(Circulation. 1997;96:3180-3191.)
© 1997 American Heart Association, Inc.
Articles |
Inhibitory Role of Plasminogen Activator Inhibitor-1 in Arterial Wound Healing and Neointima Formation
A Gene Targeting and Gene Transfer Study in Mice
From the Center for Transgene Technology and Gene Therapy (P.C., L.M., D.C., R.D.G.), Flanders Interuniversity Institute for Biotechnology, University of Leuven, Leuven, Belgium; Laboratory of Experimental Cardiology (S.J.), University of Leuven, Leuven, Belgium; Vascular Biology Laboratory (F.L.), Thrombosis Research Institute, London, UK; and Department of Biochemistry and Internal Medicine (R.D.G.), University of Texas Southwestern Medical Center, Dallas, Tex.
Correspondence to P. Carmeliet, MD, PhD, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
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Abstract |
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Background Plasminogen-deficient mice display impaired vascular wound healing and reduced arterial neointima formation after arterial injury, suggesting that inhibition of plasmin generation might reduce arterial neointima formation. Therefore, we studied the consequences of plasminogen activator inhibitor-1 (PAI-1) gene inactivation and adenoviral PAI-1 gene transfer on arterial neointima formation.
Methods and Results Neointima formation was evaluated in PAI-1–deficient (PAI-1-/-) mice with perivascular electric or transluminal mechanical injury. PAI-1 deficiency improved vascular wound healing in both models: the cross-sectional neointimal area was 0.001±0.001 mm2 in PAI-1+/+ and 0.016±0.008 mm2 in PAI-1-/- mice within 1 week after electric injury (P<.02) and 0.055±0.008 mm2 in PAI-1+/+ and 0.126±0.006 mm2 in PAI-1-/- mice within 3 weeks after mechanical injury (P<.001). Proliferation of smooth muscle cells was not affected by PAI-1 deficiency. Topographic analysis of arterial wound healing after electric injury revealed that PAI-1-/- smooth muscle cells, originating from the uninjured borders, more rapidly migrated into the necrotic center of the arterial wound than wild-type smooth muscle cells. On the basis of immunostaining, PAI-1 expression was markedly upregulated during vascular wound healing. There were no genotypic differences in reendothelialization of the vascular wound. When PAI-1-/- mice were intravenously injected with replication- defective adenovirus expressing human PAI-1 (AdCMVPAI-1), plasma PAI-1 antigen levels increased in a dose-dependent fashion up to to 61±8 µg/mL with 2x109 plaque-forming units (pfu) virus. Luminal stenosis was 35±13% in control AdRR5-treated (2x109 pfu) and suppressed to 5±5% in AdCMVPAI-1–treated (6x108 pfu) PAI-1-/- mice (P<.002).
Conclusions By affecting cellular migration, PAI-1 plays an inhibitory role in vascular wound healing and arterial neointima formation after injury, and adenoviral PAI-1 gene transfer reduces arterial neointima formation in mice.
Key Words: plasminogen • angioplasty • stenosis • genetics • viruses
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Introduction |
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The plasminogen system has been implicated in several cardiovascular disorders, including wound healing after vascular reconstructions.1–12 The inactive proenzyme plasminogen is converted to its active derivative plasmin by two physiological PAs, t-PA and u-PA.13 Their action is controlled by irreversible PAIs, of which PAI-1 appears to be the predominant inhibitor.5 Plasmin has been implicated in wound healing via proteolysis of extracellular matrix components or activation of growth factors.13–15
Luminal stenosis, resulting from arterial neointima formation, limits the success of vascular reconstructions for treatment of atherothrombosis.6 Although vascular remodeling appears to be a major determinant of luminal stenosis after balloon angioplasty, intimal thickening can also contribute to the luminal narrowing, for example after placement of intraluminal stents. Intimal thickening results from accumulation of smooth muscle cells that proliferate in the media, migrate across the elastic membrane and subsequently contribute to intimal thickening by increased proliferation and matrix deposition.6,16 In a recent study, we have demonstrated that vascular wound healing and arterial neointima formation are significantly impaired in mice with a disruption of the plasminogen gene.17 In addition, u-PA was found to be the predominant activator mediating migration of smooth muscle cells.18 The role of PAI-1 in these studies was, however, not defined.
The present study in mice in which the gene encoding PAI-1 has been inactivated (PAI-1-/-)19 provides direct evidence, using two models of arterial injury, for a significant role of PAI-1 in arterial neointima formation via regulation of smooth muscle cell migration. In addition, we demonstrate that overexpression of PAI-1 in the plasma of mutant mice by intravenous injection of replication-defective adenovirus expressing human PAI-1 is able to suppress arterial neointima formation. These data might have relevance for the design of strategies to treat restenosis in patients but also suggest caution in the therapeutic use of PAI-1 inhibitors to improve thrombolysis and reduce atherosclerosis.
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Methods |
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Arterial Injury Model
Six- to 8-week-old PAI-1+/+ and PAI-1-/- mice19 of either sex with a 75% C57Bl6/25% 129 genetic background were used. Groups of 8 to 14 animals were studied. Perivascular electric injury of left femoral arteries and analysis of neointima were performed as described elsewhere.20 Although neointima formation occurs in both femoral and carotid arteries, the response in the femoral arteries was analyzed because it occurs somewhat more rapidly and vigorously (P. Carmeliet, L. Moons, D. Collen, unpublished observations). Intraluminal mechanical injury of the carotid arteries was performed according to a recently described method by Lindner et al,21 with minor modifications. Briefly, the left internal carotid artery was exposed by blunt-end dissection, tied off distally, and looped proximally with 7–0 Mersilene nylon suture for vascular control during the procedure. A transverse arteriotomy was made in the proximal portion of the internal carotid artery, and a 380-µm flexible guide wire (C-SF 15–15, Cook Belgium NV) was passed to the aortic arch over ~15 mm and withdrawn three times with a rotating motion. After removal of the guide wire, the proximal portion of the internal carotid artery was tied off, and the skin incision was closed as described above. This protocol allowed reproducible injury, resulting in rupture of the internal elastic lamina and neointimal accumulation of
-smooth muscle actin–positive cells within 3 weeks after injury.
The carotid artery was used because of a technical limitation with
insertion of the guide wire in the femoral artery.
Tissue Harvest, Histology, and Immunocytochemistry
Tissue harvest, fixation, embedding, and sectioning were
performed as described previously.17,20 Smooth
muscle and inflammatory cells were immunostained for smooth
muscle -actin or CD45 as described
previously.20 Macrophages were visualized
with the use of a rat monoclonal antibody against Mac-3 (Pharmingen).
Replication was determined by labeling cells with
5'-bromo-2'-deoxyuridine and immunostaining as
described previously.20 Vitronectin
was stained with a biotinylated cross-reacting anti-human mouse
monoclonal antibody.22
PAI-1 staining was performed with a polyclonal rabbit anti-murine PAI-1
or a rabbit anti-human PAI-1 antiserum (with similar
results).19 Colocalization of PAI-1 with smooth
muscle cells or macrophages was established through the use of
a double immunofluorescence labeling approach as
described previously.23 With this procedure,
PAI-1–positive cells appear green, -actin–or Mac-3–positive cells
appear red, and cells containing PAI-1/-actin or PAI-1/Mac-3 appear
yellow.
Morphometric Analysis and Reendothelialization
Morphometric measurements of cross-sectional areas, cell counts
and proliferation rates, and topographic analysis of
neointima formation in electrically injured arteries were
performed as described previously.17,20
Morphometric analysis of mechanically injured arteries was
performed on five sections, equally spaced across the injured segment.
Reendothelialization was evaluated after staining of
the denuded blood vessels with Evans blue as described
previously.20
Zymographic Analysis
Zymographic analysis of PA activities in
arterial extracts was performed as described
previously.17 In situ zymography on 7-µm
cryosections of arteries was performed by fibrin overlay using a gel
prepared by clotting a mixture of human fibrinogen (final
concentration, 4 mg/mL), plasminogen (final
concentration, 10 µg/mL), and agarose (final concentration,
0.5%) with thrombin (final concentration, 0.3 NIH U/mL). The fraction
of the lysis due to t-PA or u-PA activity was determined by including
in the fibrin gel 20 µg/mL polyclonal neutralizing rabbit
anti-murine t-PA–and/or u-PA–specific IgG,
respectively.24 Purified murine t-PA and u-PA
were used as standards.24 The amount of lysis of
the gels (areaxintensity) was quantitatively analyzed using
the Quantimed 600 image analysis software and expressed in
arbitrary units.
Adenovirus-Mediated PAI-1 Gene Transfer
The adenovirus precursor pACCMVPAI-1 was generated by ligating
the 1.4-kb EcoRI/BglII fragment of
pPAI-1RBR25 containing the entire coding sequence
of human PAI-1 into EcoRI/BamHI–digested
pACCMVpLpA. In this plasmid, the PAI-1 cDNA is positioned
between the human CMV immediate-early enhancer/promoter and the SV40
t-antigen intron/polyadenylation signal. AdCMVPAI-1, control AdRR5
adenovirus (without transgene),26 and AdCMVLacZ
virus (expressing the Escherichia coli ß-galactosidase
gene) were generated according to standard procedures and purified to
stocks of >1010 pfu/ml as described
previously.27
Primary human umbilical vein endothelial cells were cultured in serum and growth factors as described previously,25 infected for 1 hour with recombinant adenovirus stocks at a multiplicity of infection of 20 pfu/cell (2.5x106/6-cm plate), and subsequently cultured in culture medium for 48 hours. Cells were refed with 2 mL of Dulbecco's modified Eagle's medium/Medium 199 (1:1) supplemented with 0.5% bovine serum albumin, and the conditioned medium (4 hours at 37°C) was assayed for PAI-1 activity.28
Three days after electric injury, anesthetized mice were intravenously injected with 200 µL of the indicated dose of control (AdRR5) or recombinant adenovirus (AdCMVPAI-1, AdCMVLacZ) using a 2F catheter (Portex green, Baxter). On the seventh day (eg, 4 days after virus injection), 100 µL blood was sampled from the retroorbital plexus from virus-transduced mice in a recipient containing 0.1 vol of EDTA (100 µL of 10 mg/mL stock solution). PAI-1 antigen and activity were measured as described previously.29 On day 14, the mice were killed, and the injured arteries were analyzed as described above. In separate experiments, PAI-1-/- mice were injected with AdRR5, AdCMVLacZ, or AdCMVPAI-1 (1 to 2x109 pfu), and the liver and the injured left and uninjured right femoral arteries were harvested 4 days after infection for immunohistochemical staining of PAI-1 antigen or LacZ staining as described previously.27
Statistical Analysis
Experimental values are expressed as mean±SEM. Statistical
significant differences between groups were calculated by ANOVA
followed by Bonferroni's correction or Wilcoxon's signed rank
analysis, as indicated in the text.
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Results |
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Vascular Wound Healing in Wild-Type Mice
Electric Injury Model
Because the electric injury model differs from the mechanical injury model,21 we briefly discuss the characteristics. Fig 1
schematically
represents the healing response to electric injury, which
denudes the endothelium and destroys all cells in the
media (<1% surviving medial cells).20 This
resulted in a wound-healing response characterized by transient
thrombosis, removal of necrotic debris,
reendothelialization, repopulation of the media by
smooth muscle cells, and the formation of a neointima with
only a few layers within 1 week (Fig 2b)
and multiple layers of smooth muscle cells within 3 weeks after injury
(Fig 2d and 2e).20 A neoadventitia formed that
was rich in leukocytes and fibroblast-like cells.
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Mechanical Injury Model
Mechanical injury resulted in less severe medial cell necrosis
(52±8% surviving medial cells, P<.05 versus uninjured
arteries) than electric injury. Because residual viable smooth muscle
cells persisted across the entire injured segment,
neointima formation occurred more uniformly across the
injured segment, as described previously for similar mechanical injury
models.21 Fig 2g
and 2h displays a
representative neointimal lesion containing
smooth muscle cells in a wild-type carotid artery within 3 weeks after
mechanical injury. Because the electric injury model permits the
differentiation between proliferation and migration of smooth muscle
cells and is technically easier than the mechanical injury
model,20 it was used more extensively throughout
this study.
Vascular Wound Healing in PAI-1-/- Mice
Electric Injury Model
After electric injury, the removal of the necrotic debris,
repopulation of the media by smooth muscle cells, and accumulation of
neointimal cells were accelerated in
PAI-1-/- mice during the first 2 weeks after
electric injury. Compared with PAI-1+/+ arteries,
which revealed only a small neointima at the uninjured
borders within 1 week after injury (Fig 2b), a larger
neointima containing more cells was already present in
PAI-1-/- arteries at the uninjured borders (Fig 2c) and progressed further into the necrotic center (see below). In
addition, the adventitia appeared larger and hypercellular (not shown).
Quantitative measurements of the cross-sectional neointimal
area within the boundary of the internal elastic lamina and the lumen
(Fig 3a), intima-to-media ratio (Fig 3b),
percent luminal stenosis (Fig 3c), and number of
neointimal cells (Fig 3d) revealed that
neointima formation was significantly accelerated in
PAI-1-/- mice compared with
PAI-1+/+ mice within the first weeks after
electric injury. There were no significant genotype-specific
differences in the neointimal area or cell counts beyond 3
weeks after injury (Fig 3), indicating that PAI-1 deficiency
accelerated but did not increase the extent of neointima
formation in the electric injury model.
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Mechanical Injury Model
PAI-1 deficiency also improved neointima formation
after mechanical injury. On histological
analysis, the neointima appeared much larger and
contained more cells (Fig 2i). Morphometric analysis revealed
that within 3 weeks after injury, the neointimal
cross-sectional area (Fig 4a),
intima-to-media ratio (Fig 4b), percent luminal stenosis (Fig 4c), and number of neointimal cells (Fig 4d) were higher in
PAI-1-/- than PAI-1+/+
arteries. The mechanisms underlying the improved neointima
formation in PAI-1-/- mice were further
investigated using the electric injury model.
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Proliferation of Smooth Muscle Cells
To evaluate whether deficiency of PAI-1 affects cellular
proliferation, incorporation of 5'-bromo-2'-deoxyuridine into
replicating cells was determined (Table 1). Proliferation of cells in
electrically injured arteries 2 weeks after injury is a better index of
smooth muscle cell proliferation because the media and
neointima are largely devoid of leukocytes beyond 1 week
after injury.20 As shown in Table 1, medial and
neointimal cell proliferation did not different
significantly between PAI-1+/+ and
PAI-1-/- arteries.
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Migration of Smooth Muscle Cells
After electric injury, smooth muscle cells
migrated within the media and alongside the lumen from the uninjured
borders into the necrotic center, resulting in the formation of a
neointima, first at the uninjured borders and subsequently
with progression into the center of the
wound.20 Progression of the neointima
from the borders to the center was quantified through measurement of
the luminal narrowing (percent stenosis) at equally spaced
positions across the injured segment. Within 1 week after injury,
significant neointima formation was initiated at the
borders of the injury in PAI-1-/- arteries, but
formation was only minimal in PAI-1+/+ arteries
(P<.05 versus PAI-1+/+ by ANOVA) (Fig 5a). Within 3 weeks after injury, the
neointima had progressed into the center of the injury in
both PAI-1-/- and
PAI-1+/+ arteries (P=NS) (Fig 5b).
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Medial cell repopulation was evaluated by counting the medial cell
nuclei at the left border, center, and right border of the injured
segment (locations 1, 5, and 10 in the arteries of Fig 1), revealing
that the cells migrated further and were found at earlier times after
injury in the center of the wound in PAI-1-/-
than in PAI-1+/+ arteries (Table 2). This process is schematically
represented in Fig 1.
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Reendothelialization
Electric injury completely denuded the injured segment of intact
endothelium as revealed by Evans blue staining
immediately after injury.20
Reendothelialization was initiated from the uninjured
borders, progressed into the necrotic center, and almost complete
within 1 week after injury in PAI-1+/+ mice (n=7
mice) (Fig 6).
Endothelial regrowth was not affected, either in extent
or in rate, by PAI-1 deficiency (n=5 mice) (P=NS).
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Thrombosis
Because deficiency of PAI-1 protects against thrombosis and
facilitates lysis of 125I-fibrin-labeled
pulmonary plasma clots,19 the incidence
and extent of thrombosis were semiquantitatively evaluated. At 1 week
after injury, 5 of 34 sections (15%) from wild-type arteries (n=7)
contained residual mural thrombus that stenosed the lumen for 5% to
25%. In contrast, only 2 of 40 PAI-1-/-
arteries (n=8; 5%) contained a mural thrombus (P=.08 versus
wild-type by Wilcoxon's signed rank analysis),
indicating a trend toward increased clot lysis in
PAI-1-/- mice.
Expression of PAI-1 During Arterial Wound
Healing
PAI-1 staining was evaluated in uninjured and electrically injured
arteries (n=3) within 1 and 2 weeks after injury, which is the time
frame of active cellular migration. Fig 7a summarizes the
semiquantitative analysis and indicates the topographic
locations across the injured arteries.
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Uninjured Arteries
In most uninjured arteries, PAI-1 immunoreactivity was
undetectable (Fig 7b); only occasionally could a low level of PAI-1
immunoreactivity be detected in medial smooth muscle cells of quiescent
arteries.
Injured Arteries
At 1 week after injury, expression of PAI-1 was induced in cells
that accumulated in the media, neointima, and, to a lesser
degree, adventitia. In sections adjacent to the injury in which smooth
muscle cells start to proliferate and migrate into the wound (location
I in Fig 7a), PAI-1 immunoreactivity was present in smooth muscle
cells in the media (Fig 7c). At the site of maximal
neointima formation (location II), PAI-1 staining was
present in most of the cells in the intima and the media (Fig 7d).
Regenerating endothelial cells expressed low levels of
PAI-1. PAI-1 staining was also detectable, albeit at a lower level, in
fibroblast-like cells in the enlarged adventitia closest to the
external elastic lamina (Fig 7d) and in areas of leukocyte infiltration
(cfr infra). A similar PAI-1–staining pattern was observed at the
leading front of migrating cells (location III) (not shown). Double
immunofluorescence staining revealed colocalization
(yellow) of PAI-1 (green) within -actin smooth muscle cells (red) in
the media and neointima (Fig 7e) and within Mac-3–positive
macrophages (red), predominantly located in the adventitia (Fig 7f). There was no PAI-1 immunoreactivity detected in injured
PAI-1-/- arteries (Fig 7g) or in wild-type
arteries when the primary antibodies were omitted (not shown). Overall,
a similar staining pattern and intensity profile were observed in
injured arteries within 2 weeks after injury. Immunoreactivity for
vitronectin, which interacts with
PAI-1,30,31 was detected in the adventitia of
electrically injured arteries of either genotype only within 2
and 3 weeks after injury. This suggests that PAI-1 and
vitronectin in the media and intima did not colocalize,
either spatially or temporally, during cell migration (not shown).
Taken together, these data indicate that during vascular wound healing,
PAI-1 is produced by smooth muscle, endothelial,
inflammatory, and fibroblast-like cells that repopulate the vascular
wound. Induction of PAI-1 in activated
endothelial, inflammatory, and smooth muscle cells
during wound healing has been reported
previously.2,32,33
Zymographic Analysis of PAs in PAI-1-/-
Mice
Uninjured Arteries
To evaluate whether deficiency of PAI-1 caused any changes in t-PA
or u-PA expression in uninjured arteries, t-PA and u-PA activities were
zymographically quantified in extracts of
PAI-1+/+ and PAI-1-/-
arteries. There was a slight increase in t-PA–mediated lysis in
PAI-1-/- arteries (0.99±0.06; n=3) compared
with PAI-1+/+ arteries (0.60±0.03; n=5;
P<.05 by ANOVA), whereas u-PA–mediated lysis in
PAI-1+/+ arteries (1.18±0.13; n=5) was similar
to that in PAI-1-/- arteries (0.90±0.12; n=3;
P=NS).
Injured Arteries
PA activities of injured arteries were evaluated by in situ
zymography on sections, allowing precise discrimination between
uninjured and injured regions as deduced from
histological analysis on adjacent sections.
Because the absolute levels of lysis varied between experiments and the
specific activities of t-PA and u-PA in this assay were significantly
different (due to their different affinity for and activation by
fibrin), PA activities were compared by expressing them as a ratio of
lysis present in injured versus noninjured segments per artery.
Lysis was quantified within 1 week after injury, when genotypic
differences in neointima formation were significant.
The ratio of lysis observed in injured versus noninjured sections within 1 week after injury was 1.02±0.09 (n=17) in PAI-1+/+ arteries and 2.3±0.3 (n=17; P<.05 versus PAI-1+/+) in PAI-1-/- arterial sections. Because lysis of the fibrin overlay was inhibited >95% by inclusion of neutralizing t-PA antibodies in the overlay, t-PA activity after injury was similar in PAI-1+/+ arteries and slightly increased in PAI-1-/- arteries. Accurate quantification of u-PA activity could not be reliably performed due to incomplete immunoneutralization of t-PA activity, which lyses fibrin overlays much more efficiently than u-PA (data not shown).
Adenoviral PAI-1 Gene Transfer
Rationale and Study Protocol
To confirm the inhibitory role of PAI-1 on
neointima formation, we studied the effect of adenoviral
PAI-1 gene transfer on arterial
neointima formation. PAI-1-/- mice
were used as recipients for PAI-1 gene transfer for two
reasons: (1) it permits a greater genetic shift in the proteolytic
balance without possible interference by endogenous PAI-1,
and (2) neointima formation is greater and occurs at
earlier times after injury in PAI-1-/- than in
PAI-1+/+ mice (7 to 14 days), coincident with the
time window of transgene expression after adenoviral gene transfer
using first-generation adenovirus vectors (7 to 10 days). Systemic
injection of AdCMVPAI-1 virus was used instead of local
PAI-1 gene transfer in the injured artery, for technical
reasons and because it allows the study of the effect of increased
plasma PAI-1 levels on neointima formation (see below).
Therefore, PAI-1-/- mice were injected
intravenously with AdCMVPAI-1 or control AdRR5 virus 3
days after electric injury (eg, at a time just before the first cells
start to migrate), and neointima formation was quantified
after 2 weeks. Plasma PAI-1 levels were determined 4 days after viral
infection, which corresponds to the time of maximal PAI-1
gene expression (as revealed from initial experiments).
PAI-1 Expression After Adenoviral PAI-1 Gene Transfer
Intravenous injection of 109 pfu
AdCMVLacZ virus in PAI-1-/- mice (n=5) resulted
in preferential expression of LacZ in the liver (as revealed by the
widespread blue staining; Fig 8a),
consistent with numerous previous
studies,34 In contrast, transduced cells were not
observed in the uninjured aorta (Fig 8b) or femoral artery (Fig 8c), or
in the femoral artery that was electrically injured 3 days before virus
injection (Fig 8d). Thus, systemic virus injection failed to locally
transduce vascular cells but preferentially targets expression of the
transgene to the liver.
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AdCMVPAI-1 efficiently infected cultured human umbilical vein
endothelial cells; synthesis of active PAI-1 was <7.5
fmol/h when they were infected with control AdRR5 virus and 14
pmol/h (1870-fold increase) when infected with AdCMVPAI-1 virus.
Intravenous injection of PAI-1-/-
mice with 2 x 109 pfu AdCMVPAI-1 resulted
in PAI-1 immunoreactivity in ~30% of hepatocytes but not
in uninfected control animals or animals infected with AdRR5 (Fig 7h).
Production of human PAI-1 in the liver significantly increased
plasma PAI-1 levels; human plasma PAI-1 antigen levels increased from
undetectable (<0.8 ng/mL) after AdRR5 adenovirus injection to
0.8±0.2, 13±7, and 61±8 µg/mL at 4 days after
intravenous injection with 2x108,
6x108, and 2x109 pfu
AdCMVPAI-1. These plasma levels of human PAI-1 are significantly higher
than the usual murine PAI-1 levels in the plasma from wild-type mice
(~2 ng/mL).19,35 Active PAI-1 levels in
mice injected with 2x108,
6x108, and 2x109 pfu
AdCMVPAI-1 were 80±8, 130±80, and 3000±140 ng/mL,
respectively, whereas they were undetectable in uninfected or
AdRR5- infected mice (<0.8 ng/mL). Through
immunostaining, human PAI-1 immunoreactivity was
detected within 1 week after electric injury in the
neointima from PAI-1-/- mice that
received a viral dose of 109 pfu AdCMVPAI-1 but
not of AdRR5 (Fig 8e and 8f). Taken together, systemic injection of
AdCMVPAI-1 did not transduce the vascular cells directly but increased
production of PAI-1 in the transduced hepatocytes,
with resultant augmented levels of PAI-1 in the plasma and its
deposition in the extracellular matrix of the neointima in
the injured artery.
No side effects of liver inflammation (histologically analyzed) or toxicity (revealed by measurements of the liver enzymes alanine aminotransferase and aspartate aminotransferase) resulted from the highest dose of adenoviral PAI-1 gene transfer (not shown). There also were no signs of thrombosis in AdCMVPAI-1–infected mice.
Inhibition of Neointima Formation by PAI-1 Gene
Transfer
PAI-1-/- mice were injected
intravenously with increasing doses of AdCMVPAI-1 or AdRR5
at 3 days after electric injury, and neointima formation
was quantified after 2 weeks. Intravenous injection of
AdCMVPAI-1, at doses ranging from 2x108 to
2x109 pfu, inhibited neointima
formation in PAI-1-/- mice in a dose-related
manner (Fig 8g). Half-maximal and maximal inhibition rates were
obtained at a dose of 2x108 and
6x108 pfu AdCMVPAI-1, respectively, which are
1 order of magnitude lower than the toxic dose of adenovirus
(>1010 pfu/mouse). Neointima
formation was reduced by >85% by comparison with untreated or
AdRR5-treated control mice, whereas the administration of an equivalent
dose of AdRR5 was ineffective. A representative example
of an AdCMVPAI-1–treated artery is shown in Fig 2f.
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Discussion |
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Role of PAI-1 in Smooth Muscle Cell Migration and Tissue Remodeling
The present study demonstrates that PAI-1 is an inhibitory regulator of arterial neointima formation during vascular wound healing and functions by affecting migration rather than proliferation of smooth muscle cells (Fig 1
). This hypothesis is consistent with
previous reports that both the plasmin inhibitor tranexamic
acid and metalloproteinase inhibitors reduce smooth muscle
cell migration in the rat carotid artery without affecting smooth
muscle cell proliferation.7,36 Apart from
controlling cellular migration, PAI-1 may also affect tissue remodeling
during arterial wound healing. Removal of the necrotic
debris and formation of a neoadventitia were impaired in
u-PA–deficient18 and plasminogen-deficient
arteries17 but accelerated in PAI-1–deficient
arteries (present study), implicating a role for u-PA/PAI-1 in
remodeling of the vessel wall. That both wild-type and
PAI-1-/- arteries eventually develop a similar
degree of neointima may suggest that the PA/PAI-1 system is
not the sole proteinase system involved in smooth muscle cell
migration. To what extent matrix metalloproteinases participate in this
process remains to be determined. PAI-1 could affect vascular wound healing by inhibiting u-PA–mediated pericellular plasmin proteolysis or by inhibiting u-PAR–31 or integrin-30 mediated cell adhesion and migration through interaction with vitronectin. The precise contribution of each of these mechanisms is at present unknown. Although u-PAR is expressed by migrating smooth muscle cells in the electrically injured artery, its deficiency apparently does not affect cellular migration or neointima formation (P. Carmeliet, L. Moons, M. Dewerchin, S. Rosenberg, J.-M. Herbert, F. Lupu, and D. Collen, unpublished observations). In addition, although vitronectin is present in the injured artery, it is mostly present in the adventitia and is more abundant beyond the second week after injury (eg, after maximal cell migration has occurred; not shown). Thus, although we cannot exclude that PAI-1 inhibits cellular migration by affecting cell adhesion,30,31 its poor temporal and spatial colocalization with vitronectin, the apparent redundancy of u-PAR, and the finding that deficiency of plasminogen17 also impairs neointima formation to the same extent as deficiency of u-PA18 argue for a role of PAI-1 in controlling proteolysis rather than adhesion.
Expression of PAI-1 During Vascular Wound Healing
PAI-1 expression was minimal in the uninjured femoral artery but
significantly induced during wound healing by cells at the migrating
front and at sites of tissue remodeling. This is somewhat surprising
because migrating cells require increased pericellular proteolysis.
This apparent paradox can be explained by the observation that
expression of u-PA mRNA and immunoreactivity and, more
importantly, that net u-PA–dependent fibrinolytic activity in injured
arteries increased by >100-fold. This therefore indicates that u-PA
synthesis increased more than PAI-1. Possibly, the increased PAI-1
levels in the vascular wound may limit excessive pericellular
proteolysis, which might result from uncontrolled u-PA expression.
Zymographic analysis indicated that t-PA activity was somewhat higher in PAI-1-/- than in PAI-1+/+ arteries before and after injury. However, it is unlikely that t-PA is responsible for the facilitated cellular migration in the mutant mice. Indeed, u-PA increased much more (>100-fold) than t-PA (less than twofold) after injury, and a deficiency of u-PA, but not t-PA, impaired smooth muscle migration and arterial neointima formation.18 It is at present unclear what role t-PA plays in vascular wound healing, although it is presumed to participate in passivation of the injured vessel lumen.1,2
Role of PAI-1 in Endothelial Cell
Regeneration
Endothelial cell regrowth was not accelerated by
PAI-1 deficiency. This is somewhat surprising in view of the induced
expression of PAs and PAIs in regenerating endothelium
in vivo and in vitro after injury (present study and References 2,
33, and 372 33 37 ). This may suggest that the plasminogen system
is redundant, possibly because other proteinase systems (maybe matrix
metalloproteinases) play a more important role. Alternatively,
endothelial cells may require more (plasmin)
pericellular proteolysis when they cross anatomic barriers (eg, during
the formation of new blood vessels) than when they migrate alongside
the internal elastic lamina of a denuded artery. Indeed, pathological
neovascularization during tumor formation appears to be impaired in
mice lacking u-PA–mediated plasmin
proteolysis.38
Arterial Injury Models in Mice: Relevance for
Human Restenosis?
PAI-1 inhibited neointima formation in both the
electric and mechanical injury models. The rate versus the extent of
neointima formation in electrically versus mechanically
injured PAI-1-/- arteries may relate to the
amount of injury and cell death, the degree and timing of proliferation
or migration, thrombus formation, inflammation, and the amount and
composition of the deposited matrix, which may differ significantly
between the two models.20 Although the mechanical
injury model more closely reflects the injury sustained by vessels in
human subjects after angioplasty, the electric injury model may better
reflect the healing process during conditions of more severe
inflammation, cell death, or tissue necrosis. Leukocytes, cell death,
and matrix deposition are characteristic features of advanced
atherosclerotic plaques and have previously been suggested to determine
the cellular responses during
restenosis.6 The topographic healing and
cellular migration pattern in electrically injured arteries are similar
to those observed after venous grafts, laser-induced thermal injury,
necrotizing transluminal ligation injury, and end-to-end
microvascular anastomosis (see Reference 2020 for references).
It remains to be determined how PAI-1 gene transfer will affect arterial wound healing in other animal models or in patients. Indeed, its overall effect may depend on the level, timing, and cellular distribution of recombinant PAI-1 gene expression; presence of inflammation and necrosis and the expression pattern of endogenous plasminogen activators in the injured vessel; degree to which intimal thickening depends on cellular migration; and cellular requirement for pericellular plasmin proteolysis. Intravascular seeding of retrovirally transduced smooth muscle cells overexpressing PAI-1 in balloon-injured rat carotid arteries inhibits neointima formation (Clower A.W. et al, personal communications), whereas a virus-encoded serine proteinase inhibitor, SERP-1 (inhibiting plasminogen activators and plasmin), also blocks atherosclerotic plaque development after balloon angioplasty in the rabbit femoral artery.39 Furthermore, although most currently available (gene) therapies aim to reduce intimal cell accumulation to improve blood flow, the level of PAI-1–mediated inhibition of intimal thickening may have to be controlled because insufficient repopulation may leave the arterial wound weak and unstable. Conversely, our finding that inhibition of PAI-1 may aggravate intimal thickening cautions against the (excessive) use of PAI-1 inhibitors used to improve thrombolysis and reduce atherosclerosis during periods of healing after vascular reconstructions.
In conclusion, this study provides evidence for an inhibitory role of PAI-1 in vascular wound healing and arterial neointima formation due to inhibition of cellular migration.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Flanders Interuniversity Institute for Biotechnology and by NIH SCOR grant HL-17669. The authors gratefully acknowledge the technical assistance of B. Amarneh, A. Bouché, I. Cornelissen, O.J. Cooper, M. De Mol, B. Hermans, G. Luyckx, A. Manderveld, and A. Van den Boomen and the help of M. Deprez for artwork.
Received March 25, 1997; revision received July 23, 1997; accepted August 14, 1997.
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References |
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J. Orbe, J. A Rodriguez, A. Calvo, A. Grau, M. S Belzunce, D. Martinez-Caro, and J. A Paramo Vitamins C and E attenuate plasminogen activator inhibitor-1 (PAI-1) expression in a hypercholesterolemic porcine model of angioplasty Cardiovasc Res, February 1, 2001; 49(2): 484 - 492. [Abstract] [Full Text] [PDF]
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P. H. A. Quax, M. L. M. Lamfers, J. H. P. Lardenoye, J. M. Grimbergen, M. R. de Vries, J. Slomp, M. C. de Ruiter, M. M. Kockx, J. H. Verheijen, and V. W. M. van Hinsbergh Adenoviral Expression of a Urokinase Receptor-Targeted Protease Inhibitor Inhibits Neointima Formation in Murine and Human Blood Vessels Circulation, January 30, 2001; 103(4): 562 - 569. [Abstract] [Full Text] [PDF]
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S. Konstantinides, K. Schafer, T. Thinnes, and D. J. Loskutoff Plasminogen Activator Inhibitor-1 and Its Cofactor Vitronectin Stabilize Arterial Thrombi After Vascular Injury in Mice Circulation, January 30, 2001; 103(4): 576 - 583. [Abstract] [Full Text] [PDF]
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E. M. Redmond, J. P. Cullen, P. A. Cahill, J. V. Sitzmann, S. Stefansson, D. A. Lawrence, and S. S. Okada Endothelial Cells Inhibit Flow-Induced Smooth Muscle Cell Migration : Role of Plasminogen Activator Inhibitor-1 Circulation, January 30, 2001; 103(4): 597 - 603. [Abstract] [Full Text] [PDF]
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V. A. Ploplis, I. Cornelissen, M. J. Sandoval-Cooper, L. Weeks, F. A. Noria, and F. J. Castellino Remodeling of the Vessel Wall after Copper-Induced Injury Is Highly Attenuated in Mice with a Total Deficiency of Plasminogen Activator Inhibitor-1 Am. J. Pathol., January 1, 2001; 158(1): 107 - 117. [Abstract] [Full Text] [PDF]
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 D. T. Eitzman, R. J. Westrick, Z. Xu, J. Tyson, and D. Ginsburg Plasminogen activator inhibitor-1 deficiency protects against atherosclerosis progression in the mouse carotid artery Blood, December 15, 2000; 96(13): 4212 - 4215. [Abstract] [Full Text] [PDF]
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 H. P. Kohler and P. J. Grant Plasminogen-Activator Inhibitor Type 1 and Coronary Artery Disease N. Engl. J. Med., June 15, 2000; 342(24): 1792 - 1801. [Full Text] [PDF]
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H. R. Lijnen, B. Van Hoef, M. Dewerchin, and D. Collen {alpha}2-Antiplasmin Gene Deficiency in Mice Does Not Affect Neointima Formation After Vascular Injury Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1488 - 1492. [Abstract] [Full Text] [PDF]
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A. A. Stoop, F. Lupu, and H. Pannekoek Colocalization of Thrombin, PAI-1, and Vitronectin in the Atherosclerotic Vessel Wall : A Potential Regulatory Mechanism of Thrombin Activity by PAI-1/Vitronectin Complexes Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 1143 - 1149. [Abstract] [Full Text] [PDF]
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M. Koziczak, W. Krek, and Y. Nagamine Pocket Protein-Independent Repression of Urokinase-Type Plasminogen Activator and Plasminogen Activator Inhibitor 1 Gene Expression by E2F1 Mol. Cell. Biol., March 15, 2000; 20(6): 2014 - 2022. [Abstract] [Full Text]
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D. Hasenstab, H. Lea, and A. W. Clowes Local Plasminogen Activator Inhibitor Type 1 Overexpression in Rat Carotid Artery Enhances Thrombosis and Endothelial Regeneration While Inhibiting Intimal Thickening Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 853 - 859. [Abstract] [Full Text] [PDF]
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S. Lopez, F. Peiretti, B. Bonardo, I. Juhan-Vague, and G. Nalbone Tumor Necrosis Factor alpha Up-regulates in an Autocrine Manner the Synthesis of Plasminogen Activator Inhibitor Type-1 during Induction of Monocytic Differentiation of Human HL-60 Leukemia Cells J. Biol. Chem., February 4, 2000; 275(5): 3081 - 3087. [Abstract] [Full Text] [PDF]
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W. Dichtl, A. Stiko, P. Eriksson, I. Goncalves, F. Calara, C. Banfi, M. P. S. Ares, A. Hamsten, and J. Nilsson Oxidized LDL and Lysophosphatidylcholine Stimulate Plasminogen Activator Inhibitor-1 Expression in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 3025 - 3032. [Abstract] [Full Text] [PDF]
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B. H. Strauss, H. K. Lau, K. A. Bowman, J. Sparkes, R. J. Chisholm, M. B. Garvey, L. L. Fenkell, M. K. Natarajan, I. Singh, and J. M. Teitel Plasma Urokinase Antigen and Plasminogen Activator Inhibitor-1 Antigen Levels Predict Angiographic Coronary Restenosis Circulation, October 12, 1999; 100(15): 1616 - 1622. [Abstract] [Full Text] [PDF]
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C. Rosnoblet, U. M. Vischer, R. D. Gerard, J.-C. Irminger, P. A. Halban, and E. K. O. Kruithof Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells Arterioscler Thromb Vasc Biol, July 1, 1999; 19(7): 1796 - 1803. [Abstract] [Full Text] [PDF]
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M. Cesari and G. P. Rossi Plasminogen Activator Inhibitor Type 1 in Ischemic Cardiomyopathy Arterioscler Thromb Vasc Biol, June 1, 1999; 19(6): 1378 - 1386. [Full Text] [PDF]
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B. E. Sobel Increased Plasminogen Activator Inhibitor-1 and Vasculopathy : A Reconcilable Paradox Circulation, May 18, 1999; 99(19): 2496 - 2498. [Full Text] [PDF]
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N. Nagai, M. De Mol, H. R. Lijnen, P. Carmeliet, and D. Collen Role of Plasminogen System Components in Focal Cerebral Ischemic Infarction : A Gene Targeting and Gene Transfer Study in Mice Circulation, May 11, 1999; 99(18): 2440 - 2444. [Abstract] [Full Text] [PDF]
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A. C Newby and A. B Zaltsman Fibrous cap formation or destruction -- the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation Cardiovasc Res, February 1, 1999; 41(2): 345 - 360. [Abstract] [Full Text] [PDF]
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J. L. Bouchie, H. Hansen, and E. P. Feener Natriuretic Factors and Nitric Oxide Suppress Plasminogen Activator Inhibitor-1 Expression in Vascular Smooth Muscle Cells : Role of cGMP in the Regulation of the Plasminogen System Arterioscler Thromb Vasc Biol, November 1, 1998; 18(11): 1771 - 1779. [Abstract] [Full Text] [PDF]
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P. Carmeliet, L. Moons, and D. Collen Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis Cardiovasc Res, July 1, 1998; 39(1): 8 - 33. [Abstract] [Full Text] [PDF]
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P. Carmeliet, L. Moons, M. Dewerchin, S. Rosenberg, J.-M. Herbert, F. Lupu, and D. Collen Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice J. Cell Biol., January 12, 1998; 140(1): 233 - 245. [Abstract] [Full Text] [PDF]
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F. Peiretti, S. Lopez, P. Deprez-Beauclair, B. Bonardo, I. Juhan-Vague, and G. Nalbone Inhibition of p70S6 Kinase during Transforming Growth Factor-beta 1/Vitamin D3-induced Monocyte Differentiation of HL-60 Cells Allows Tumor Necrosis Factor-alpha to Stimulate Plasminogen Activator Inhibitor-1 Synthesis J. Biol. Chem., August 17, 2001; 276(34): 32214 - 32219. [Abstract] [Full Text] [PDF]
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