Atherosclerosis 159 (2001) 297– 306
www.elsevier.com/locate/atherosclerosis
Urokinase plasminogen activator augments cell proliferation and
neointima formation in injured arteries via proteolytic mechanisms
Olga Plekhanova a, Yelena Parfyonova a,*, Robert Bibilashvily a, Sergei Domogatskii a,
Victoria Stepanova a, Dietrich C. Gulba b, Alex Agrotis c, Alex Bobik c,
Vsevolod Tkachuk a
a
b
Molecular Endocrinology Laboratory, Institute of Experimental Cardiology, Cardiology Research Center, Moscow, 121552, Russia
Franz-Volhard Clinic at Charite, Clinic of Humboldt Uni6ersity, Max-Delbruech Centrum for Molecular Medicine, Berlin, Germany
c
Cell Biology Laboratory, Baker Medical Research Institute, Alfred Hospital, Melbourne, 3181, Australia
Received 18 November 2000; accepted 15 March 2001
Abstract
Urokinase plasminogen activator (uPA) has been implicated in the healing responses of injured arteries, but the importance of
its various properties that influence smooth muscle cell (SMC) proliferation and migration in vivo is unclear. We used three
recombinant (r-) forms of uPA, which differ markedly in their proteolytic activities and abilities to bind to the uPA receptor
(uPAR), to determine, which property most influences the healing responses of balloon catheter injured rat carotid arteries. After
injury, uPA and uPAR expression increased markedly throughout the period when medial SMCs were rapidly proliferating and
migrating to form the neointima. Perivascular application of uPA neutralizing antibodies immediately after injury attenuated the
healing response, significantly reducing neointima size and neointimal SMC numbers. Perivascular application of r-uPAwt (wild
type uPA) or r-uPA/GDF (r-uPA with multiple mutations in its growth factor-like domain) doubled the size of the neointima.
Four days after injury these two uPAs nearly doubled neointimal and medial SMC numbers in the vessels, and induced greater
reductions in lumen size than injury alone. Proteolytically inactive r-uPA/H/Q (containing glutamine rather than histidine-204 in
its catalytic site) did not affect neointima or lumen size. Also, in contrast to the actions of proteolytically active uPAs, tissue
plasminogen activator (tPA) did not affect the rate of neointima development. We conclude that uPA is an important factor
regulating the healing responses of balloon catheter injured arteries, and its proteolytic property, which cannot be mimicked by
tPA, greatly influences SMC proliferation and early neointima formation. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Vessel injury; Plasminogen activators; Proteolysis; Neointima; Proliferation
1. Introduction
Percutaneous coronary artery angioplasty and intraluminal stenting are common procedures used to increase blood flow to an ischaemic myocardium.
However, restenosis due to severe lumen narrowing
greatly limits their usefulness, necessitating repeat interventions in up to 30% of cases [1,2]. The mechanisms
responsible for this narrowing can involve vessel remodeling and/or the development of a large neointima
[3]. The cellular responses to vessel injury include exces* Corresponding author. Tel.: + 7-095-4146712; fax: + 7-0954146712.
E-mail address: yeparfyon@cardio.ru (Y. Parfyonova).
sive smooth muscle cell (SMC) proliferation and migration, resulting in an occlusive neointima [4,5]. Both
metalloproteinases [6] and the plasminogen activator
system (PAS) have been implicated in these processes
[7].
The PAS is composed of two activators, a tissue-type
PA (tPA) and a urokinase-type PA (uPA), specific uPA
cell surface receptors (uPAR) and multiple plasminogen
activator inhibitors (PAIs) [8]. In uninjured arteries,
tPA appears important for maintaining vessel patency,
whilst a haemostatic barrier is provided by SMC
derived PAI-1 [9]. After balloon catheter injury to a
vessel, tPA and uPA expression by the medial SMCs is
rapidly increased, with high uPA expression during
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 1 5 0 ( 0 1 ) 0 0 5 1 1 - 1
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SMC mitogenesis and tPA expression greatest during
migration [10]. Despite these associations, studies with
uPA and tPA knockout mice indicate that uPA
rather than tPA participates in SMC migration and
neointima formation after vessel injury [11]. The role
of uPAR in responses to injury is less defined, despite
its ability to influence the migration of cultured
SMCs [12]. For example, the expected mitogenic and
chemotactic effects of fibroblast growth factor-2
(FGF-2), a growth factor implicated in the early proliferative and migratory responses of SMCs to injury
[13] are absent in uPAR-deficient cells, or when cultured SMCs are exposed to uPAR antibodies [14].
Similarly, the presence of the growth factor-like domain of uPA, a region that specifically interacts with
uPAR is essential for its chemotactic action on cultured SMCs [15]. Other studies indicate that the ability of uPA to stimulate mitogenesis and cell migration
is not dependent on plasmin generation [16], a key
factor in the activation of tissue proteolysis.
In this study, we determined the extent to which
such uPA-dependent proteolytic and non-proteolytic
mechanisms affect neointima formation after balloon
catheter injury to the rat carotid artery. By examining
the effects of recombinant forms of native (i.e. wild
type) and mutated uPAs on SMC proliferation and
the size of the developing neointima, we find that the
predominant structural region of uPA responsible for
regulating SMC proliferation, neointima formation
and probably SMC migration is the proteolytic domain.
2. Materials and methods
2.1. Animals and experimental design
Male Wistar-Kyoto rats (4– 5 months old) were obtained from a colony maintained at the Cardiology
Research Center, Moscow, Russia. Their left common
carotid artery was subjected to balloon catheter injury
using surgical procedures approved by the Cardiology
Research Center’s Animal Experimentation Committee.
Initially we determined the time course of changes
in uPA/uPAR expression in relation to SMC proliferation and neointima formation during the ensuing 4
days after balloon catheter injury. These were examined 6, 24, 48 and 96 h after vessel injury and compared with uninjured vessels. Assessment of the
significance of uPA’s proteolytic domain and the domain, which interacts with uPAR, for SMC proliferation, migration and vessel structure after injury was
investigated by applying to the adventitial side of
seven to eight arteries 20 nmol/kg of either, (1) recombinant wild type uPA (r-uPAwt); (2) recombinant
uPA with mutations in the growth factor-like domain
(r-uPA/GFD), which prevents uPAR-dependent cultured SMC migration [15]; (3) proteolytically inactive
recombinant uPA, in which glutamine replaced histidine in position-204 (r-uPA/H/Q); (4) recombinant
tPA (r-tPA). We also examined the effects of applying a uPA neutralizing monoclonal IgG antibody (500
mg per vessel) or control non-specific mouse IgG (500
mg per vessel). The peptides were dissolved in saline
containing 40% gel F-127 (Pluronic, BASF) [17,18].
Four days later the animals were culled and isolated
arteries subjected to immunohistochemical and morphometric analyses.
2.2. Surgery
Rat carotid arteries were injured with an inflated
balloon catheter [17,18]. Briefly, after anesthetizing
the rats with ketamine (100 mg/kg body wt., i.p.,
Pfaffen – Schwabenheim), a midline incision was made
in the neck to expose the left external carotid artery.
A 2 F Fogarty arterial embolectomy catheter (Baxter
Healthcare) was introduced into this artery through
an arteriotomy and passed into the common carotid
artery to the aortic arch. The balloon was inflated
and then slowly rotated while pulling the catheter
back towards the external carotid artery. This was
repeated three times and then the external carotid
artery was ligated. The contralateral right carotid
artery as well as uninjured left carotid arteries from
sham-operated rats served as controls. To peri-adventitially administer various agents, the arteries were
gently dissected free of their surrounding connective
tissue and then 0.5 ml of the desired Pluronic solution was placed around the vessel as described earlier
[17,18]. The incisions were closed and the animals
were allowed to recover.
2.3. Tissue collection and processing
The animals were deeply anesthetized with sodium
pentobarbital (100 mg/kg body wt.; Sanofi, Sante Animale), and Evans blue (60 mg/kg body wt.; IV) administered, so that removal of endothelium in the
damaged vessels could be confirmed. Then the animals were perfused (120 mmHg) with saline solution,
followed by 4% formaldehyde solution for 10 min
[18,19]. Left and right common carotid arteries were
removed, cleaned of extraneous material and cut into
three equal segments before embedding in paraffin.
Cross-sections (5 mm for immunohistochemistry and
10 mm for morphometry) were cut from each block,
at 100– 200 mm intervals. The vessels for mRNA
analyses were obtained from animals perfused only
with saline. These were placed in ice cold saline and
stripped of adventitia before storing at − 72°C.
O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
2.4. Morphometry
Morphometry was carried out as described earlier
[18,19]. Briefly, cross-sectional areas of the lumen,
neointima, media and area, encompassed by the external elastic laminae (EEL), of formaldehyde fixed toluidine blue stained sections were measured by a blinded
histologist, using a Zeiss microscope coupled to a ProgRess-3008 camera (Kontron Elektronik) and computer
with an ‘Optimas’ morphometric program (Optimas
Corporation). Morphometry was performed on multiple sections of the distal and proximal segments of the
arteries, and the data for each artery averaged.
2.5. Immunohistochemistry
Sections were deparaffinized with xylene, rehydrated
and treated with 3% hydrogen peroxide to quench
endogenous peroxidase. Serial sections were used to
localize uPA and uPAR immunoreactive peptides
[17,19]. The sections were incubated in 10% serum
(ICN) from the same species as the secondary biotinylated antibodies, and then with either the anti-uPA
monoclonal antibody (10 mg/ml), the anti-uPAR polyclonal antibody (10 mg/ml), the anti-PCNA monoclonal
antibody (4.8 mg/ml), the anti-alpha smooth muscle
actin monoclonal antibody (3.6 mg/ml) or the appropriate control non-immune IgGs (either mouse or rabbit)
in concentrations, coinciding with those of each immune IgG, for 1 h in a humidified chamber. After
multiple washings in physiological buffered saline (PBS,
pH 7.4), the sections were incubated with a biotinylated
anti-rabbit or anti-mouse antibody (15 mg/ml). Antigens
were detected using the ABC method (Vector laboratories Inc.) and the chromogen 3,3%-diaminobenzidine tetrahydrochloride, before lightly staining the sections
with haematoxylin.
2.6. RT-PCR analysis
uPA and uPAR mRNA in vessels were determined
using a semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) procedure as described earlier [17,19,20]. Oligonucleotide primer pairs
for detection by PCR of uPA and uPAR cDNA fragments were designed to the following criteria, GC content 45–55%, melting point (m.p.) 76– 83.5°C, filtering
hairpins and 3%-homologies. The sequences for the uPA
and uPAR primers were based on GenBank accession
nos. X65651 and X71899, respectively. The ‘uPA
primers’ were sense: 5%-TCGTGAATCAGCCAAAGAAGGAAGAGTACG-3% (bp 680–709 in the cDNA)
and antisense: 5%-CAACTGACATTTTCAGGTTC-3%
(bp 993–1012 in the cDNA), to theoretically amplify a
333 bp cDNA fragment. The ‘uPAR primers’
were
sense:
5%-CAGAACACTGTATTGAAGTG-
299
GTGACGCTCC-3% (bp 431–460 in the cDNA), antisense: 5%-TCCAAGCACTGATTCATTGGTCCCCG-3%
(bp 712– 737 in the cDNA) to amplify a 307 bp cDNA
fragment. Oligonucleotide primers for L7 were those
earlier used by us [17].
Amplification parameters for semi-quantitative PCR
were 94°C for 30 s, 60°C for 1 min and 72°C for 2 min
[17,19]. The optimum number of cycles for uPA was 28
and 26 for uPAR. These numbers were chosen to
ensure that the product amplification-RNA relationship
was always in the log-linear range [17]. Confirmation of
identical amounts of RNA for each RT-PCR was carried out by amplifying in 23 PCR cycles the L7 cDNA
fragment; the L7 cDNA encodes a non-inducible cell
cycle-independent ribosomal protein [17]. PCR products were electrophoresed in 3% agarose gels containing
ethidium bromide, photographed under ultraviolet light
using positive/negative film (Polaroid 665), and then the
intensities of the bands were quantitated using laser
densitometry (LKB 2202 Ultrascan Bromma, LKB).
Those for uPA and uPAR were normalized to the L7
band, and the results expressed as fold increases above
values from uninjured vessels.
2.7. Recombinant mutated uPAs, antibodies and
reagents
A recombinant wild type human uPA (r-uPAwt) and
a recombinant uPA form with a mutated growth factorlike domain (r-uPA/GFD) were produced as described
earlier [15,20– 22]. Recombinant-uPA/GFD differs
from r-uPAwt in the first 24 amino acids, SNELHQVPSNCDCLNGGTCVSNKY, of the N-terminus, which
were replaced with ITPSLHACRSTLD [15]. Recombinant-uPA/GFD does not stimulate cultured rat aortic
SMCs to migrate, contrasting with the stimulatory activity of r-uPAwt [15]. After activation with plasmin the
proteolytic activities of r-uPAwt and r-uPA/GFD
ranged from 1 to 1.2× 105 U/mg protein [15]. A proteolytically inactive uPA, r-uPA/H/Q was prepared by
mutating His-204 within the catalytic center to Gln
[21,22]. Recombinant-uPA/H/Q was mapped with antibodies for different uPA epitopes. It did not possess
proteolytic activity, measured using S2444 (Chromogenix). The ability of r-uPA/H/Q to bind to human
uPAR is identical to r-uPAwt and it was as effective as
r-uPAwt in displacing 125I-r-uPAwt bound to cultured
rat aortic medial SMC (IC50’s20 nM) [21].
All the recombinant forms of uPA appeared as single
proteins on SDS-electrophoresis, with apparent molecular weights ranging from 40 to 43 kDa. Their purity
was greater than 95%.
An anti-uPA monoclonal IgG1 antibody was prepared by immunizing mice with human urokinase,
purified from urine. The antibody has a high affinity for
human and rat uPA and recognized all forms of uPA.
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O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
It is capable of neutralizing human recombinant uPA
activity (1 mg/ml neutralizes 0.8 nM uPA). In rat tissues, it detects uPA with an apparent molecular weight
of 48 kDa. Non-specific total mouse IgG from pooled
serum (Inpharm Inc, Russia) was used as control for
peri-adventitial anti-uPA antibody. An anti-uPA monoclonal mouse IgG (Inpharm Inc, Russia) raised against
human uPA was used for immunohistochemistry. It
also detects uPA in rat tissues. A specific uPAR
rabbit polyclonal IgG antibody that detects
glycosylated forms of human uPAR (39– 66 kDa) [23]
and rat uPAR (35– 58 kDa, Stepanova V., unpublished), was purchased from American Diagnostica
Inc. A specific PCNA mouse monoclonal IgG antibody
(PC 10) was purchased from Santa Cruz Biotechnology.
The anti-a-smooth muscle actin (a-SM actin) monoclonal antibody was from Dako. Control nonimmune rabbit and mouse IgGs, a biotinylated horse
anti-mouse rat adsorbed IgG antibody and a biotinylated goat anti-rabbit IgG were purchased from Vector
Laboratories. Recombinant tissue-type plasminogen activator (tPA) was from Boehringer Ingelheim Pharma
KG.
2.8. Assessment of efficacy of peri6ascular
administration
The efficacy of perivascular delivery of the uPA
neutralizing antibody was assessed using a rat-adsorbed
biotinylated horse anti-mouse antibody, a polyclonal
horse anti-goat antibody as negative control and an
avidin –biotin immunoperoxidase kit. The delivery of
control non-specific total mouse IgG was also assessed.
The efficacy of perivascular delivery of the recombinant
forms of uPA was assessed 2 and 4 days after their
application to the arteries, using r-uPA forms conjugated to biotin. Recombinant-uPAs applied to the vessels were later detected using (a) Western blots and
NeutrAvidin (Pierce) and (b) immunohistochemistry
with the Vector Laboratories’ streptavidin-peroxidase
kit. All substances were present in the media and
adventitia of the arteries 2 days after their application,
but by 4 days they were no longer detectable (not
shown).
2.9. Data analyses
The intensities of immunohistochemical stains detecting uPA and uPAR were graded in a semi-quantitative
blinded manner by two histologists, using a scale of
0 –4, with ‘0’ indicating no staining (background); ‘+
1’, low and variable staining in a specific region; ‘ +2’,
weak staining; ‘ +3’, consistent positive staining; ‘+ 4’,
intense staining. The neointima (region between the
endothelium and the internal elastic laminae) and media (region between the internal and external elastic
laminae) were assessed in this manner (three sections
per rat) [24,25]. PCNA positive cells and total cell
numbers (i.e. hematoxylin stained nuclei in the media
and neointima) were determined by counting (three
sections per rat). A PCNA labeling index was calculated using the equation, PCNA labeling index=
[PCNA −positive cells per three cross sections/total
cells per three cross sections]× 100. The percentage of
a-SM actin positive cells in the neointima was determined in the same manner. All results are means9
standard error of mean (S.E.M.). Comparisons between
multiple groups were performed using the one-way
analysis of variance (ANOVA) and the Student–Newman – Keuls test for multiple comparisons. Single comparisons were made using Student’s t-test. A value of
PB 0.05 was considered statistically significant. All
statistical analyses were performed using ‘Jandel
SigmaStat’.
3. Results
3.1. uPA and uPAR expression after experimental
angioplasty
Urokinase PA immunoreactive peptides were readily
detected in the media of uninjured carotid arteries and
localized predominantly to SMCs (Fig. 1). About 48 h
after injury, uPA was elevated nearly two-fold (Fig. 2,
top panel) and distributed uniformly throughout the
media (Fig. 1). This elevation became apparent as early
as 6 h after injury and persisted for at least 96 h (Figs.
1 and 2, top panel), i.e. throughout the time when
SMCs proliferate rapidly in the media and migrate to
form the neointima [10]. At 96 h uPA was also highly
expressed in the developing neointima (Figs. 1 and 2,
top panel). There were also associated increases in uPA
mRNA, indicated by the greater abundance of RTPCR transcripts for uPA (333 bp fragment), compared
with those obtained from uninjured vessels, 2 and 4
days after the injury (Fig. 3).
Increases in uPAR immunoreactive peptides were
also apparent after injury (Fig. 1). They were more
gradual than for uPA (Fig. 2, bottom panel), with peak
levels being reached at 48 h nearly three times higher
than those in the uninjured vessels (Fig. 2, bottom
panel). Immunohistochemistry on serial sections indicated a similar distribution pattern for uPAR and uPA
in the media and in the neointima (Figs. 1 and 2,
bottom panel). The injured vessels also contained
higher levels of uPAR mRNA than uninjured vessels,
detected by RT-PCR as a 307 bp cDNA fragment, 2
and 4 days after injury (Fig. 3). At 4 days 979 1% of
the cells in the media and 9792% in the developing
neointima expressed a-SM actin.
O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
3.2. Urokinase PAs and early neointima de6elopment
Since uPA and its receptor are expressed early after
injury, when the neointima commences to develop and
the media is being repaired, we next investigated, (1)
how elevations in uPA affect neointima formation and
vessel structure; (2) the importance of uPA’s receptor
binding property and (3) the importance of its proteolytic property in modulating vessel structure.
Periadventitial application of r-uPAwt doubled the
size of the neointima in the carotid arteries 4 days after
injury (PB 0.05 from control; Fig. 4, top panel). The
size of the lumen was reduced (P B0.05 from control;
Fig. 4, middle panel). There was also a tendency for the
size of the media to increase (P\ 0.05; Fig. 4, lower
panel). Periadventitial application of r-uPA/GFD also
increased neointima size (P B0.05), to the same level as
achieved with r-uPAwt (Fig. 4, top panel). Recombi-
301
nant-uPA/GFD reduced lumen size after injury (P B
0.05 from injured control vessels; Fig. 4, middle panels).
Here, there was also a tendency for medial cross sectional area to be increased, although not statistically
significant (P \0.05; Fig. 4, lower panel). In contrast,
peri-adventitial application of r-uPA/H/Q, which lacks
proteolytic activity, did not increase neointima size
after balloon catheter injury (P for difference \0.05
from vehicle treated vessels; Fig. 4, top panel). Recombinant-uPA/H/Q also had no effect on lumen or media
size (P\ 0.05; Fig. 4, middle and lower panels). Periadventitial application of the uPA neutralizing antibody attenuated neointima size 4 day after vessel
injury, by about 35% (P B0.05; Fig. 4, top panel).
Although there was a tendency for lumen size to be
reduced, as well as for medial cross-sectional area, these
effects were not statistically significant (P \0.05; Fig. 4,
middle and lower panels). Peri-adventitial application
Fig. 1. Top panels: expression of uPA and uPAR immunoreactive peptides in serial sections of uninjured carotid arteries; controls shown represent
non-immune rabbit IgG controls for the uPAR polyclonal antibody; identical results (not shown) were obtained with the control mouse IgG for
the uPA monoclonal antibody. Center panels: expression 48 h after balloon catheter injury. Bottom panels: expression 96 h after injury.
Immunoreactive peptides are represented by the brown coloration. L represents the lumen. Elastic laminae are stained blue, cell nuclei are stained
with haematoxylin. Scale bar 50 mm. Magnifications × 450.
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O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
sis [14,15], we compared the effects of the recombinant
forms of uPA on SMC accumulation in the neointima.
As reported earlier [26], the number of SMCs in the
neointima increased significantly 4 days after injury
(PB 0.05). Perivascular r-uPAwt doubled the number
of SMCs in the developing neointima (P B0.05; Fig. 5,
top panel). The number of SMCs in the healing media
also increased by nearly 50% (P B0.05; Fig. 5, bottom
panel). The increases in neointima SMC numbers were
also observed after r-uPA/GFD (P B0.05; Fig. 5, top
panel). However, the small increase ( 25%) in SMC
numbers in the media was not statistically significant
(P \0.05; Fig. 5; bottom panel). Since r-uPA/GFD
Fig. 2. Time course of changes in the expression of uPA and uPAR
during the 96 h after balloon catheter injury to the rat carotid artery.
Control represents values obtained in uninjured arteries. The intensity
of staining was graded from 0 to +4 as described in Section 2 and
the results for each animal averaged. Results are the means 9 S.E.M.
of four animals in each group. * PB 0.05 from control.
of control non-specific mouse IgG did not induce
changes in vessel structure compared with vehicletreated injured arteries (not shown).
To determine whether the proteolytic dependent effects of uPA on vessel structure were specific to uPA,
we examined the effects of tPA. Four days after its
application to the balloon catheter injured arteries, the
size of the neointima was identical to that observed in
vehicle-treated injured arteries (tPA: 0.01390.001
mm2; vehicle: 0.01490.001 mm2; P\ 0.05).
3.3. Urokinase PAs and neointimal SMC numbers
Since uPA stimulates cultured SMCs to proliferate
and migrate by mechanisms not dependent on proteoly-
Fig. 3. Panel A, typical agarose gels demonstrating the effects of
injury to the carotid artery on mRNAs encoding uPA, uPAR and L7.
Top bands represent uPA RT-PCR transcripts obtained from the
RNA of injured (I) left carotid and uninjured (U) right carotid
arteries of the same animals 48 and 96 h after the balloon catheter
injury. Middle and bottom bands, RT-PCR transcripts of uPAR and
L7, obtained from the same RNA. Panel B, magnitude of the changes
in uPA and uPAR mRNAs observed 48 and 96 h after injury. Results
are the average from four experiments,* PB 0.05.
O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
303
mouse IgG did not affect neointimal nor medial SMC
numbers compared with vehicle-treated arteries (data
not shown).
3.4. uPAs and neointima SMC cycle status
Both proliferating and non proliferating SMCs are
known to migrate from the injured media to form the
neointima [27]. To determine whether uPA increased
the number of neointimal and medial cells, entering the
cell cycle, we compared the proportion of PCNA positive SMC in the neointima and media of uPA and
vehicle treated arteries 4 days after injury. Urokinase
PA increased the frequency of PCNA expressing SMCs
in the neointima from 26.791.4 to 36.99 2.3% (P B
0.05), while PCNA labeling index in the media was not
affected compared with vehicle-treated injured arteries
(P \0.05).
4. Discussion
Fig. 4. Bar graphs summarizing the effects of perivascularly applied
r-uPAwt, r-uPA/GFD, r-uPA/H/Q and uPA neutralizing antibody on
neointima cross sectional area (upper panel), lumen area (middle
panel) and media area (bottom panel) 4 days after injury to carotid
arteries. Control represents vessels, which treated only with Pluronic
gel/saline. All injured vessels were compared with their corresponding
right uninjured carotid artery in the same animals and the differences
calculated. Results are the mean 9 S.E.M. of six to seven animals in
each group. * P B 0.05 from control.
does not stimulate SMC migration in vitro [15], its
ability to increase neointima SMC numbers appears
mostly due to increases in SMC proliferation. In contrast, perivascular r-uPA/H/Q did not increase neointimal SMC numbers; rather there tended to be a small
but not statistically significant decrease (P\ 0.05; Fig.
5, top panel). It also did not affect SMC numbers in the
media (P \0.05 from control; Fig. 5, lower panel).
Perivascular application of the uPA neutralizing antibody reduced neointimal SMC numbers by approximately 45% (P B0.05; Fig. 5, top panel), without
affecting SMC numbers in the media (P\ 0.05; Fig. 5,
bottom panel). Peri-adventitial control non-specific
It is well accepted that the uPA system can regulate
vascular SMC migration as well as participate in
neointima growth in injured vessels and the development of various human vascular disorders
[10,11,15,16,18,28– 30]. Although cell culture studies indicate that uPA stimulates SMC DNA biosynthesis and
migration by mechanisms apparently not dependent on
proteolysis or uPAR occupancy [21,31– 33], in balloon
catheter injured arteries the proteolytic property of
uPA, independent of the interaction with uPAR, is the
prime mechanism by which uPA enhances neointima
growth. Enhanced neointimal SMCs proliferation appears mostly responsible for the increase in neointima
size; effects due to an increase in SMC migratory
activity appear to be less evident. This proteolytic-dependent effect of uPA on the size of the neointima of
the injured arteries could not be induced by tPA.
Two lines of evidence indicate that the proteolytic
property of uPA alone, despite the early expression of
uPAR in the balloon catheter injured arteries, appears
to be the major determinant influencing SMC proliferation and possibly migration. First, proteolytically inactive r-uPA/H/Q did not increase early neointima
formation, contrasting markedly with the effects of
proteolytically active recombinant uPAs. Second, proteolytically active r-uPA/GFD, which does not stimulate
SMC migration in vitro [15], is similar to r-uPAwt in its
ability to augment neointima size and neointimal SMC
numbers. Differences in environmental conditions of
cultured SMCs and SMCs in the injured vessels could
account for the different responses of SMCs in arteries
and cell culture to the recombinant forms of uPA
[31,32]. However, the probable explanation is that the
proteolytic property of uPA predominantly regulates
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O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
SMC proliferation in vivo. Our conclusion on the significance of interactions of uPA with uPAR for SMC
proliferation, early neointima formation and possibly
migration are consistent with recent observations on
the healing responses of injured arteries in uPAR
knockout mice [12]. Whilst uPAR can be involved in
SMC migration, in its absence other non-uPAR dependent mechanism appears to take over [34]. Whether this
latter mechanism is the consequence of uPA interacting
with yet to be characterized receptors in the vascula-
ture, such as those reported on platelets [35] and rat
parenchymal liver cells [36] is not known.
Correlations have been reported between the early
expression of uPA and SMC proliferation and tPA and
SMC migration in balloon catheter injured arteries [10].
Also, other studies in knockout mice [11] and primate
arterial tissue [37] suggest that uPA participates in
neointima development. Our study indicates that uPA
markedly enhances neointimal SMCs proliferation. Exogenous r-uPAwt considerably enhanced cell prolifera-
Fig. 5. Bar graphs summarizing the effects of r-uPAwt, r-uPA/GFD, r-uPA/H/Q and a uPA neutralizing antibody on the number of smooth
muscle cells in the neointima (top panel) and the media (bottom panel) of carotid arteries 4 days after injury. Control represents injured vessels
to which only Pluronic solution was applied. Results are the mean 9 S.E.M. of six to seven animals in each group. * PB 0.05 from control.
O. Plekhano6a et al. / Atherosclerosis 159 (2001) 297–306
tion and neointima formation, whilst uPA neutralizing
antibody attenuated neointima formation in the injured
arteries. In uPA-knockout mice, the reduction in
neointimal SMC numbers after vessel injury was similar
to those reported here (45%) [11]. Our finding that
the elevation in uPA is a significant contributor to the
early SMC proliferation and neointima formation
might be related with the demonstrated role for FGF-2
in the healing of mechanically injured vessels. FGF-2 is
rapidly released from SMCs in arteries following balloon catheter injury [13,38]. Also, in cultured SMCs
FGF-2 elevates uPA levels four to five-fold [14]. Urokinase PA also appears necessary for FGF-2 to exert its
maximal mitogenic and chemotactic effects on vascular
smooth muscle [14]. We have already shown that the
ability of PDGF-BB and FGF-2 to stimulate cultured
SMC to migrate is dependent on uPA [15].
Proteolysis initiated via uPA in injured arteries has
the potential to stimulate SMC proliferation and migration by a number of highly coordinated mechanisms.
Rapid up-regulation of metalloproteinases (MMPs) occurs after balloon catheter injury to the vessel wall,
coinciding with high rates of medial SMC proliferation,
and rapid SMC migration into the intima [39]. The
conversion of pro-MMPs to active MMPs is dependent
on urokinase-generated plasmin activities, which in turn
degrade extracellular matrix (ECM) proteins including
collagens [29]. Matrix bound growth factors, such as
FGF-2 and latent transforming growth factor-beta
(TGF-b) are released during ECM degradation [40,41],
and these have the potential to further augment SMC
proliferation and cell migration. FGF-2 is a potent
mitogen in injured vessels [13,14], and TGF-b1 also
augments neointima formation after balloon catheter
injury to carotid arteries [42]. Whilst these effects might
be considered largely due to actions on SMCs, adventitial fibroblasts can also migrate through arterial layers
after balloon catheter injury and contribute to
neointima growth [43]. It is possible that similar actions
on adventitial fibroblasts might also contribute to
uPA’s ability to augment neointima growth.
To summarize, we have shown that the proteolytic
activity of uPA is the major contributor to its ability to
augment neointima formation early after balloon
catheter injury to the rat carotid artery. Our experiments suggest that this property of uPA, which can not
be mimicked by tPA, contributes to neointima growth,
SMC accumulation and proliferation in the neointima
of healing arteries after balloon catheter injury. Urokinase PA may be a specific functional target for attenuating lesion growth.
Acknowledgements
Dr Ye. Parfyonova and Dr O. Plekhanova have
305
participated in the Russian/Australian Exchange Program in Medicine and Public Health. These studies
have in part been funded by the National Health and
Medical Research Council (NHMRC) of Australia and
Russian Basic Research Foundation, grant no. 99-0448772. The authors are grateful for assistance with
research of Svetlana Mukhina, Alexey Poliakov,
Marina Solomatina and Sergei Ragozin.
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