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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Lack of ␣2-antiplasmin promotes re-endothelialization via over-release
of VEGF after vascular injury in mice
Hiroyuki Matsuno, Akira Ishisaki, Keiichi Nakajima, Kiyotaka Okada, Shigeru Ueshima, Osamu Matsuo, and Osamu Kozawa
We here report that the arterial blood flow
after endothelial injury in mice deficient
in ␣2-antiplasmin (␣2-APⴚ/ⴚ mice) was
well maintained compared with that of
wild-type mice. Moreover, the development of neointima 4 weeks after injury in
␣2-APⴚ/ⴚ mice was significantly decreased. Histologic observations showed
a prompt recovery of endothelial cells
with a much higher proliferating index in
repaired endothelium in ␣2-APⴚ/ⴚ mice.
The amount of secreted vascular endothelial growth factor (VEGF) by explanted
vascular smooth muscle cells (SMCs)
from ␣2-APⴚ/ⴚ mice was significantly increased. In separate experiments using a
human endothelial cell (EC) line, we could
demonstrate that plasminogen binds to
ECs and that this binding can be prevented by ␣2-AP. Finally, an injection of
either an anti-VEGF receptor-1 antibody
or ␣2-AP reduced the prompt endothelial
healing. ␣2-AP is the main inactivator of
plasmin, which cleaves extracellular matrix-bound VEGF to release a diffusible
proteolytic fragment. Lack of ␣2-AP, there-
fore, could lead to a local over-release of
VEGF by the continuously active plasmin
in the injured area, which could result in a
prompt re-endothelialization after vascular injury. Our results provide new insight
into the role of ␣2-AP and VEGF in the
pathogenesis of re-endothelialization following vascular injury. (Blood. 2003;102:
3621-3628)
© 2003 by The American Society of Hematology
Introduction
The integrity of the endothelial surface is essential for maintaining
homeostasis between blood and surrounding tissues. Vascular
endothelial growth factor (VEGF) is a potent mitogen with high
specificity for endothelial cells.1 It plays a major role in angiogenesis,2 and indeed mice lacking 1 of the 2 VEGF alleles die before
birth and show defects in the development of the cardiovascular
system.3 Moreover, VEGF mediates vascular permeability,4,5 endothelial chemotaxis,6 endothelium-derived relaxing factor-dependent vasodilatation,7 and thrombogenesity.8 Four different molecular isoforms of VEGF exist, having 121, 165, 189, and 206 amino
acids, respectively (VEGF121, VEGF165, VEGF189, and
VEGF206). Native VEGF is a basic heparin-binding glycoprotein
of 4.5 kDa.9 These properties correspond to those of VEGF165, the
major isoform. VEGF121 is a weakly acidic polypeptide that fails
to bind to heparin.10 VEGF189 and VEGF206 are more basic and
bind to heparin with greater affinity than VEGF165. Interestingly,
the longer forms VEGF189 and VEGF206 are almost completely
sequestered in the extracellular matrix and may be released
by plasmin.10
␣2-Antiplasmin (␣2-AP) is a serpin (serine protease inhibitor)
and is the main physiologic inhibitor of the fibrinolytic plasmin in
mammalian plasma. It is synthesized in the liver and is present in
plasma at a concentration of about 1.0 nmol/mL.11 Human and
murine ␣2-AP with molecular weight of 65 to 70 kDa12 rapidly
inactivate plasmin, resulting in the formation of a stable inactive
complex, plasmin–␣2-AP.13 Apart from the removal of fibrin, the
fibrinolytic system also plays a pivotal role in phenomena such as
embryogenesis, ovulation, intima formation, proliferation, migration, tumorigenesis, and metastasis.14 It has been reported that the
levels of plasmin–␣2-AP complex in plasma are elevated in acute
stroke, myocardial infarction, unstable angina, and arterial fibrillation.15,16 These findings may reflect a self-defense system against
the risk of ischemic events, which are caused by vascular stenosis
after vascular injury. Endothelial cell proliferation and migration to
repair the vascular surface and vessel wall follow vascular injury.
Studies have shown that VEGF is highly expressed in smooth
muscle cells after endothelial denudation and that it accelerates
re-endothelialization.17,18 However, plasmin plays a role to cleave
and release VEGF from smooth muscle cells (SMCs).9 We,
therefore, investigated the role of ␣2-AP, a physiologic plasmin
inhibitor, in vascular remodeling by using mice deficient in ␣2-AP.
Here, we report for the first time a crucial role of ␣2-AP following
endothelial injury.
From the Department of Pharmacology, Gifu University School of Medicine,
Gifu, Japan; Department of Physiology II, Kinki University School of Medicine,
Osakasayama City, Japan.
Culture, Sports, Science and Technology.
Submitted March 7, 2003; accepted July 13, 2003. Prepublished online as
Blood First Edition Paper,July 31, 2003; DOI 10.1182/blood-2003-03-0700.
Supported by a Grant for Scientific Research (no. 15590223) from Ministry of
Education, Science, Sports and Culture of Japan and by Hitech research grant
at Kinki University, Graduate School of Medicine from the Ministry of Education,
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
Materials and methods
Animals
Deficient mice were generated by homologous recombination in embryonic
stem cells, as described previously.19,20 All experiments were performed in
accordance with institutional guidelines.
Reprints: Hiroyuki Matsuno, Department of Pharmacology, Gifu University
School of Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan; e-mail:
leuven@cc.gifu-u.ac.jp.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
3621
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3622
MATSUNO et al
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
Reagents
Electron microscopic analysis
Recombinant murine VEGF, antimouse VEGF, antimouse VEGF receptor-1
(Fit-1), and anti-VEGF receptor-2 (Flk-1) were purchased from R&D
Systems (Minneapolis, MN). The other chemical substances were obtained
from Sigma Chemical (St Louis, MO).
Mice (n ⫽ 3 each) were killed by injection of an overdose of pentobarbital
48 hours after the initiation of endothelial injury. At the time of death, mice
were exsanguinated and 2 mL phosphate-buffered saline (PBS) was
injected into the right jugular vein to perfuse the whole body. Carotid
arteries were removed and then transferred into 4% formaldehyde or 2%
glutaraldehyde for 24 hours and next into in 50 mM sodium phosphate
buffer. The formaldehyde-fixed samples were paraffin embedded, cut in
butterfly-shaped sections of 5-␮m thickness, placed on glass slides, and
stained with hematoxylin and eosin (HE). The other glutaraldehyde-fixed
samples were cut open longitudinally to allow visual inspection for
scanning electron microscopy (SEM) as described.24
Experimental endothelial injury in mice
The experimental procedure to induce an endothelial injury has been
described in detail previously.21,22 Mice (n ⫽ 8, each group) were anesthetized by intraperitoneal injection of 44 mg/kg sodium pentobarbital. In
brief, the right common carotid artery, the left jugular vein, and the right
femoral artery were exposed under anesthesia with pentobarbital. Catheters
(internal diameter ⫽ 0.5 mm; outer diameter ⫽ 0.8 mm, polyethylene sp3;
Natume, Tokyo, Japan) were connected to the left jugular vein and to the
right femoral artery for the injection of Rose Bengal (50 mg/kg; Sigma
Chemical) and for monitoring blood pressure and pulse rate using a
pressure transducer (AP601G; Nihon Koden, Tokyo, Japan) during experiments on day 0. Blood flow in the carotid artery was continuously
monitored using a Doppler flow probe (Model PDV-20; Crystal Biotech,
Tokyo, Japan) positioned proximally to the injured area of the carotid artery.
Irradiation by green light (540 nm) proximal to the flow probe was started,
and then Rose Bengal was injected as a bolus 10 minutes after the
observation of control blood flow. The irradiation was continued for 15
minutes after the injection of Rose Bengal. This procedure results in
destruction of endothelial cells in the irradiated area by oxygen radicals
induced by the photochemical reaction between Rose Bengal and green
light. Our previous histologic observations have revealed that under such
conditions, a platelet-rich thrombus including fibrin was formed.17 The flow
probe was removed after the first observation (day 0) and replaced on each
consecutive observation day (day 1, 2, and 3). The presence of an occlusive
thrombus was detected when blood flow was zero. After recovery from
anesthesia, the animals were kept in individual cages and fed standard chow
(RC4; Oriental Yeast, Osaka, Japan).
Quantitative analysis of neointima formation
Four weeks after the endothelial injury, the mice (n ⫽ 5, each time point)
were anesthetized by sodium pentobarbital (44 mg/kg, intraperitoneally),
and the common carotid artery was excised, rinsed with saline, and frozen.
After removal of the artery, the animal was killed by an intraperitoneal
injection of an overdose of sodium pentobarbital. The frozen sections were
cut transversely into 20 sections at 100-␮m intervals, followed by staining
with hematoxylin and eosin (Sigma Chemical) after perfusion fixation at
constant physiologic pressure. The total areas within the internal elastic
lamina (IELA) and lumen (LA) were measured by using a computerized
image graphic analysis system. For this analysis, 5 consecutive carotid
artery cross sections (4-5 ␮m thick) were taken at 100-␮m intervals from
the bifurcation of the carotid artery. The intima area (IA ⫽ IELA ⫺ LA)
was then expressed proportional to IELA by averaging the 3 measurements
performed for each cross section.23
Proliferation index in vivo
In separate experiments, proliferating SMCs were identified by the
thymidine analog 5-bromo-2-deoxy-Uridine (BrdU) in both types of
mice.24 BrdU tests were performed at day 1, 3, 7, and 14 after injury (n ⫽ 4
each time point). BrdU (50 mg/kg) was injected subcutaneously 1, 8, 16,
and 24 hours prior to removal of the carotid arteries. Following removal of
the arteries, frozen cross sections were prepared from these arteries.
BrdU-positive cells were stained with a murine monoclonal antibody
(Sigma), followed by goat antimouse immunoglobulin antibodies conjugated to peroxidase and detected with diaminobenzidine (DAB). Sections
were also stained for background with hematoxylin. The numbers of
positive and negative nuclei were counted in the media and newly formed
intima. The BrdU-labeling index was calculated by using the following
formula: (the number of positive nuclei stained with DAB)/(the number of
total nuclei stained with hematoxylin) ⫻ 100.
Immunohistochemical staining of VWF
Staining for von Willebrand factor (VWF) was used to detect repairing
endothelial cells in the injured area of the murine vessels. Mice (n ⫽ 4 each
time point) were killed by injection of an overdose of pentobarbital before
and 2 hours, 48 hours, and 4 weeks after the initiation of endothelial injury.
At the time of death, mice were exsanguinated, and 2 mL saline was
injected into the right jugular vein to perfuse the whole body. Following
removal of the carotid artery, frozen cross sections were prepared.
Endothelial cells were stained with a peroxidase-conjugated monoclonal
anti-VWF antibody (P 0226; DAKO Japan, Kyoto, Japan) and detected
with DAB. Sections were also stained for background with hematoxylin.
Binding study of plasminogen to endothelial cells
A real-time biomolecular interaction assay system, an optical biosensor
(IAsys Auto⫹; Affinity Sensors, Cambridge, United Kingdom) was used
for the binding assay.25 In this cuvette-based resonant mirror instrument a
change in the refractive index is obtained when a molecule binds to the
sensing surface, resulting in a shift in the resonant angle. The angle change
detected by the instrument is displayed in arc seconds; higher arc seconds
mean greater binding. Plasminogen or ␣2-AP (100 ␮g/mL, 50 ␮L volume)
was immobilized on the surface of an IAsys carboxylate cuvette (Affinity
Sensors) via an aminohexanoic acid linker according to the manufacturer’s
protocol. Briefly, after equilibration and obtaining a stable baseline with
PBS, carboxylate on the cuvette surface was activated with 200 nmol/L
N-hydroxysuccimide (NHS) and 50 nmol 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) for 7 minutes. The activation solution was
removed and washed with PBS, and then 1 mol/L aminohexanoic acid
(pH ⫽ 8.5) was added for 5 minutes to immobilize. After removal of the
aminohexanoic acid solution and washing with PBS, the carboxylate of the
immobilized aminohexanoic acid was activated with 200 nmol/L NHS and
50 nmol/L EDC. After washing with PBS, 50 ␮g/mL plasminogen or
␣2-AP was added for 15 minutes. Finally, after removal of the solution and
washing with PBS, the sensor surface was blocked with 1 mg/mL bovine
serum albumin. The immobilization of plasminogen or ␣2-AP on the
cuvette surface was determined from the increasing arc seconds. A baseline
was established with PBS. Binding of endothelial cells (5 ⫻ 104 to 5 ⫻ 105
cell/mL in PBS) to immobilized plasminogen or ␣2-AP was monitored. For
competing studies, plasminogen that had been preincubated with ␣2-AP
was used to determine the binding of the endothelial cells. All experiments
in the IAsys instrument were carried out at 25°C.
Spontaneous secretion of VEGF in primary cultured cells
Vascular SMCs were obtained from the thoracic aorta of wild-type mice and
mice deficient in ␣2-AP.26 The cultured cells (1 ⫻ 105) were seeded into
35-mm–diameter dishes and maintained in 2 mL Dulbecco modified Eagle
medium (DMEM) containing 10% fetal calf serum (FCS) at 37°C in a
humidified atmosphere of 5% CO2/95% air. After 6 days, the medium was
exchanged for serum-free DMEM. The cells were used for experiments
after 48 hours. VEGF in conditioned medium was measured by enzymelinked immunosorbent assay.
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BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
ROLE OF ␣2-AP IN RE-ENDOTHELIALIZATION
3623
Table 1. Alteration of arterial blood flow after injury
␣2-APⴙ/ⴙ mice
␣2-APⴚ/ⴚ mice
11.3 ⫾ 1.2
16.1 ⫾ 4.1
Reperfusion
56.3 ⫾ 8.4
66.2 ⫾ 7.2
Day 1
49.9 ⫾ 9.1
70.3 ⫾ 8.3*
Day 2
59.4 ⫾ 8.4
75.3 ⫾ 4.6*
Day 3
63.3 ⫾ 6.3
82.5 ⫾ 3.6*
Time to occlusion, min
Mean blood flow, %
Blood flow is represented as a percentage of control blood flow (before the
initiation of injury).
*Indicates P ⬍ .05 versus corresponding time point in ␣2-AP⫹/⫹ mice.
Results
Lack of ␣2-AP well maintains the blood flow after
endothelial injury
Figure 1. Alteration of arterial blood flow after endothelial injury. Vascular
patency after spontaneous reperfusion in the carotid artery of ␣2-AP⫹/⫹ mice (A) and
␣2-AP⫺/⫺ mice (B). The time profile of vascular patency after the endothelial injury
was schematically illustrated for 120 minutes at the first observation (day 0) and for
30 minutes at day 1, 2, or 3. The black and open columns indicate the periods of
vascular occlusion and of blood flow (more than 10% of the blood flow obtained
before the initiation of vascular injury), respectively.
Treatment of ␣2-APⴚ/ⴚ mice with anti-VEGF antibodies
An anti-VEGF antibody (25 ␮g per body) was injected as a bolus 5 minutes
before the initiation of endothelial injury in ␣2-AP⫺/⫺ mice (n ⫽ 3, each
group). In separate mice (n ⫽ 3, each group), antibodies against VEGFR-1
(Flt-1) or VEGFR-2 (KDR), which are expressed almost exclusively on
endothelial cells,27 were also injected as a bolus, and then endothelial injury
was induced in the carotid artery of ␣2-AP⫺/⫺ mice. Two days after the
injury, the injured artery was removed and treated for electron microscopic
analysis and for the measurement of BrdU-positive cells.
Treatment of ␣2-APⴙ/ⴙ mice with VEGF
To more directly define the effect of VEGF after endothelial injury, VEGF
(2, 4, or 8 ng per body, n ⫽ 3 each group) was injected as a bolus in
wild-type mice a few minutes before the initiation of injury. Two days after
the injury, the injured artery was removed and treated for electron
microscopic analysis and for the measurement of BrdU-positive cells.
Treatment of ␣2-APⴚ/ⴚ mice with ␣2-AP
␣2-AP (75 ␮g per body) was injected as a bolus 5 minutes before the
initiation of endothelial injury in ␣2-AP⫺/⫺ mice (n ⫽ 3). Two days after
the injury, the injured artery was removed and treated for electron
microscopic analysis. In separate ␣2-AP⫺/⫺ mice, ␣2-AP (75 ␮g per body)
was injected as a bolus 5 minutes before the initiation of endothelial injury
via tail vein and continuously treated for 1 day, 3 days, 1 week, 2 weeks, or
4 weeks. Four weeks after the initiation of endothelial injury, vessels in each
group were removed, and neointimal area was measured as mentioned in
“Quantitative analysis of neointima formation.”
Statistical analysis
All data are expressed as the mean ⫾ SEM. The significance of the effect of
each treatment (*P ⬍ .01) was determined by analysis of variance (ANOVA)
followed by the Student Newman-Keuls test.
The time profiles of vascular patency after the initiation of
endothelial injury by the photochemical reaction are schematically
illustrated in Figure 1. The time to occlusion because of the
development of a thrombus after endothelial injury in ␣2-AP⫺/⫺
mice (16.6 ⫾ 1.6 minutes) was slightly prolonged compared with
that of wild-type mice (13.2 ⫾ 0.9 minutes). In both types of mice,
cyclic reocclusion and reflow after spontaneous reperfusion were
clearly observed in the artery on the day of the endothelial injury
(day 0). Spontaneous reperfusion was observed in 5 of 8 wild-type
mice at the end of the first observation period 120 minutes after the
initiation of injury, which was consistently associated with cyclic
reocclusion and reflow. In the other mice, cyclic reocclusion and
reflow were also observed. Twenty-four hours later (day 1), a
persistent occlusion was seen in 4 mice; cyclic reocclusion and
reflow in 4 others. These findings gradually changed until at day 3,
persistent patency was observed in 2 mice, whereas in the others
cyclic reocclusion and reflow were still present. Following reperfusion, the mean blood flow remained less than 58% of the baseline
blood flow (Table 1). Spontaneous reperfusion was clearly observed in all arteries of ␣2-AP⫺/⫺ mice within the first observation
period, however, with prominent cyclic reocclusion/reflow. These
flow patterns clearly changed at day 1 when cyclic reocclusion/
reflow was diminished. Persistent patency was observed in all
␣2-AP⫺/⫺ mice until day 3, and reperfused blood flow recovered to
86% of baseline flow. Mean arterial blood flows and vascular
patency after spontaneous reperfusion are shown in Tables 1 and 2.
Table 2. Vascular patency after spontaneous reperfusion of artery
in ␣2-APⴙ/ⴙ mice
␣2-APⴙ/ⴙ mice
␣2-APⴚ/ⴚ mice
PO
CR
PP
PO
CR
PP
Day 0
3
5
0
1
7
0
Day 1
4
4
0
0
6
1
Day 2
1
7
0
0
1
7*
Day 3
0
6
2
0
0*
8*
Vascular patency was judged to be the state at the end of the observation period
on each time course. The carotid arterial or jugular vein patency was expressed as
persistent occlusion (PO) when no reperfusion was observed at all; as cyclic flow
reduction (CR) when the vascular reflow alternately showed stop and flows; and as
persistent patency (PP) when the vascular flow was maintained until the end of the
observation period.
*P ⬍ .05 versus each wild-type mouse on the same time course. Data
correspond to the number of vessels in each group.
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3624
MATSUNO et al
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
Figure 2. Histologic analysis of time-dependent re-endothelialization in murine carotid artery. Typical observations of endothelial cells stained by anti-VWF antibodies in
the carotid artery of ␣2-AP⫺/⫺ (B,D,F,H; original magnification, ⫻ 400) and in ␣2-AP⫹/⫹ mice (A,C,E,G; original magnification, ⫻ 400). Before the initiation of injury, an intact
endothelial cell layer was observed (A-B). Endothelial cells were removed by the injury (C-D). Regenerated endothelial cells (arrows) only partly covered the injured area in
wild-type mice (E); however, an obvious compact endothelial cell layer (arrows) developed on the injured surface in ␣2-AP⫺/⫺ mice (F) 2 days after injury. Four weeks after
injury, a neointimal thickening (ⴱ) had developed in the injured area. A regenerated endothelial cell layer (arrows) was observed on the neointima in wild-type mice (G) and in
␣2-AP⫺/⫺ mice, in which the endothelial cell layer (arrows), however, was thicker (G). Scanning electron photomicrographs of regeneration of endothelial cells in the injured
carotid artery 48 hours after injury in ␣2-AP⫹/⫹ mice (I,K) and in ␣2-AP⫺/⫺ mice (J,L) showed prompt and intense re-endothelialization in ␣2-AP⫺/⫺ mice. The white bar
represents 10 ␮m.
Time-dependent re-endothelialization
Neointima formation in response to endothelial injury
Figure 2 shows endothelial cells before and after injury in both
types of mice. Before the initiation of injury, endothelial cell layers
in both types of mice were clearly observed (Figure 2A-B). A few
endothelial cells remained on the vascular surface after injury
(Figure 2C-D). No difference between ␣2-AP⫺/⫺ mice and wildtype mice could be observed in the above conditions. In contrast,
the re-endothelialization was markedly different 48 hours after
injury. Indeed, the endothelial cell layer was much greater covering
the injured area in ␣2-AP⫺/⫺ mice (Figure 2F), whereas this
covering was only partial in wild-type mice (Figure 2E). Development of neointima was next measured 4 weeks after injury in both
types of mice (Figure 2G-H) and showed a complete reendothelialization of the newly formed intima. However, a thicker
reconstructed endothelial layer was seen in ␣2-AP⫺/⫺ mice than in
wild-type mice.
All mice developed concentric intimal lesions in response to
endothelial injury. The ratios of time-dependent vascular occlusion
by newly formed neointima are shown in Figure 3. In ␣2-AP⫺/⫺
mice, the extent of vascular occlusion 4 weeks after the initiation of
injury was significantly smaller compared with those of wild-type
mice. Typical examples of newly formed neointima in both types of
mice were shown in Figure 2G-H.
Electron microscopic observation
SEM at 2 days after injury confirmed that the injured vascular
surface was not completely covered by repairing endothelial cells
in wild-type mice (Figure 2I-J). However, in ␣2-AP⫺/⫺ mice,
saturation of endothelial cells was observed on the injured vascular
surface (Figure 2K). The repaired endothelial surface of ␣2-AP⫺/⫺
mice, surprisingly, was not smooth as expected but quite rough
with extruding cells (Figure 2L).
Figure 3. Time-dependent neointima formation. The development of neointima in
␣2-AP⫺/⫺ (F) and ␣2-AP⫹/⫹ mice (E) is shown as the percentage of luminal stenosis
by newly formed intima (n ⫽ 6 each time point). * indicates P ⬍ .05. Data represents
mean ⫾ SEM.
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BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
ROLE OF ␣2-AP IN RE-ENDOTHELIALIZATION
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Proliferation index in vivo
Figure 4 shows the percentage of BrdU-positive cells in regenerating endothelial cells or in newly formed intima on day 1, 3, 7, or 14
after vascular injury. The lack of ␣2-AP caused a significant
increase of BrdU-positive cells in recovered endothelial cells.
Typical observations of BrdU-positive cells 1 day after injury are
shown in Figure 4C-D.
Spontaneous secretion of VEGF in cultured cells of ␣2-APⴚ/ⴚ
and ␣2-APⴙ/ⴙ mice
Spontaneous release of VEGF was observed in vascular SMCs
from both mice types. VEGF in the conditioned medium, measured
by ELISA in function of time (Figure 5), showed that the levels of
VEGF from vascular smooth muscle cells of ␣2-AP⫺/⫺ mice were
about 2.5 times higher than those of ␣2-AP⫹/⫹ mice.
Binding and competitive inhibition assay of ␣2-AP
to endothelial cells
Immobilized plasminogen binds to endothelial cells, and the
intensity of the signal was dependent on the cell number (Figure
6A). However, immobilized ␣2-AP binds to plasminogen but not to
endothelial cells (Figure 6B).
Effect of injection of an anti-VEGF antibody in ␣2-APⴚ/ⴚ mice
Intravenous injection of an anti-VEGF antibody 5 minutes before
the initiation of endothelial injury in ␣2-AP⫺/⫺ mice markedly
changed the recovering endothelial surface in ␣2-AP⫺/⫺ mice,
which now was almost the same as in the wild-type mice (Figure
7A). Moreover, the action of VEGF is mediated by a particular
family of receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2
(KDR), which are expressed almost exclusively on endothelial
cells.27 The regenerating endothelial surface of ␣2-AP⫺/⫺ mice
Figure 4. Proliferative index in vivo. Cell proliferation (n ⫽ 4, for each time point)
measured as the BrdU index (percentage) of recovered endothelial cells (䡺) and
SMCs in media (f) following vascular injury in ␣2-AP⫹/⫹ (A) or ␣2-AP⫺/⫺ mice (B).
Data represents mean ⫾ SEM. Typical observations of BrdU-positive cells in the
injured carotid artery of ␣2-AP⫺/⫺ (C) and in ␣2-AP⫹/⫹ mice (D). Twenty-four hours
after injury, BrdU-positive cells (arrows) were clearly observed in the recovered
endothelial layer in ␣2-AP⫺/⫺ mice (C). However, BrdU-positive cells were present
mainly in media (SMCs) in ␣2-AP⫹/⫹ mice (D). Original magnification, ⫻ 400.
Figure 5. VEGF secretion in vitro. Spontaneous secretion of VEGF is increased in
vascular SMCs from ␣2-AP⫺/⫺ (F) as compared with those from ␣2-AP⫹/⫹ mice (E).
Each point represents the mean of duplicate cultures. Data represents mean ⫾ SEM.
treated with an inhibitory anti–VEGFR-1 antibody was not different from the one of the wild-type mice (Figure 7B), whereas all 4
␣2-AP⫺/⫺ mice treated with the anti–VEGFR-2 antibody showed a
prompt endothelial healing after injury (Figure 7C). BrdU-positive
cells were also counted (Figure 7D). The number of positive cells
in ␣2-AP⫺/⫺ mice treated with either VEGF antibody or anti–
VEGFR-1 antibody did not significantly differ as compared with
that of wild-type mice. However, the treatment with an anti–
VEGFR-2 antibody in ␣2-AP⫺/⫺ mice did not affect BrdU intake in
vivo. Four weeks after the vascular injury, a neointima had
developed in both types of mice, and the stenosis area of both types
of mice was not significantly different (data not shown).
Figure 6. Binding of endothelial cells to immobilized plasminogen or ␣2-AP.
(A) Binding of 5 ⫻ 105 (1), 2.5 ⫻ 105 (2), or 1 ⫻ 105 endothelial cells/mL (3) to
immobilized plasminogen was measured by biosensor. Immobilized ␣2-AP (B)
readily binds to plasminogen (4) but fails to bind endothelial cells when 5 ⫻ 105
cells/mL are added (5).
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3626
MATSUNO et al
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
Figure 7. Effects of VEGF antibodies in ␣2-APⴚ/ⴚ mice. Scanning electron photomicrographs of the regenerated endothelial surface of a carotid artery 48 hours after injury
in ␣2-AP⫺/⫺ mice. When either VEGF antibody or anti–EGFR-1 antibody was administered to ␣2-AP⫺/⫺ mice, the regeneration of the endothelium was normalized (A-B,
respectively). ␣2-AP⫺/⫺ mice treated with an anti–VEGFR-2 antibody showed a prompt and dense re-endothelialization (C). Cell proliferation (n ⫽ 4) measured as the BrdU
index (percentage) of recovered endothelial cell layer (䡺) and SMCs in media (f) following vascular injury in ␣2-AP⫺/⫺ mice (D). An anti-VEGF antibody and antibodies against
VEGFR-1 (Flt-1) or VEGFR-2 (KDR), (25 ␮g per body) were injected as a bolus 5 minutes before the initiation of endothelial injury in ␣2-AP⫺/⫺ mice (n ⫽ 3, each group). BrdU
(50 mg/kg) was injected subcutaneously 1, 8, 16, and 24 hours prior to removal of the carotid arteries. Two days after the injury, the injured artery was removed and stained for
the measurement of BrdU-positive cells. ⴱ indicates P ⬍ .05 control. Scale bars represent 10 ␮m.
Effect of injection of VEGF in ␣2-APⴙ/ⴙ mice and of ␣2-AP
in ␣2-APⴚ/ⴚ mice
When the highest dose of VEGF was injected in wild-type mice with
endothelial injury, the endothelial layer of all mice was slightly
thickened (Figure 8A). BrdU-positive cells in regenerating endothelial
cells increased in a dose-dependent manner (Figure 8B). However, the
development of the neointima 4 weeks after injury was not decreased
(data not shown). However, intravenous injection of ␣2-AP 5 minutes
before the initiation of endothelial injury in ␣2-AP⫺/⫺ mice markedly
changed, and the recovering endothelial surface (48 hours after injury)
in ␣2-AP⫺/⫺ mice now was almost the same as the one in the wild-type
mice (Figure 8C). When ␣2-AP was continuously administered for
more than 1 week, development of neointimal area was almost the same
as that of wild-type mice (Figure 8D). On the contrary, when ␣2-AP was
treated for 1 day or 3 days, development of neointimal area 4 weeks after
injury was still reduced compared with that of wild-type mice.
Discussion
The present study demonstrated that lack of ␣2-AP, a physiologic
inhibitor of plasmin, resulted in the improvement of vascular patency
after experimental endothelial injury in mice and indicates that ␣2-AP is
essential for the preservation of the regenerating endothelial cells via
regulation of local VEGF secretion. This effect could be explained
mainly by the prompt re-endothelialization because of the over-release
of VEGF by continuous local activation of plasmin.
Mice deficient in ␣2-AP were described, but no major physiologic
dysfunction could be observed when they were not challenged.28 In our
first experiment, we could demonstrate that the arterial blood flow after
spontaneous reperfusion was well maintained in mice deficient in
␣2-AP, even though the time to occlusion was slightly prolonged, albeit
not statistically significant, compared with that of wild-type mice.
Moreover, the area of the neointima was significantly decreased in
␣2-AP⫺/⫺ mice compared with that of wild-type mice. Time-dependent
profiles of the BrdU index showed that proliferating cells were densely
located in the newly formed intima 24 hours after injury in ␣2-AP⫺/⫺
mice. These results made us speculate that the lack of ␣2-AP may play a
key role in the regeneration of endothelial cells following endothelial
injury. Immunohistochemical stain of endothelial cells using anti-VWF
antibodies clearly indicated that regeneration of endothelial cells in
␣2-AP⫺/⫺ mice was more rapid and dense than in ␣2-AP⫹/⫹ mice.
Additionally, electron microscopic observations showed that the vascular surface after endothelial injury in ␣2-AP⫺/⫺ mice was markedly
different from that of wild-type mice in which repairing endothelial cells
did not yet completely cover the injured area 24 to 48 hours after injury.
We previously reported that local adhering microthrombi, including
activated platelets, were observed in wild-type mice until a few days
after injury.22 However, in ␣2-AP⫺/⫺ mice, a large number of endothelial cells overcrowded the injured area, whereas no thrombi were
Figure 8. Effect of VEGF in ␣2-APⴙ/ⴙ mice and of ␣2-AP in ␣2-APⴚ/ⴚ mice. Scanning electron photomicrographs of the regenerated endothelial surface of a carotid artery
48 hours after injury in ␣2-AP⫹/⫹ mice treated with VEGF at a dose of 8 ng per body (A) and treated with ␣2-AP at a dose of 75 mg per body (C). Cell proliferation (n ⫽ 4)
measured as the BrdU index (percentage) of recovered endothelial cell layer (䡺) and SMCs in media (f) following vascular injury in ␣2-AP⫹/⫹ mice (B). VEGF (2, 4, 8 ng/body)
was injected as a bolus before the initiation of endothelial injury. BrdU (50 mg/kg) was injected subcutaneously 1, 8, 16, and 24 hours prior to removal of the carotid arteries. Two
days after the injury, the injured artery was removed and stained for the measurement of BrdU-positive cells. The development of neointima in ␣2-AP⫺/⫺ mice is shown as the
percentage of luminal stenosis by newly formed intima (D). ␣2-AP was injected as a bolus via tail vein for 1 day, 3 days, 1 week, 2 weeks, or 4 weeks after endothelial injury, and
the injured carotid artery was removed 4 weeks after injury (n ⫽ 4 each). ␣2-AP⫹/⫹ mice (wild type [WT]) were treated with ␣2-AP for 4 weeks. * indicates P ⬍ .05 versus control
(without ␣2-AP). Scale bar represents 10 ␮m.
From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
observed. These results indicate that a significant endothelial regeneration is rapidly induced in ␣2-AP⫺/⫺ mice following experimental
endothelial denudation.
It is well known that VEGF is a major regulator of endothelial
cell production under both physiologic and pathologic conditions.29
Previous studies carried out in a variety of animal species have
repeatedly shown that extensive endothelial denudation of the
arterial wall leads to neointimal thicking.30,31 Local delivery of
VEGF to the site of vascular injury resulted in expeditious
reendothlialization30 and reduced the development of a neointima.31,32 In the present study, the plasma concentrations of VEGF
in both types of mice were almost the same before and after the
initiation of endothelial injury when blood samples were taken
from the jugular vein (data not shown). However, we confirmed
that spontaneous release of VEGF by cultured vascular SMCs from
␣2-AP⫺/⫺ mice was significantly higher than that by wild-type
mice cells. Plasmin plays a role in the cleavage of VEGF from
SMCs.9 ␣2-AP and plasminogen, but not plasmin, are secreted
mainly by the liver into the circulation,13 indicating that lack of
␣2-AP is expected to have a local effect but not a systemic effect.
Our binding studies on the one hand showed that plasminogen
binds readily to endothelial cells and on the other hand indicated
that ␣2-AP prevents the binding between plasminogen and endothelial cells dose dependently. Therefore, we speculated that lack of
␣2-AP could induce conditions that allow for easy binding of
plasminogen to endothelial cells. This plasminogen then can be
activated by plasminogen activators, such as tissue-type plasminogen activator secreted by endothelial cells, resulting in local
production of plasmin, as in the above situation, in the absence of
␣2-AP, as in ␣2-AP⫺/⫺ mice. This phenomenon might continuously stimulate the secretion of VEGF.
To further define the physiologic relation between ␣2-AP and
VEGF in vascular remodeling, we performed several additional
experiments using ␣2-AP⫺/⫺ and ␣2-AP⫹/⫹ mice. When ␣2-AP⫺/⫺
mice were supplemented with ␣2-AP for 1 day or 3 days, the
vascular surface was not completely covered with regenerating
endothelial cells until 2 days after the injury. Under those conditions, the vascular patency and the development of neointimal area
were different from those of wild-type mice. However, when
␣2-AP⫺/⫺ mice were continuously supplemented with ␣2-AP for
over 1 week after injury, the development of neointimal area was
almost the same as that of wild-type mice. These results directly
prove that lack of ␣2-AP, especially in the early phase of the
recovery process from vascular injury, results in the maintenance of
vascular patency and in the reduction of neointima formation after
injury in mice. Second, when a high dose of VEGF (8 ng per body)
was injected as a bolus in wild-type mice before the injury, the
number of endothelial cells in the injured area was only slightly
elevated, and the stenosis area at 4 weeks after injury was not
significantly diminished even if BrdU-positive cells in the recovering endothelial layer increased in a dose-dependent manner. The
half-life of recombinant VEGF in the circulation is only minutes,
and, indeed, administration of recombinant VEGF, also in humans,
was shown to be ineffective.34 These findings clearly indicate that
only local release of a high concentration of VEGF can promote a
ROLE OF ␣2-AP IN RE-ENDOTHELIALIZATION
3627
prompt and intense regenerating of endothelial cells after vascular
injury. Additionally, when administration of an anti-VEGF antibody to ␣2-AP⫺/⫺ mice resulted in normalization of the repair
process with the condition of the vascular surface not different
from that of wild-type mice, the proliferating endothelial cell
number was decreased. This rescue was also detected when
␣2-AP⫺/⫺ mice were treated with an anti-VEGF receptor-1 antibody, but not with an anti-VEGF receptor-2 antibody. This result
supports the previous observation that the effect of VEGF is
mediated mainly via VEGFR-1 (Flt-1). Indeed, our data show that
also proliferation of endothelial cells in mice in vivo is regulated
mainly by VEGFR-1, but not by VEGFR-2.
To the best of our knowledge, the present report is the first to
describe an essential role of ␣2-AP in vivo in the recovery after
vascular injury. The physiologic scenario of such a response might
be as follows: first, VEGF is released mainly from vascular SMCs
after endothelial injury. VEGF physiologically stimulates the
production of plasminogen activators, and then plasminogen is
converted into the active plasmin on endothelial cells. Plasmin
further stimulates the release of VEGF.10 During this process,
␣2-AP plays an inhibitory action on the mutual induction of VEGF
and plasmin, and ␣2-AP prevents plasminogen to bind to endothelial cells. Therefore, lack of ␣2-AP locally induces over-release of
VEGF after vascular injury, which extremely changes the rate of
endothelial healing mainly via activation of VEGFR-1 (Flt-1).
Additionally, ␣2-AP deficiency enhances activation of plasmin
leading to fibrinolysis.21 Both the prompt healing of endothelial
cells by over-release of VEGF and the enhancement of the
fibrinolytic action are beneficial for vascular repairing. VEGF is
also known as a vascular permeability factor on the basis of its
ability to induce vascular leakage.9 Therefore, local highly elevated
VEGF levels in ␣2-AP⫺/⫺ mice may in addition induce vascular
permeability and subsequent VEGF oversecretion from SMCs
stimulated by plasmin. Indeed, our previous findings showed
that oversecretion of VEGF in experimental acute myocardial
infarction in ␣2-AP⫺/⫺ mice increased the vascular permeability.5 This observation may indicate a link between plasmin
generation and SMCs.
In conclusion, lack of ␣2-AP improves the vascular patency
after endothelial injury which is mainly because of the enhancement of endothelial cell healing via an over-release of VEGF as a
result of the exaggerated activity of plasmin no longer tempered by
␣2-AP. Moreover, the increased fibrinolytic potential in addition
would reduce thrombotic vessel reocclusion. This dual effect might
regulate the neointimal thickening after endothelial injury. We,
therefore, concluded that ␣2-AP has an important local physiologic
role in vascular remodeling. The findings in this report have
identified a new target for the development of new therapeutics for
the clinical therapy of cardiovascular diseases.
Acknowledgment
We thank Prof Hans Deckmyn (K. U. Leuven, Campus Kortrijk,
Belgium) for his help.
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From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
2003 102: 3621-3628
doi:10.1182/blood-2003-03-0700 originally published online
July 31, 2003
Lack of α2-antiplasmin promotes re-endothelialization via over-release
of VEGF after vascular injury in mice
Hiroyuki Matsuno, Akira Ishisaki, Keiichi Nakajima, Kiyotaka Okada, Shigeru Ueshima, Osamu
Matsuo and Osamu Kozawa
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