© 2000 American Heart Association, Inc.
Vascular Biology |
2-Antiplasmin Gene Deficiency in Mice Does Not Affect Neointima Formation After Vascular Injury
From the Center for Molecular and Vascular Biology (H.R.L., B.V.H., D.C.), University of Leuven, and the Center for Transgene Technology and Gene Therapy (M.D., D.C.), Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.
Correspondence to H. R. Lijnen, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail roger.lijnen{at}med.kuleuven.ac.be
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Abstract |
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Abstract—The hypothesis that
2-antiplasmin (2-AP), the main
physiological plasmin inhibitor, plays
a role in neointima formation was tested with use of a
vascular injury model in wild-type (2-AP+/+)
and 2-AP–deficient
(2-AP-/-) mice. The neointimal
and medial areas were similar 1 to 3 weeks after electric injury of the
femoral artery in 2-AP+/+ and
2-AP-/- mice, resulting in comparable
intima/media ratios (eg, 0.43±0.12 and 0.42±0.11 2 weeks after
injury). Nuclear cell counts in cross-sectional areas of the intima of
the injured region were also comparable in arteries from
2-AP+/+ and
2-AP-/- mice (78±19 and 69±8). Fibrin
deposition was not significantly different in arteries of both
genotypes 1 day after injury, and no mural thrombosis was
detected 1 week after injury. Fibrinolytic activity in femoral
arterial sections, as monitored by fibrin zymography, was
higher in 2-AP-/- mice 1 week after injury
(P<0.001) but was comparable in both genotypes
2 and 3 weeks after injury. Staining for elastin did not reveal
significant degradation of the internal elastica lamina in either
genotype. Immunocytochemical analysis revealed a
comparable distribution pattern of -actin–positive smooth muscle
cells in both genotypes. These findings indicate that the
endogenous fibrinolytic system of
2-AP+/+ mice is capable of preventing fibrin
deposition after vascular injury and suggest that 2-AP
does not play a major role in smooth muscle cell migration and
neointima formation in vivo.
Key Words: neointima • restenosis • transgenic mice • 2-antiplasmin
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Introduction |
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Neointima formation, a wound healing process in response to vascular injury, may contribute to luminal stenosis.1 It involves migration of smooth muscle cells (SMCs) from the media across the internal elastic lamina underneath the endothelium and deposition of extracellular matrix.2 3 4 Proteinases of the plasminogen/plasmin and matrix metalloproteinase systems play a role in SMC migration by degrading the extracellular matrix that prevents migration.5 6 7 8 9 10 A crucial role of the plasminogen/plasmin system was suggested by the observation that in vivo SMC migration in the rat was inhibited by the plasmin inhibitor tranexamic acid.11 More direct evidence of a role of plasmin was provided by the findings that arterial neointima formation after vascular injury was significantly impaired in urokinase-deficient and plasminogen-deficient mice.12 13 The perivascular electric injury model used in these studies also causes development of transient mural thrombosis early after injury.14 The contribution of thrombosis to the subsequent neointima formation, however, is unclear.
The plasminogen/plasmin (fibrinolytic) system contains a
proenzyme, plasminogen, that is activated to the
active enzyme plasmin by tissue-type (tPA) or urokinase-type (uPA)
plasminogen activator. Inhibition occurs
through neutralization of tPA or uPA by type 1 plasminogen
activator inhibitor (PAI-1) or through
inhibition of plasmin by 2-antiplasmin
(2-AP).15 Recently, mice were
generated with inactivation of the
2-antiplasmin gene, the main
physiological plasmin
inhibitor.16 As expected, these animals
displayed a higher endogenous fibrinolytic potential than
wild-type littermates, as revealed by a higher spontaneous lysis rate
of experimental pulmonary emboli and decreased fibrin
deposition in the kidneys after endotoxin injection.16 In
the present study, these mice were used to study the role of
plasmin inhibition by 2-AP in thrombosis early
after electric injury and the subsequent effect on
neointima formation.
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Methods |
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Animals
2-AP–deficient
(2-AP-/-) and
wild-type (2-AP+/+) mice
(overall 50% C57/BL6 to 50% 129SVj genetic background) were obtained
by intercrossing germline offspring from 2-AP
chimeras mated with wild-type C57/BL6 females without further
backcrossing into the C57/BL6 or 129SVj background, and their
genotypes were determined as described
elsewhere.16 Mice were kept in microisolation cages on a
12-hour-day/12-hour-night cycle and fed regular chow. Animals were
anesthetized by intraperitoneal injection
of 60 mg/kg Nembutal (Abbott Laboratories), and all experiments were
performed in accordance with the guiding principles of the American
Physiological Society and the International Society
on Thrombosis and Haemostasis.17 Adult mice 9 to 12 weeks
old (males and females) were used. Mean body weight (±SEM) was
23±0.64 g for 2-AP+/+
mice (n= 28) and 23±0.65 g for
2-AP-/- mice (n= 30).
t tests were used for statistical analysis.
Vascular Injury Model
Perivascular electric injury to the femoral artery was induced
essentially as described elsewhere.14 In brief, arteries
were exposed by blunt-end dissection and injured by electric current at
distances of 1 mm over a total length of 2 or 3 mm. Control
arteries were removed from noninjured mice, and injured arteries were
collected 1 to 3 weeks after injury. Vessel segments were fixed in 1%
paraformaldehyde or directly embedded in ornithine
carbamyl transferase (Tissue-Tek), snap-frozen in precooled
2-methylbutane, and stored at -80°C. In addition, some animals were
killed 1 day after injury and immediately perfused for 15 to 30 minutes
with saline; arteries were removed and postfixed in paraffin.
Seven-micron-thick sections were made throughout the whole arteries for
histological and immunocytochemical studies. Blood was
collected on 0.01 mol/L citrate, and platelet-poor plasma was
prepared by centrifugation at 4000g for 5
minutes. Longitudinally alongside the artery, different positions
corresponding to noninjured sections (positions 1 and 5), to borders of
the injury (positions 2 and 4), and to the center of the injury
(position 3) were identified (Figure
,
inset).
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Histological and Immunocytochemical
Studies
Morphometric measurements of cross-sectional areas were made and
cell counts were performed in a blinded manner on fixed transverse
arterial sections (stained with hematoxylin and eosin)
using a computer-assisted image analysis system as described
elsewhere.14 Measurements were made at equally spaced
positions (140 µm apart) along the artery. Areas in the injured
region of the artery (positions 2 to 4) were then averaged for each
artery. Data are the mean±SEM of these average values obtained for all
arteries analyzed in each group.
For immunostaining of SMCs, biotinylated mouse
anti-human smooth muscle -actin (clone A14, Sigma Chemical Co) was
used as primary antibody in combination with the Vectastain system (ABC
Elite kit, Vector Laboratories Inc). Immunostaining for
fibrin and fibrinogen was performed by incubating the sections with
goat antiserum against murine fibrin (Nordic) in 0.01 mol/L Tris-HCl,
pH 7.6, containing 0.9% NaCl and 0.1% Triton X-100 for 3 hours at
room temperature. After rinsing, sections were incubated consecutively
for 60 minutes with biotinylated rabbit anti-goat immunoglobulin G
(IgG; Dako) and with peroxidase-labeled avidin-biotin complex (Dako).
Peroxidase activity was developed by incubating sections in 0.05 mol/L
Tris-HCl buffer, pH 7.0, containing 0.06% 3,3'-diaminobenzidine and
0.01% H2O2, followed by
counterstaining with Harris’ hematoxylin. Staining specificity was
confirmed by omission of the primary antibody or by replacement of the
antibody with equivalent amounts of isotype-matched nonimmune IgG or
serum. Staining for elastin was performed with use of Verhoeff’s and
von Gieson’s reagents.
Protein Assays and Zymographic Analysis
For extraction, femoral arteries dissected free of tissue were
pulverized under liquid nitrogen and extracted for 1 hour at 4°C with
60 µL of 10 mmol/L sodium phosphate buffer, pH 7.2, containing
150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium
deoxycholate, and 0.2% sodium azide. After extensive vortexing and
centrifugation at 13 000 rpm for 5 minutes, the
protein concentration of the supernatants was determined (BCA protein
assay, Pierce).
For zymographic analysis of plasminogen activator activity (tPA and uPA), arterial extracts were electrophoresed on a 12.5% acrylamide gel cast with 1% nonfat dry milk and 5 µg/mL human plasminogen under nonreducing conditions.18 Lysis of the substrate gel (area by intensity) was measured using Quantimed 600 image analysis software (Leica) and expressed in arbitrary units (AU) of lysis obtained per milligram of protein in the extract.
Fibrinolytic activity was monitored by fibrin overlay on 7-µm cryostat sections (nonfixed) of arteries at 37°C for 2 hours. The lysis area (mm2) was normalized to the total area of the section.
2-AP and plasminogen antigen
levels were measured by ELISA using polyclonal antibodies against the
purified murine proteins raised in rabbits.16
Plasmin–2-AP complexes in plasma were
measured using microtiter plates coated with polyclonal rabbit
anti-murine 2-AP antibodies; after incubation
of diluted plasma samples, bound complex was detected with
HRP-conjugated polyclonal rabbit anti-murine plasminogen
antibodies. For calibration, murine plasma fully activated for
90 minutes at 37°C with uPA (50 nmol/L final concentration) was
used.
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Results |
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Histological and Immunocytochemical Studies
Hematoxylin and eosin staining of femoral arterial sections taken from noninjured arteries (control animals) showed comparable pictures for
2-AP+/+ and
2-AP-/- mice; the
adventitial and medial areas were comparable, and no significant
neointima was detectable (Table 1 and Figure
). Nuclear cell counts in the
media were comparable for both genotypes (Table 2), as were counts for intimal (mainly
endothelial) cells (19±2 in both
genotypes).
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Staining of sections obtained 1 to 3 weeks after injury at equally
spaced locations (positions 2 to 4) throughout the damaged area showed
the formation of a neointima in both
2-AP+/+ and
2-AP-/- mice (Figure).
Overall, the intimal and medial areas were comparable, resulting in
intima/media ratios that were not significantly different between both
genotypes. However, 1 week after injury the intimal area and
intima/media ratio appeared to be higher in
2-AP+/+ mice (Table 1). Nuclear cell counts revealed a comparable cell population in
media of normal sections (positions 1 and 5) of injured arteries (data
not shown) and in control noninjured arteries (cf above). In the
injured area (positions 2 to 4), cell counts were overall comparable
over time in the media and intima of both genotypes (Table 2).
Immunostaining for -actin showed a similar picture
for uninjured control arteries of
2-AP+/+ and
2-AP-/- mice, with 2
or 3 layers of -actin–immunoreactive cells in the media but none in
the intima or adventitia (Figure). One week after injury, virtually no
-actin–positive cells were detected in the intima of both
genotypes, whereas they represented <20% of the
cell population in the media in both genotypes (not shown). Two
weeks after injury, -actin–positive SMCs in the intima of both
genotypes were more abundant at the borders of the injury than
in the center, whereas at 3 weeks after injury, 
70% of cells in the
injured area were -actin–positive (Figure). Staining for elastin
did not reveal significant degradation of the internal elastica lamina
in either genotype.
In separate experiments, femoral arteries were removed 1 day after
electric injury and used for immunostaining with an
antiserum against murine fibrin and fibrinogen. No fibrin was detected
in sections of noninjured control arteries of
2-AP+/+ or
2-AP-/- mice (n=4
each; not shown). One day after injury, no intravascular thrombosis in
the lumen was observed in either genotype, whereas weak
positive immunostaining for fibrin was observed in the
adventitia of the damaged area in 4 of 4
2-AP+/+ mice and 2 of 3
2-AP-/- mice (not
shown). Plasma levels of plasmin–2-AP complex
in 2-AP+/+ mice were
1300±280 ng/mL in control animals (n= 8) and 1500±240 ng/mL in
injured animals 1 day after injury (mean±SEM, n= 6).
Protein Assays and Zymographic Analysis
In situ zymographic analysis was performed by fibrin
overlay of arterial sections. This assay detects primarily
tPA activity, as shown by the finding that lysis of the fibrin gel was
virtually abolished on addition of anti-tPA antibodies (final
concentration 200 µg/mL gel) but was not significantly affected by
anti-uPA antibodies (not shown). When the fibrinolytic activity (lysis
zone) was normalized to the section area, fibrinolytic activities in
control arteries of
2-AP-/- mice were
somewhat, but not significantly (P=0.11), higher than those
in 2-AP+/+ mice (Table 3). One week after vascular injury,
activity was significantly higher in
2-AP-/- arteries,
whereas no significant difference was observed 2 to 3 weeks after
injury.
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Determination of fibrinolytic parameters in femoral
arterial extracts revealed somewhat higher tPA activity in
2-AP-/- arteries 1
week after injury and somewhat higher uPA activity in control arteries
of 2-AP-/- mice (Table 4). At other time points, both activities
were comparable for both genotypes. Plasminogen
antigen levels were also comparable. 2-AP
antigen levels were enhanced 1 and 2 weeks after injury compared with
controls (P=0.07).
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Discussion |
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Neointima formation plays a role in the pathogenesis of atherosclerosis, restenosis after angioplasty, and late vein graft failure.19 It involves degradation of extracellular matrix by proteinases of the plasminogen/plasmin or matrix metalloproteinase system, with migration of SMCs from the media to the intima.2 3 We used a perivascular electric injury model14 in
2-AP+/+ and
2-AP-/- mice to study
the role of 2-AP, the main
physiological plasmin inhibitor, in
neointima formation. In this model, wound healing is
initiated from the adjacent uninjured borders, progresses into the
necrotic center, and is associated with migration of SMCs. One to 2
weeks after injury, the intimal cell population is
heterogeneous, consisting not only of -actin–positive
SMCs but also of leukocytes and occasionally
macrophages.12 13 14 As in other models in the rat
and pig, mural thrombosis early after injury is frequently
observed.14 20 21 Mural thrombosis is also frequently
observed after vascular reconstruction in humans, and it has been
suggested that colonization of such thrombi may contribute to the
development of intimal lesions.1 2 22 23 24 Therefore, we
investigated a potential relation between early thrombosis and
subsequent neointima formation. However, 1 day after
vascular injury, fibrin deposition did not appear to be significantly
different between femoral arteries of
2-AP+/+ and
2-AP-/- mice and mural
thrombosis was not observed. One week after injury, all thrombi were
cleared in both genotypes. This finding contrasts with results
of previous studies using this model, which reported mural thrombosis
occluding the lumen by <25% in about one quarter of wild-type
mice.12 13 It is not clear whether this difference is due
to a difference in genetic background of the mice (overall 75% C57/BL6
and 25% 129SVPas versus overall 50% C57/BL6 and 50% 129SVj in this
study). In any case, in this model mural thrombi appear to be lysed
before accumulation of SMCs and neointima formation
occur.14 No significant difference in neointima formation was observed in both genotypes 1 to 3 weeks after injury. This suggests that neointima formation after electric injury does not develop from colonization of thrombi. This observation is in agreement with general clinical observations in patients after vascular interventions, in whom thrombi can be lysed or limited by use of thrombolytic agents without affecting the occurrence of restenosis. In contrast, efficient antithrombotic strategies (eg, glycoprotein IIb/IIIa antagonists) appear to be effective in limiting restenosis.25 26
The similar neointima formation in
2-AP+/+ and
2-AP-/- mice also
indicates that the physiological plasmin
inhibitor does not play a major role. This is somewhat
surprising in view of previous studies showing impaired
neointima formation in uPA and in
plasminogen-deficient mice,12 13 thus
suggesting a key role of plasmin. The aim of this study, however, was
not to study the role of plasmin directly but to evaluate the role of
2-AP, 1 (albeit the
physiologically most relevant) of the plasmin
inhibitors in plasma.
In contrast to the present findings on the role of
2-AP, we previously showed that PAI-1, the
main physiological plasminogen
activator inhibitor, plays an
inhibitory role in vascular wound healing and
neointima formation after injury. PAI-1 apparently
functions by affecting SMC migration, most likely as a result of
inhibition of uPA-mediated proteolysis.27
It was suggested previously that other serine proteinase
inhibitors in the murine system may compensate for the lack
of 2-AP. This may explain the absence of a
bleeding phenotype in
2-AP-/-
mice16 in contrast to homozygous
2-AP–deficient patients. However, in injured
sections of wild-type mice, significant plasmin activity can be
generated locally at the sites of injury, as shown by fibrin
zymography, which revealed significant fibrinolytic activity. This was
higher in arteries from
2-AP-/- mice 1 week
after injury (Table 3), consistent with higher tPA
activity levels measured by casein zymography with arterial
extracts (Table 4). However, 2 to 3 weeks after injury there
were no significant differences. Expression of
2-AP after vascular injury in wild-type mice
is confirmed by the 2-AP levels in femoral
arterial extracts. Activation of the fibrinolytic system 1
day after vascular injury, however, seems to be limited, as evidenced
by unaltered levels of plasma plasmin–2-AP
complex in comparison with control animals.
Together these findings indicate that the endogenous
fibrinolytic system of
2-AP+/+ mice is capable
of preventing mural thrombosis after vascular injury and suggest that
2-AP does not play a major role in SMC
migration and neointima formation in vivo.
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Acknowledgments |
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The skillful technical assistance of A. De Wolf, L. Frederix, G. Lemmens, and I. Vanlinthout is gratefully acknowledged. This study was supported by grants from the Flemish Fund for Scientific Research (contract G.0293.98) and from the Interuniversitaire Attractiepolen (contract P4/34).
Received October 28, 1999; accepted December 20, 1999.
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