter were cultured in DMEM containing 10% FBS (GIBCO) in triplicate and then at various time points were trypsinized and counted in a hemocytometer.
SMC Seeding In Vivo
Male Fischer 344 rats (250 to 300 g) were
anesthetized,18 and the left common artery was
surgically exposed and stripped of endothelium by the
passage of a balloon catheter. Transduced SMCs were seeded onto the
luminal surface as previously described.10 SMCs
(105) were infused into the carotid artery and
allowed to attach for 10 minutes.
At various times, rats were killed, and the carotid arteries 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 No. 86-23, revised 1985). One day before the rats were killed, one 5-bromo-2'-deoxyuridine (BrdU) tablet (50 mg, Boehringer-Mannheim) was implanted subcutaneously to measure proliferation.
PAI Activity in Carotid Extracts
Plasmin generation was measured as an indicator of net PAI/PA
activity in seeded carotid arteries essentially as described
previously.19 20 Carotid arteries seeded with PAI-1
(LPAISN) or control SMCs (LXSN) were excised and
homogenized in cold buffer (50 mmol/L Tris, pH 9.0).
Protein (10 µg) was added to human Glu-type plasminogen
(0.5 µmol/L final, American Diagnostica), fibrinogen
(100 µg/mL final, American Diagnostica), and a
chromogenic substrate of plasmin S-2251 (400 nmol/L final,
Chromogenix), and the optical density at 405 nm was measured after 30
minutes.
Tissue Preparation, Immunohistochemistry, Morphometry, and
Endothelial Staining
At various time points, PAI-1–overexpressing and control
cell–seeded carotid arteries were flushed clear of blood with lactated
Ringer’s solution (Baxter) and perfusion-fixed with 4% formalin in
PBS (Fisher Scientific) at 120 mm Hg pressure.
Endothelial regeneration was visualized by Evans blue
staining. Evans blue dye (60 mg/kg, E-2129, Sigma) was injected in the
tail vein 60 minutes before the animals were killed.21 The
blue-white boundary defines the limit of endothelial
ingrowth. Endothelial ingrowth was measured from the
proximal and distal ends of the injured carotid by using the carotid
bifurcation and proximal tie as reference markers. When excised
for measurement, the injured region of the carotid artery is 10
mm long. Vessels were embedded in paraffin and cross-sectioned for
histology and immunohistochemistry.
Proliferating SMCs were quantified on histological cross sections after immunostaining for BrdU. BrdU was detected immunohistochemically and reported as percent BrdU-positive cells in the intima.22
Morphometric analysis of the lumen, intima, and media was performed by using a camera lucida linked to a computer-driven digitizing pad and software (Opelco)
Electron Microscopy
Carotid arteries were flushed with Ringer’s solution and then
perfusion-fixed at 120 mm Hg with 4%
paraformaldehyde for 3 minutes. Tissue intended for
transmission electron microscopy was embedded in Epon (Polysciences)
for sectioning and subsequent transmission electron microscopy
analysis on a JEOL 100B (Japan Optics Electron Laboratory) at
60 kV. Tissue intended for scanning electron microscopy was pinned out
to expose the luminal surface and fixed in 2% osmium tetroxide before
sputter coating (n=3 in each group at 1 and 2 weeks, n=2 in each group
at 4 weeks.) The entire seeded area and several millimeters of carotid
proximal to the seeded area were examined.
Representative areas of minimal and maximal
platelet accumulation were photographed.
Quantification of cell volume–to–matrix ratios were performed on cross sections of PAI-1 and control cell–seeded carotid arteries as described previously.23
Statistics
All values are expressed as mean±SD. Comparisons between PAI-1
and control groups were made by Mann-Whitney nonparametric
tests (SPSS, version 8.0.0). Statistical significance was set at
P
0.05 by 2-tailed tests.
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Results |
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Total RNA from PAI-1–overexpressing and control cells was analyzed by Northern blotting to verify expression of the PAI-1 cDNA (Figure
I, published online at http://atvb.
ahajournals.org/cgi/content/full/20/3/853/DC1). A 4.2-kb PAI-1
transcript was easily identified in PAI-1–overexpressing cells. This
transcript was of the expected size and is composed of 3.1 kb of the
retroviral vector sequence and 1.1 kb of rat PAI-1 open reading frame.
The control vector alone and PAI-1–overexpressing SMCs show a band at
3.0 kb corresponding to the endogenous message.
Endogenous PAI-1 mRNA is diminished when serum is withdrawn
(data not shown). Clonal isolates of PAI-1–overexpressing SMCs had a
similar expression of PAI-1 compared with pooled groups of transduced
SMCs. Pooled groups of PAI-1–overexpressing SMCs were used for all
experiments in the present study.
PAI-1–overexpressing SMCs have the same morphological and growth
properties as control vector alone–transduced SMCs and passage-matched
primary arterial SMC isolates. There were no significant
differences in the growth rate of PAI-1–overexpressing SMCs compared
with SMCs transduced with the control vector (Figure II, published
online at
http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1).
PAI-1 activity in cultured cells was measured by their ability to
reduce plasmin generation in a reverse zymogram. There was an 4-fold
increase in PA inhibitory activity in the
PAI-1–overexpressing SMCs compared with control SMCs (Figure III,
published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1).
SMC migration through matrix is dependent on proteolytic
activity.24 25 26 Therefore, we assayed the ability of
PAI-1–overexpressing SMCs to migrate through Matrigel in a modified
Boyden chamber. No significant differences were observed in migration
over a 5-hour time period or in attachment to Matrigel (Figure IV,
published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1). In longer
migration assays in which SMCs migrate into a collagen gel for 48
hours, PAI-1–overexpressing SMCs had reduced levels of migration
compared with control SMCs. This reduced migration was reversed by the
addition of a PAI-1–blocking antibody (Figure 2). PAI-1–blocking antibodies did not
significantly increase control SMC migration.
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Effects of PAI-1 Overexpression on Neointima
PAI-1–overexpressing SMCs were seeded into the lumen of
balloon-injured carotid arteries. Increased PAI-1 activity was
independently confirmed by measuring net plasmin generation ex vivo. We
have measured the residual active PA and amount of plasmin generated in
the seeded carotid arteries and found that PAI-1 overexpression reduces
local PA activity and subsequent plasmin generation in the carotid by
50%, as measured by chromogenic substrate S-2251
(Figure 3). The extraction buffer used
does not activate latent PAI-1, and the direct plasmin
generation assay without SDS present gives us confidence that PAI-1
produced by the seeded SMCs is biologically active.
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Carotids seeded with PAI-1–overexpressing SMCs have increased PAI-1
immunoreactivity compared with uninjured or control cell–seeded
carotid arteries (Figure 4). The media of
all vessels shows little PAI-1 immunoreactivity. The
neointima is composed of seeded and endogenous
SMCs. The even distribution of PAI-1 immunoreactivity in the
neointima suggests that PAI-1–overexpressing SMCs have
migrated throughout the neointima and that not all PAI-1
immunoreactivity is cell-associated.
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There was increased PA inhibitory activity in the
PAI-1–seeded vessels at 1 week as measured by reverse zymography
(Figure 5, lanes 1 and 2). At earlier
development times, the reverse zymograph showed faint PAI-1 activity in
the control lane (data not shown). There was an 8-fold increase in
PAI-1 activity in the PAI-1–overexpressing carotid extracts compared
with control cell–seeded carotid arteries. Carotid arteries
overexpressing PAI-1 did show an increase in a low molecular weight
caseinolytic activity compared with control cell–seeded vessels
(Figure 5, lanes 3 and 4). This 28-kDa caseinolytic activity
might be a fragment of uPA in view of the fact that it was inhibited
with 1 mmol/L amiloride.
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PAI-1 overexpression resulted in increased mural thrombus compared with
either control cell–seeded carotid arteries (Figure 6) or regions beyond the zone of PAI-1
seeding (data not shown). Areas of mural thrombus formation with
clearly identifiable fibrin accumulation were seen only in
PAI-1–overexpressing carotid arteries (Figure 6D). This
increase in platelet adhesion and mural thrombus persisted for 1
week. At 2 and 4 weeks, platelets and fibrin were not visible on
the luminal surface.
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PAI-1 overexpression decreased neointimal areas by 30% and
25% at 1 week and 2 weeks, respectively (Figure 7). By 1 month, the intimal areas of the
PAI-1–overexpressing and control carotid arteries were not
significantly different. Medial areas and internal elastic lamina
lengths were not significantly different at any time tested. There were
also no significant differences in proliferation rates in vivo at 1, 7,
and 14 days after seeding (Figure V, published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1). To
determine whether the loss of reduced intimal thickening at 1 month was
due to a loss of gene expression, PAI-1 activity was measured by
reverse zymography at 1 month. There was no apparent loss of elevated
PAI-1 expression compared with control cell–seeded vessels or vessels
receiving injury alone (data not shown).
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10) and day 14
(*P=0.037, n
Changes in the proteolytic balance could result in changes in
extracellular matrix composition or volume. At early time points after
cell seeding, we found differences in platelet and fibrin
accumulation. There were no significant differences in the ratio of
matrix to cell volume in the neointima between control
cell– and PAI-1–seeded carotid arteries at 1 week (Figure VI,
published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1).
Ultrastructural morphology within the neointima did not
show any clear differences between control cell– and PAI-1–seeded
carotid arteries, although the luminal surface of the
PAI-1–overexpressing carotid arteries at 4 days had more platelets
(data not shown).
Endothelial Regeneration
Reendothelialization was accelerated in
PAI-1–overexpressing neointimas. At early time points, the
entire area of the PAI-10–overexpressing neointima was
covered by patches of platelet-rich mural thrombus (Figure 6). At later time points (2 weeks and 1 month), the surface was
free of platelets, and the PAI-1–overexpressing
neointima was rapidly reendothelialized
(Figure 8). In the injured rat carotid
artery, endothelial cells migrate and proliferate from
the proximal and distal uninjured regions adjoining the denuded areas.
PAI-1–overexpressing carotid arteries had a 2-fold increase in
endothelial coverage of the seeded area at 2 weeks and
4 weeks compared with control cell–seeded vessels. The control
cell–seeded vessel was reendothelialized at a rate
similar to that of the unseeded balloon-injured vessel (data not
shown).
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Discussion |
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Localized and stable overexpression of PAI-1 was achieved by using a novel cell-based gene transfer technique involving SMCs seeded onto denuded carotid arteries. Increased PAI-1 expression altered the development of the neointima after arterial injury. In the present study, we report that PAI-1 overexpression in the neointima enhanced early thrombosis, reduced intimal thickening, and accelerated endothelial regrowth. These results support the conclusion that PAI-1 plays a direct role in regulating intimal formation in injured arteries and may play a role in atherosclerosis and restenosis.
Effects of PAI-1 Overexpression on Thrombosis
At early times after arterial injury in the rat, there
is some platelet adherence along with limited fibrin deposition on
the luminal surface that clears after 1 day. PAI-1 is transiently
induced during the first 24 hours after injury and may serve to limit
fibrinolysis.17 PAI-1 overexpression
during this early repair period extended the duration of platelet
adhesion and fibrin accumulation through 1 week.
Increased platelet and fibrin accumulation does not persist beyond 1 week, even though plasmin generation remains suppressed. This result suggests either that increased levels of PAI-1 are able only to slow the removal of fibrin or that compensatory increases in PA activity counteract increased PAI-1. We detected no changes in uPA or tissue PA in PAI-1–overexpressing carotid arteries; however, we did see the appearance of a 28-kDa caseinolytic activity. This 28-kDa caseinase is likely to be a low molecular weight fragment of uPA, because it is inactivated by amiloride. We conclude that a sustained increase in PAI-1 is sufficient to delay but not prevent the removal of fibrin on the luminal surface.
Endothelial Regeneration
PAI-1 overexpression in the injured rat carotid artery accelerated
reendothelialization. The mechanism for accelerated
regrowth is not known, but several previous publications have addressed
this question. Lindner et al27 compared gentle filament
denudation with balloon catheter injury and found that gentle
denudation increased platelet adherence and accelerated
endothelial regrowth. Cell-based tissue factor
overexpression increases platelet and fibrin accumulation and also
accelerates endothelial regrowth, suggesting that
platelets or fibrin could stimulate endothelial
regrowth.27a However, Carmeliet et al8
compared endothelial regrowth rates in PAI-1–deficient
mice and mice with normal PAI-1 levels and did not find any differences
at 1 week after injury. This result suggests that PAI-1 does not have a
direct role in endothelial regrowth, at least in the
murine electric injury model. Increased PAI-1 expression in vitro
inhibits endothelial migration.28 Taking
these potentially conflicting results together, we believe that
increased PAI-1 could accelerate endothelial regrowth
by increasing the duration or amount of platelet adherence to the
vessel wall or by increasing the amount of fibrin on the luminal
surface after injury. The lack of visible platelets or fibrin at
any time after 1 week in either tissue factor–overexpressing,
PAI-1–overexpressing, or gently denuded carotid arteries combined with
prolonged increases in endothelial migration suggests
that fibrin breakdown products or platelet factors released
early after injury could continue to drive endothelial
migration at later time points.
Intimal Hyperplasia
Intimal areas were significantly reduced at 1 and 2 weeks after
injury in PAI-1–overexpressing carotid arteries. At these times, SMCs
are migrating from the media to the intima. It is interesting to note
that treatments that reduce migration through a wide variety of methods
tend to block intimal growth for 2 weeks after injury. After this
time, the intimas seem to catch up with controls. Carotid arteries
overexpressing PAI-1 also follow this same pattern, which might be
attributed to a decrease in migration followed by compensatory low
level increases in proliferation or matrix accumulation over a
several-week period. Proliferation indices (BrdU labeling) at 1, 7, and
14 days were the same in PAI-1–overexpressing and LXSN control
cell–seeded arteries (Figure V). This finding is consistent
with other experiments in which inhibitors of PAs or matrix
metalloproteinases (tranexamic acid, tissue inhibitor of
metalloproteinases type 1, and BB-94) significantly inhibited
SMC migration and reduced neointimal formation up to 2
weeks but did not sustain this effect at later times.29 30
In our short-term in vitro migration assays, PAI-1–overexpressing SMCs
migrated at the same rate as did LXSN control SMCs. In longer term
experiments, PAI-1–overexpressing SMCs exhibited decreased rates of
migration, and this reduced migration was reversed when PAI-1 was
inhibited with a blocking antibody.
Increased PAI-1 expression could alter the proteolytic balance in the arterial wall, resulting in changes in the ratio of cells to the surrounding matrix. PAI-1–overexpressing carotid arteries were examined at 1 week by transmission electron microscopy and histochemistry for relative changes in cell and matrix components. At this time point, we did not detect any changes in cell-to-matrix ratios or extracellular matrix composition between PAI-1–overexpressing SMCs and LXSN control cell–seeded carotid arteries.
Our results from overexpressing PAI-1 in the rat carotid artery confirm previous reports by Carmeliet and colleagues8 9 showing that PAI-1 can inhibit intimal formation after electrical arterial injury in mice. However, our experiments also demonstrate significant differences, including the degree of inhibition and effects on endothelial regrowth. These differences may be due to the systemic release of PAI-1, lack of sustained expression in adenovirus-mediated PAI-1 expression in mice, and the increased severity of the electrical injury.
Relevance of the Rat Model to Advanced Atherosclerosis
In the present study, we demonstrate that localized
overexpression of PAI-1 can modulate intimal biology. Our studies might
also help resolve the dilemma posed in the introduction.6
Overexpression of PAI-1 in the intima models several aspects of the
human lesion, including prolonged elevation of PAI-1 localized to the
intima and increased mural thrombus. PAI-1 might perform the same
functions in the atherosclerotic plaque as in the intima generated by
cell seeding. It might also contribute to plaque fragility by
preventing SMC migration into the fibrous cap.6 After the
fibrous cap ruptures, it would be expected to prevent dissolution of
the thrombus formed at the site of plaque rupture. In addition, it
might encourage endothelial regeneration over the
disrupted intima but suppress SMC migration and intimal hyperplasia. In
summary, PAI-1 expression might encourage rapid repair and limit
scarring at the risk of increasing thrombotic complications at sites of
vascular injury.
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Acknowledgments |
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This study was supported by National Institutes of Health (NIH) grant HL-52459. D.H. was supported by NIH training grant T32 HL-07312. We thank Dr Thomas D. Gelehrter (University of Michigan, Ann Arbor) for the rat PAI-1 cDNA.
Received August 25, 1999; accepted October 7, 1999.
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References |
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