© 1997 American Heart Association, Inc.
Articles |
Urokinase but Not Tissue Plasminogen Activator Mediates Arterial Neointima Formation in Mice
From the Center for Transgene Technology and Gene Therapy (P.C., L.M., R.L., D.C.), Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium; the Haemobiology Research Department (J.-M.H., ), Sanofi Recherche, Toulouse, France; and the Vascular Biology Laboratory (J.C., F.L.), the Thrombosis Research Institute, London, UK.
Correspondence to P. Carmeliet, MD, PhD, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
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Abstract |
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Abstract To define the role of the plasminogen activators (PAs) tissue PA (t-PA) and urokinase PA (u-PA) in vascular wound healing, neointima formation and reendothelialization were evaluated after electric or mechanical arterial injury in mice with a single or combined deficiency of t-PA (t-PA-/-) and/or u-PA (u-PA-/-). In both models, neointima formation and neointimal cell accumulation were reduced in u-PA-/- and in t-PA-/-/u-PA-/- arteries but not in t-PA-/- arteries. The electric injury model was used to characterize the underlying cellular mechanisms. Topographic analysis of vascular wound healing in electrically injured wild-type and t-PA-/- arteries revealed a similar degree of migration of smooth muscle cells from the noninjured borders into the necrotic center. In contrast, in u-PA-/- and t-PA-/-/u-PA-/- arteries, smooth muscle cells accumulated at the uninjured borders but failed to migrate into the necrotic center. Cultured u-PA-/- but not t-PA-/- smooth muscle cells also failed to migrate in vitro after scrape wounding. Proliferation of smooth muscle cells was not affected by PA deficiency. Reendothelialization after electric injury was similar in all genotypes. In situ analysis revealed markedly elevated u-PA zymographic activity, mRNA, and immunoreactivity in smooth muscle cells, endothelial cells, and leukocytes within 1 week after injury, eg, when cells migrated into the wound. Thus, u-PA plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting cellular migration.
Key Words: plasminogen activator • neointima • restenosis • mouse • blood vessel
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proteinases have been implicated in luminal stenosis, complicating vascular reconstructions for the treatment of atherothrombosis.1 2 3 4 5 Although vascular remodeling appears to be a major determinant of luminal stenosis after balloon angioplasty, intimal thickening can also contribute to the luminal narrowing, especially when vascular remodeling is prevented by placement of intraluminal stents. The latter results from accumulation of smooth muscle cells that proliferate in the media, migrate across the elastic membrane, and contribute to matrix deposition.3 4 5 Recent indirect evidence suggests an involvement of two proteinase systems, the plasminogen and metalloproteinase systems, in the process of smooth muscle cell migration.1 2 3 4 5 6 7 8 9 10 11
The plasminogen system is composed of an inactive proenzyme, plasminogen, that is converted to active plasmin by two physiological PAs, t-PA and u-PA.12 Their action is controlled by PAIs, of which PAI-1 appears to be the predominant physiological inhibitor. Whereas t-PA is primarily involved in clot dissolution, u-PA, which binds to a membrane-anchored receptor (u-PAR), has been implicated in pericellular proteolysis during cell migration or tissue remodeling.13 Plasmin has been presumed to play a role in tissue remodeling during wound healing and inflammation in a variety of processes, including glomerulonephritis, skin healing, atherosclerosis, and arterial neointima formation, via proteolysis of extracellular matrix components or activation of growth factors.14 15 16 Recent observations further suggest that the role of plasmin may largely depend on the breakdown of fibrin.17
In an uninjured artery, t-PA production by quiescent endothelial cells may promote vascular patency, whereas PAI-1 synthesis by medial smooth muscle cells has been proposed to provide a hemostatic barrier.1 10 After injury, expression of t-PA, u-PA, and u-PAR by smooth muscle, endothelial, and inflammatory cells is significantly induced, suggesting that a hyperfibrinolytic response may participate in the migration and/or proliferation of these cells.7 8 9 11 13 18 19 Indeed, smooth muscle cells use proteinases to degrade the extracellular matrix that encages them, preventing them from migration into the wound.1 3 Plasmin may trigger this process, since it can directly degrade fibrin and matrix and also activate other matrix-degrading proteinases, including the metalloproteinases.1 14 Indirect evidence has been provided that the plasminogen system participates in vascular wound healing in several species, including humans.20 21 22 In addition, when a perivascular electric injury model was used in plasminogen-deficient mice, it was demonstrated that plasmin proteolysis was essential for normal arterial wound healing and contributed significantly to the formation of a neointima.23 Studies in the rat with the plasmin inhibitor tranexamic acid have also suggested a role for plasmin in cellular migration following balloon injury of the carotid artery.8 Although increased expression of both plasminogen activators suggested their involvement in this process,1 7 8 9 direct functional proof for a causal and possible differential role in neointima formation remained to be determined. In addition, it was unknown whether t-PA and/or u-PA are essential for reendothelialization.
Using two models of arterial injury, the present study involving mice with inactivation of the genes encoding t-PA (t-PA-/-) and/or u-PA (u-PA-/-)24 provides direct genetic proof, of a significant role of u-PA–mediated but not t-PA–mediated plasmin proteolysis in arterial neointima formation, most likely via regulation of smooth muscle cell migration.
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Materials and Methods |
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Arterial Injury Model
Six- to 8-week-old WT, t-PA-/-, or u-PA-/- mice24 of either sex with a 75% C57Bl6 and 25% 129 genetic background were used. Groups of 8 to 14 animals were studied. Perivascular electric injury of left femoral arteries and analysis of the neointima was performed as described elsewhere.25 Although neointima formation occurs in both femoral and carotid arteries, the response in the femoral arteries was analyzed, since it occurs somewhat more rapidly and extensively (authors' unpublished data, 1996). Intraluminal mechanical injury of the carotid arteries was performed according to a recently described method by Lindner et al,26 with minor modifications. Briefly, the left internal carotid artery was exposed by blunt-end dissection, tied off distally, and looped proximally with 7.0 Mersilene nylon suture for vascular control during the procedure. A transverse arteriotomy was made in the proximal portion of the internal carotid artery, and a 380-µm flexible guide wire (C-SF 15-15, Cook Belgium NV) was passed toward the aortic arch over
15 mm and withdrawn three times
with a rotating motion. After removal of the guide wire, the proximal
portion of the internal carotid artery was tied off, and the skin
incision was closed. This protocol allowed reproducible injury,
resulting in rupture of the internal elastic lamina and
neointimal accumulation of 
-smooth muscle
actin–positive cells within 3 weeks after injury. The carotid artery
was used because of technical limitations to insert the guide wire in
the smaller femoral artery.
Tissue Harvest, Histology, and Immunocytochemistry
Tissue harvest, fixation, embedding, and sectioning were
performed as described.25 For t-PA or u-PA
immunostaining, the injured vessels were
perfusion-fixed with 1% phosphate-buffered
paraformaldehyde (pH 7.0) for 10 minutes without
postfixation and cryoembedded. Smooth muscle and inflammatory cells
were immunostained for smooth muscle -actin or CD45 as
described.25 Macrophages were visualized using a
rat monoclonal antibody against Mac-3 (Pharmingen). Replication was
determined by labeling cells with BrdU and
immunostaining as described.25 For t-PA
staining, a polyclonal rabbit anti-murine t-PA24 or goat
anti-human t-PA (10 µg/mL, American Diagnostica
No. 387/1) antiserum was used with similar results. For u-PA staining,
a biotinylated mouse anti-murine u-PA monoclonal antibody (clone No.
H77A10, 30 µg/mL),27 a rabbit anti-murine
u-PA,24 a goat anti-human u-PA (100
µg/mL, American Diagnostica No. 398), or
rabbit anti-human u-PA (100 µg/mL, American
Diagnostica No. 389) antiserum was used with similar
results, although background staining was somewhat different.
Colocalization of PAs with smooth muscle cells or macrophages
was established by use of a
double–immunofluorescence labeling approach as
previously described.28 With this procedure, PA-positive
cells appear green, -actin– or Mac-3–positive cells appear red,
and cells containing PA and -actin or PA and Mac-3 appear
yellow.
Morphometric Analysis and Reendothelialization
Morphometric measurements of cross-sectional areas, cell counts,
and proliferation rates and topographic analysis of
neointima formation in electrically injured arteries were
performed as described.25 Morphometric analysis of
mechanically injured arteries was performed on five sections equally
spaced across the injured segment. Reendothelialization
was evaluated after staining the denuded blood vessels with Evans blue
as previously described.25
Zymographic Analysis
Zymographic analysis of PA activities in
arterial extracts was performed as
described.23 In situ zymography on 7-µm
arterial cryosections was performed by fibrin overlay,
using a gel of fixed thickness prepared by clotting a mixture of human
fibrinogen (final concentration, 4 mg/mL),
plasminogen (final concentration, 10 µg/mL), and
agarose (final concentration, 0.5%) with thrombin (final
concentration, 0.3 NIH units/mL). The fraction of the lysis due to t-PA
or u-PA activity was determined by including in the fibrin gel 50
µg/mL polyclonal neutralizing rabbit anti-murine t-PA– and/or
u-PA–specific IgGs, respectively.24 A standard curve for
t-PA and u-PA activity was obtained by spotting different amounts of
purified murine t-PA and u-PA24 on the fibrin gels and
quantification of the lysis at different time intervals. The amount of
lysis, representing the lysis area multiplied by the
intensity of lysis and thus the total fibrinolytic activity per
section, was quantitatively analyzed using the Quantimed 600
image analysis software and expressed in arbitrary units.
In Situ Hybridization
In situ hybridization was performed as previously
described.28 The following cRNA probes were used: a 600-bp
fragment of the human u-PA cDNA or a 159-bp fragment of the murine u-PA
cDNA, subcloned in the Bluescript M13+ vector (Stratagene
Inc) or in the pGEM-7 vector (Promega), respectively, and labeled by
run-on transcription using 35S-labeled UTP (specific
activity, 1300 Ci/mmol, New England Nuclear). Sense and antisense
probes were prepared by linearizing the constructs with the appropriate
restriction endonucleases and by the use of either T7/T3 RNA or T7/SP6
RNA polymerases (Boehringer-Mannheim). RNase digestion was
carried out after probe hybridization to reduce the background. Both
human and murine u-PA probes yielded similar results.
Smooth Muscle Cell Cultures and In Vitro Migration Assay
After saline perfusion and removal of the adventitia, the
thoracic and upper part of the abdominal aorta were rinsed in saline
and cut into small pieces (1 mm2), and the fragments
were enzymatically dispersed by incubation for 16 hours at 37°C in
DMEM containing 0.15% collagenase (type II, Sigma) and 5%
fetal calf serum. Smooth muscle cells were cultured in DMEM containing
10% fetal calf serum and passaged one to three times before
analysis. Wound assays were performed in vitro as
described.29 Briefly, confluent monolayers of smooth
muscle cells, seeded 7 days earlier in DMEM containing 10% fetal calf
serum, in 35-mm dishes were wounded with a razor blade. After wounding,
the cells were washed with PBS and further incubated for 72 hours at
37°C in DMEM containing 0.1% gelatin and 10% fetal calf serum.
After fixation with absolute methanol, the cells were stained with
Giemsa. Cells that had migrated from the edge of the wound into the
denuded area were counted in seven successive 125-µm increments at
x100 magnification.
Statistical Analysis
Experimental values were expressed as mean±SEM. Statistically
significant differences between groups were calculated by ANOVA
followed by Bonferroni correction or by 
2
analysis, as indicated in the text.
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Results |
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Vascular Wound Healing in WT Mice
Electric Injury Model
Since the electric injury model differs from the mechanical injury model,25 its characteristics are briefly discussed (Fig 1
). Electric injury
denudes the endothelium and destroys all cells in the
media (<1% surviving medial cells), resulting in a wound-healing
response characterized by transient thrombosis, removal of necrotic
debris, reendothelialization, repopulation of the media
by smooth muscle cells, and the formation of a neointima
with multiple layers of smooth muscle cells within 3 weeks after injury
(Fig 2a to 2d).25 A
neoadventitia that was rich in leukocytes and fibroblast-like cells
developed.
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Mechanical Injury Model
Mechanical injury resulted in less severe medial cell necrosis
(52±8% surviving medial cells, P<.05 versus uninjured
arteries) than did electric injury. Since residual viable smooth muscle
cells persisted across the entire injured segment,
neointima formation occurred more uniformly across the
injured segment, as described previously for similar mechanical injury
models.30 In Fig 2
, panel g displays a
representative neointima from a WT carotid
artery, containing smooth muscle cells within 3 weeks after mechanical
injury. Since the electric injury model permits the differentiation
between proliferation and migration of smooth muscle cells and is
technically easier than the mechanical injury model, it was used more
extensively throughout the present study.
Vascular Wound Healing in Gene-Inactivated Mice
Electric Injury Model
After electric injury, vascular wound healing was similar in
t-PA-/- and WT arteries. In contrast, healing was
significantly impaired in u-PA-/- and
t-PA-/-/ u-PA-/- arteries, as evidenced
by the reduced accumulation of cells within the media and
neointima (Fig 2e and 2f; see below for quantification) and
by the impaired removal of the necrotic debris in the media (as judged
by microscopic analysis, Fig 2e and 2f). Compared with WT and
t-PA-/- arteries, which revealed a neointima
across the entire injured segment (from location 1 to 10 in
schematically represented arteries in Fig 1) within 3 weeks
after injury, a neointima was present only in
u-PA-/- and t-PA-/-/u-PA-/-
arteries at the uninjured borders (location 1 or 10) (Fig 2e) and
failed to progress into the necrotic center (location 5) (Fig 2f) (see
also below). Although adventitial inflammation was observed in all
genotypes, it was less extensive in u-PA-/- and
t-PA-/-/u-PA-/- arteries than in WT or
t-PA-/- arteries (compare Fig 2c with 2e).
Quantitative morphometry revealed that the neointima, as
deduced from measuring the cross-sectional area between the internal
elastic lamina and the lumen (Fig 3), and
the number of neointimal cells (Table 1) were significantly reduced in
u-PA-/- and t-PA-/-/u-PA-/-
arteries but not in t-PA-/- arteries. Within 4 weeks
after injury, the intima-to-media ratio was 1.60±0.25 in WT mice,
1.48±0.33 in t-PA-/- mice (P=NS), 0.57±0.15
in u-PA-/- mice (P=.003 versus WT mice), and
0.29±0.17 in t-PA-/-/u-PA-/- mice
(P=.011 versus WT mice). Repopulation of the media was also
impaired in u-PA-/- and in
t-PA-/-/u-PA-/- mice compared with WT and
t-PA-/- mice (Table 1).
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Mechanical Injury Model
Deficiency of u-PA also impaired neointima formation
after mechanical injury, as revealed by the
histological analysis in Fig 2h and 2i. Within
3 weeks after injury, the neointimal cross-sectional area,
the luminal stenosis, the intima-to-media ratio, and the number
of neointimal cells were lower in u-PA-/-
than in WT or t-PA-/- mice (Table 2). Thus, deficiency of u-PA impaired
vascular wound healing and neointima formation in both
injury models. The mechanisms underlying the improved
neointima formation in u-PA-/- mice were
further investigated using the electric injury model.
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Proliferation of Smooth Muscle Cells
To evaluate whether deficiency of t-PA or u-PA affects cellular
proliferation, incorporation of BrdU into replicating cells was
determined (Table 3). Within 1 week after
electric injury, the total BrdU-labeling index represents
proliferation of leukocytes and, to a lesser extent, smooth muscle
cells, whereas within 2 weeks after injury, the total BrdU-labeling
index reflects proliferation of smooth muscle cells, since they
constitute >95% of the intimal and medial cell
population.25 As shown in Table 3, medial and
neointimal cell proliferation were not significantly
different between the different genotypes, except for a
somewhat lower proliferation rate of medial smooth muscle cells in
u-PA-/- arteries within 1 week after injury.
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Migration of Smooth Muscle Cells In Vivo
Smooth muscle cells after electric injury migrated within the
media and alongside the lumen from the uninjured borders into the
necrotic center.25 This process was quantified by
measuring the luminal narrowing (percent stenosis) and cell
accumulation at equally spaced positions across the injured segment.
Within 3 weeks after injury, a significant neointima was
present throughout the entire injured segment in WT and
t-PA-/- arteries (Fig 4). In contrast, a neointima
was initiated at the borders of the injury in u-PA-/- and
t-PA-/-/u-PA-/- arteries but failed to
progress into the necrotic center (Fig 4).
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These findings were extended by counting the medial and
neointimal cell nuclei at three equally spaced positions
across the injured segment (locations 1, 5, and 10 in the arteries of
Fig 1) within the first 2 weeks after injury, when cells start to
migrate and accumulate in the intima. As shown in Table 4, cells accumulated at the borders and
progressed subsequently into the center of the injured segment in WT
and t-PA-/- arteries. In contrast, cells accumulated at
the borders but failed to migrate into the center of the injured
segment of u-PA-/- arteries (Table 4). Thus, these data
indicate that smooth muscle cells failed to migrate as far in
u-PA-/- arteries as they did in t-PA-/- or
WT arteries. This process is schematically represented in
Fig 1.
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Migration of Smooth Muscle Cells In Vitro
To substantiate the role of u-PA in smooth muscle cell migration,
cultured smooth muscle cells from WT, t-PA-/-,
and u-PA-/- mice were scrape-wounded, and their
accumulation into the denuded area was quantified. WT and
t-PA-/- but not u-PA-/- cells accumulated
into the denuded area within 72 hours after wounding (Fig 5). In addition, accumulation of WT cells
was reduced in the presence of neutralizing u-PA antibodies (50
µg/mL of the polyclonal rabbit anti-murine u-PA) but not of
t-PA antibodies (50 µg/mL of the polyclonal rabbit anti-murine
t-PA). Within 6 hours after wounding, 25±4 WT and 19±1
t-PA-/- but only 3±0.5 u-PA-/- cells
(P<.05 versus WT cells) accumulated in the denuded area.
Since this occurs before the cells have a chance to divide (estimated
to be 8 to 12 hours in these cultured murine smooth muscle cells), the
reduced accumulation of cells is most likely due to impaired cellular
migration.
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Reendothelialization
Electric injury completely denuded the injured segment of intact
endothelium, as revealed by Evans blue staining
immediately after injury.25
Reendothelialization was initiated from the uninjured
borders into the necrotic center and was almost complete within 1 week
after injury in WT mice. Endothelial regrowth within 7
days after injury was not affected, either in its extent or in its
rate, by deficiency of either PA (P=NS for
t-PA-/- and u-PA-/- compared with WT mice)
(Fig 6).
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Thrombosis
Electric injury of WT arteries resulted in transient mural
thrombosis during the first week after injury, ie, before the
appearance of the first smooth muscle cells in the
neointima.25 Since loss of PA function
increases the susceptibility to (venous) thrombosis and results in a
decreased ability to spontaneously lyse
125I-fibrin–labeled pulmonary plasma
clots,24 the incidence and extent of arterial
thrombosis in PA-/- arteries was semiquantitatively
evaluated. Within 1 week after injury, approximately one quarter of the
arteries contained a mural thrombus, occluding the lumen between 5%
and 25%. Within 2 weeks after injury, most WT arteries were completely
devoid of thrombus, whereas mural thrombosis persisted somewhat longer
in t-PA-/- arteries but persisted the longest in
u-PA-/- arteries (Table 5).
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Leukocytes
Electrically injured arteries become transiently infiltrated by
leukocytes during the first week after injury.25 Since the
plasminogen system significantly affects the migration of
leukocytes,31 the number of leukocytes in the media and
neointima were counted after staining for the pan-leukocyte
marker CD45. Fewer leukocytes infiltrated into u-PA-/-
than t-PA-/- or WT arteries. In the
neointima, 15±3 leukocytes (16±3% of the total number of
neointimal cells) accumulated in WT arteries, 12±4
leukocytes (23±10%) accumulated in t-PA-/- arteries
(P=NS), but only 1.3±0.3 leukocytes (4±2%) accumulated in
u-PA-/- arteries (P<.05 versus WT arteries).
Although there were too few cells present in the media for
appropriate statistical analysis, there was a similar trend of
reduced leukocyte accumulation in the media of u-PA-/-
arteries: 2±1 leukocytes (5±1% of total number of medial cells)
accumulated in WT arteries, 1±1 leukocytes (13±7%) accumulated in
t-PA-/- arteries, and only 0.2±0.2 leukocytes (1±1%)
accumulated in u-PA-/- arteries.
Zymographic Analysis of PA Expression
PA activities of arteries were evaluated by in situ zymography on
sections using fibrin overlay, since this technique allowed precise
discrimination between uninjured and electrically injured regions as
deduced from histological analysis on adjacent
sections. Lysis is expressed in arbitrary units (see "Materials and
Methods").
t-PA–Mediated Lysis
In uninjured arteries from WT mice, lysis of the fibrin gel
appeared within 2 hours (0.26±0.029, mean±SEM, n=4) and was inhibited
for 
95% (0.01±0.004) by inclusion of neutralizing t-PA antibodies
but not u-PA antibodies (0.26±0.055), indicating that lysis was due to
t-PA activity. As deduced from the immunocytochemical analysis
(see below), only endothelial cells expressed t-PA in
quiescent arteries.
In injured arteries, PA activities were expressed as a ratio of the lysis present in injured versus noninjured segments to normalize for variations in the absolute levels of lysis between experiments. In addition, since the specific activities of t-PA and u-PA in this assay were significantly different (because of their different affinity for and activation by fibrin), expressing the PA activities as ratios allowed us to compare their individual levels of induction after injury. In WT arteries, the ratio of lysis in injured versus noninjured sections was 0.28±0.06 within 2 days after injury (n=20) and restored to 1.0±0.09 (n=17) within 1 week after injury coincident with regeneration of the endothelium over the denuded surface. In u-PA-/- arterial sections, the ratio was 2.3±0.15 (n=26, P<.05 versus WT sections) within 1 week of healing. Since lysis by WT or u-PA-/- arteries at 2 and 7 days after injury was inhibited by >95% by neutralizing t-PA antibodies, lysis was essentially due to t-PA activity. There was no lysis within 2 to 5 hours over t-PA-/- sections (not shown).
u-PA–Mediated Lysis
In order to evaluate u-PA–mediated lysis, t-PA antibodies were
incorporated in the fibrin overlay to neutralize t-PA activity. Lysis
over sections from uninjured WT arteries in the presence of t-PA
antibodies appeared only after prolonged incubation (24 hours at
37°C) but was 20-fold higher than lysis in uninjured
t-PA-/- arterial sections analyzed
side by side (not shown). Thus, it is likely that the t-PA antibodies
incompletely blocked the residual t-PA in these sections upon prolonged
overlay, thereby precluding accurate quantitative analysis of
u-PA activity. Therefore, u-PA–mediated lysis of the fibrin overlay
was evaluated in t-PA-/- arteries.
In uninjured t-PA-/- arteries, u-PA–mediated lysis
appeared only after prolonged incubation (24 hours at 37°C) and was
minimal (0.002±0.0006, n=8), consistent with the minimal
expression of u-PA mRNA or immunoreactivity (see below). In sharp
contrast, within 1 week after injury, fibrin overlay on
arterial sections of t-PA-/- mice indicated
that u-PA–mediated lysis was dramatically enhanced in injured segments
compared with the noninjured border segments. Indeed, the ratio of
lysis observed in injured versus noninjured sections was 470±120
(n=19). Approximately 50% of this lysis was mediated by u-PA, as
evidenced by inclusion of neutralizing u-PA antibodies in the fibrin
overlay, suggesting that other proteinases, possibly
metalloproteinases, are responsible for the residual lysis. Lysis in
t-PA-/- arteries was present in microdissected
arterial fragments containing either the media and
neointima (primarily smooth muscle cells and some
leukocytes) (Fig 7a) or only the
adventitia (leukocytes and fibroblasts) (Fig 7b), consistent
with the in situ expression of u-PA mRNA and immunoreactive protein in
these cell types (see below). Taken together, these data demonstrate
that u-PA expression was minimal in quiescent arteries but that it
markedly increased (eg, much more than t-PA) after arterial
injury.
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In Situ Hybridization and Immunostaining
These results were extended by in situ identification of the
PA-producing cell types in WT arteries (n=3) within 1 week after injury
(eg, when cells actively migrate).
u-PA Expression
In uninjured arteries, a low level of u-PA expression was
detectable by in situ hybridization but not by immunocytochemistry
(presumably reflecting differences in sensitivity of the assays) (Figs 8a and 9b).
In contrast, within 1 week after injury, there was a marked
upregulation of u-PA mRNA and antigen expression in all layers of the
injured vessel. The uninjured border from which smooth muscle cells
start to migrate into the wound (location I in the schematically
represented artery in Fig 9a) displayed increased u-PA
staining in endothelial cells in the intima, in smooth
muscle cells in the media, and, at a lower level, in fibroblast-like
cells and leukocytes in the enlarged adventitia (Fig 9c). Significantly
induced u-PA antigen and mRNA expression were detected in
endothelial cells in the intima (Fig 8b, 8c, and 9d)
and in leukocytes (Fig 8b, 8c, and 9e) and fibroblast-like cells (Fig 8b, 8c, and 9f) in the adventitia at the site of maximal
neointima formation (location II) and at the leading front
of cellular migration (location III). In addition, double
immunostaining revealed that smooth muscle cells in the
intima and media (Fig 9h) and Mac-3–positive macrophages in
the adventitia (Fig 9i) expressed increased amounts of u-PA. u-PA
immunoreactivity was absent in u-PA-/- arteries (Fig 9g)
and in WT arteries after omission of the primary antisera (not shown).
Only background hybridization was present using a sense u-PA probe
(Fig 8d). Thus, u-PA is expressed by endothelial cells,
smooth muscle cells, leukocytes, and myofibroblast-like cells when they
repopulate the wound.
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t-PA Expression
In uninjured femoral arteries, only weak t-PA immunoreactivity was
observed in intimal endothelial cells (Fig 10b) but at a much lower level than in
endothelial cells from the aorta (insert, Fig 10b),
suggesting topographic differences in t-PA expression across the
vasculature. Immediately after injury, when the
endothelium was denuded, t-PA staining was lost in the
necrotic region (not shown), consistent with the drop in t-PA
activity as analyzed by in situ zymography. Beyond 1 week after
injury, t-PA staining was present in intimal
endothelial cells, in smooth muscle cells in the media
and intima, and in adventitial myofibroblast-like cells (Fig 10c to
10e). However, compared with the u-PA staining, the intensity of the
t-PA staining was significantly lower, and there were fewer
immunoreactive cells. In addition, topographic analysis
revealed that t-PA was present in a majority of medial smooth
muscle cells in the border region, adjacent to the injury (location I)
(Fig 10c), but that the fraction of t-PA–positive cells progressively
diminished from the uninjured border to the cellular migration front;
eg, the presence of t-PA immunoreactive cells diminished from ±50% of
cells at location II (Fig 10d) to only a minor fraction (<10%) at the
leading edge of the migration front (location III) (Fig 10e). Double
immunostaining revealed that t-PA colocalized within
-actin smooth muscle cells (Fig 10f) but not in Mac-3–positive
macrophages (not shown). The weak -actin
immunostaining within 1 week after injury is due to its
reduced expression, frequently observed in proliferating and migrating
smooth muscle cells.25
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Role of PAs in Cellular Migration and Tissue Remodeling
Deficiency of u-PA, but not t-PA, greatly reduces neointima formation, suggesting that u-PA mediates vascular wound healing after injury. In addition, combined deficiency of t-PA and u-PA resulted in a similar impairment of vascular wound healing. These data, together with our recent observations that deficiency of plasminogen also impaired arterial neointima formation,23 indicate that u-PA acts by conversion of plasminogen to plasmin.
The data further indicate that smooth muscle and possibly also
inflammatory cells require u-PA–mediated plasmin proteolysis to
migrate into the wound. Indeed, the observations, in
u-PA-/- mice, that neointima formation was
reduced, that cells accumulated at the borders but failed to progress
into the necrotic center in vivo, and that u-PA-/-
cultured smooth muscle cells failed to migrate into the denuded area
after scrape wounding in vitro indicate that u-PA deficiency impaired
smooth muscle cell and possibly also inflammatory cell migration (also
see Fig 1). Proliferation of smooth muscle cells was not affected by
deficiency of u-PA. The plasmin inhibitor tranexamic acid
and metalloproteinase inhibitors also reduce smooth muscle
cell migration in the rat carotid artery without affecting smooth
muscle cell proliferation.6 8 Thus, u-PA–mediated plasmin
proteolysis appears to play a central role in proteolytic degradation
of the extracellular matrix, allowing the cells to migrate to distant
sites. Whether u-PA affects neointima formation/smooth
muscle cell migration by modulating cell adhesion via interaction with
the vß3-integrin receptor32
remains to be defined.
Besides its role in cellular migration, u-PA may also be involved in tissue remodeling during wound healing. Indeed, electron microscopy of WT arteries has revealed that the media after electric injury consists of necrotic smooth muscle cell remnants embedded in a fibrin-rich extracellular matrix.25 Thus, healing of the necrotic media must involve proteolytic degradation and removal of this debris. Impaired migration of u-PA-/- leukocytes and fibroblasts may also have contributed to the reduced formation of a neoadventitia in u-PA-/- or combined t-PA-/-/u-PA-/- mice.
Expression of PAs During Vascular Wound Healing
t-PA was expressed by quiescent endothelial cells
but was lost immediately after denudation, similar to expression in the
balloon-injured rat carotid artery.7 Within 1 week after
injury, t-PA was expressed in regenerating endothelial
cells and in some smooth muscle cells, restoring t-PA activity in
injured regions to preinjury levels. The most significant difference in
expression was, however, observed for u-PA, which was minimal in
quiescent arteries but markedly induced during wound healing (eg, much
more than t-PA). Increased expression of u-PA during vascular wound
healing has also been observed in several other species, including
humans.7 19 20 21 Notably, net u-PA–mediated fibrinolytic
activity in injured arterial segments was >100-fold
induced. Since u-PA mRNA and immunoreactivity were maximal at the
leading edge of the migration front, a role for u-PA in cell migration
is suggested. Since smooth muscle cells produce u-PA (the present
study and References 1, 7, 9, 11, 191 7 9 11 19 -22, 33, and 34), they may control
their own migration in an autocrine way. Alternatively, inflammatory
cells in the wound (the present study and Reference 55 ) may assist
in migration of smooth muscle cells by providing u-PA (similar to the
paracrine regulation of tumor cell migration by stromal
cells35 ) or by "clearing a path" for them. In this
respect, it is noteworthy that infiltration of the vascular wound by
leukocytes was reduced in u-PA-/- mice.
Role of PAs in Thrombosis
Loss of PA gene function increases the susceptibility of mice to
injury- or inflammation-associated thrombosis, but almost exclusively
in the venous system.24 Notably, u-PA-/-
mice were more susceptible to fibrin deposition than were
t-PA-/- mice.24 The present findings of
prolonged arterial thrombosis, most significant in
u-PA-/- mice, further underline the role of u-PA in
fibrin surveillance. Endothelial and inflammatory cells
may participate in this process, since they both can produce u-PA in
vivo. Thrombosis may promote neointima formation by
providing a provisional matrix for smooth muscle cell migration and
supplying a variety of growth factors able to modulate proliferation
and migration of smooth muscle cells.3 4 5 Interestingly,
neointima formation was smaller but thrombosis was more
persistent in u-PA-/- arteries, suggesting that the
presence of a mural thrombus was not sufficient and even may have
impeded neointima formation in u-PA-/-
arteries.
Role of PAs in Endothelial Cell
Regeneration
Endothelial cell regrowth was not impaired by t-PA
or u-PA deficiency. This was not anticipated in view of the induced
expression of PAs in regenerating endothelium in vivo
and in vitro after injury (the present study and References 9 and
139 13 ). However, to date, no abnormalities in vascular development have
been described in mice with inactivated genes of
plasminogen system components during
embryogenesis,15 suggesting an accessory role for the
plasminogen system and/or compensation by other proteinase
systems in endothelial cell migration.
Endothelial cell migration alongside a denuded surface
may, however, require proteolytic mechanisms other than invasion
through anatomic barriers.
Role of t-PA in Vascular Wound Healing
Neointima formation in t-PA-/-
mice was normal and not more impaired in combined
t-PA-/-/u-PA-/- than in
u-PA-/- mice, except for a transient reduction of
neointimal cell accumulation within 1 week after injury in
t-PA-/- mice. Interestingly, t-PA expression in the
medial smooth muscle cells at the uninjured borders was increased
shortly after injury, possibly indicating that t-PA participates in the
initiation of smooth muscle cell migration. Such a role for t-PA was
suggested previously in the balloon-injured rat carotid
artery.7 8 9 However, t-PA appears to be inefficient in
mediating sustained cellular migration in contrast to u-PA, presumably
related to its lower level of expression. Unfortunately, we could not
assess whether u-PA expression was compensatorily increased in
t-PA-/- mice.
Electric Versus Mechanical Injury Model
Deficiency of u-PA impaired neointima formation,
whereas deficiency of t-PA did not affect this process in either the
electric or the mechanical injury model, indicating a general role of
u-PA in the response to arterial injury. Whereas the
mechanical injury model reflects more closely the injury inflicted in
patients undergoing balloon angioplasty,5 the electric
injury model is technically easier and has the advantage of providing a
means to quantify the migration of smooth muscle cells from the
uninjured borders into the necrotic center. Both models differ from
each other in that the electric injury induces transient thrombosis and
inflammation of the injured vessel wall. Since thrombosis and
leukocytes accompany and modulate the wound-healing response in
patients undergoing vascular reconstructions,5 the
present study on wound-healing response after electric injury may
bear some clinical relevance in our understanding of how PAs function
during this process.
In conclusion, the present study provides direct evidence that u-PA–mediated plasmin proteolysis promotes vascular wound healing and associated neointima formation in mice, most likely by promoting migration of smooth muscle cells into the wound.
|
Selected Abbreviations and Acronyms |
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Received March 24, 1997; accepted August 7, 1997.
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References |
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E. M. Redmond, J. P. Cullen, P. A. Cahill, J. V. Sitzmann, S. Stefansson, D. A. Lawrence, and S. S. Okada Endothelial Cells Inhibit Flow-Induced Smooth Muscle Cell Migration : Role of Plasminogen Activator Inhibitor-1 Circulation, January 30, 2001; 103(4): 597 - 603. [Abstract] [Full Text] [PDF]
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 T. L. Frandsen, C. Holst-Hansen, B. S. Nielsen, I. J. Christensen, J. R. Nyengaard, P. Carmeliet, and N. Brünner Direct Evidence of the Importance of Stromal Urokinase Plasminogen Activator (uPA) in the Growth of an Experimental Human Breast Cancer Using a Combined uPA Gene-Disrupted and Immunodeficient Xenograft Model Cancer Res., January 1, 2001; 61(2): 532 - 537. [Abstract] [Full Text]
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D. Godin, E. Ivan, C. Johnson, R. Magid, and Z. S. Galis Remodeling of Carotid Artery Is Associated With Increased Expression of Matrix Metalloproteinases in Mouse Blood Flow Cessation Model Circulation, December 5, 2000; 102(23): 2861 - 2866. [Abstract] [Full Text] [PDF]
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 A. HAJ-YEHIA, T. NASSAR, B. S. SACHAIS, A. KUO, K. BDEIR, A. B. AL-MEHDI, A. MAZAR, D. B. CINES, and A. A.-R. HIGAZI Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo FASEB J, July 1, 2000; 14(10): 1411 - 1422. [Abstract] [Full Text]
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H. R. Lijnen, B. Van Hoef, M. Dewerchin, and D. Collen {alpha}2-Antiplasmin Gene Deficiency in Mice Does Not Affect Neointima Formation After Vascular Injury Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1488 - 1492. [Abstract] [Full Text] [PDF]
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S. Kanda, M. Kuzuya, M. A. Ramos, T. Koike, K. Yoshino, S. Ikeda, and A. Iguchi Matrix Metalloproteinase and {alpha}v{beta}3 Integrin-Dependent Vascular Smooth Muscle Cell Invasion Through a Type I Collagen Lattice Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 998 - 1005. [Abstract] [Full Text] [PDF]
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D. Hasenstab, H. Lea, and A. W. Clowes Local Plasminogen Activator Inhibitor Type 1 Overexpression in Rat Carotid Artery Enhances Thrombosis and Endothelial Regeneration While Inhibiting Intimal Thickening Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 853 - 859. [Abstract] [Full Text] [PDF]
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S. Kojima, S. Hayashi, K. Shimokado, Y. Suzuki, J. Shimada, M. P. Crippa, and S. L. Friedman Transcriptional activation of urokinase by the Kruppel-like factor Zf9/COPEB activates latent TGF-beta 1 in vascular endothelial cells Blood, February 15, 2000; 95(4): 1309 - 1316. [Abstract] [Full Text] [PDF]
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H. R. Lijnen, P. Soloway, and D. Collen Tissue Inhibitor of Matrix Metalloproteinases-1 Impairs Arterial Neointima Formation After Vascular Injury in Mice Circ. Res., December 3, 1999; 85(12): 1186 - 1191. [Abstract] [Full Text] [PDF]
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H. R. Lijnen, B. Van Hoef, I. Vanlinthout, M. Verstreken, M.-C. Rio, and D. Collen Accelerated Neointima Formation After Vascular Injury in Mice With Stromelysin-3 (MMP-11) Gene Inactivation Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2863 - 2870. [Abstract] [Full Text] [PDF]
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B. H. Strauss, H. K. Lau, K. A. Bowman, J. Sparkes, R. J. Chisholm, M. B. Garvey, L. L. Fenkell, M. K. Natarajan, I. Singh, and J. M. Teitel Plasma Urokinase Antigen and Plasminogen Activator Inhibitor-1 Antigen Levels Predict Angiographic Coronary Restenosis Circulation, October 12, 1999; 100(15): 1616 - 1622. [Abstract] [Full Text] [PDF]
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H. K.F. Lau Regulation of proteolytic enzymes and inhibitors in two smooth muscle cell phenotypes Cardiovasc Res, September 1, 1999; 43(4): 1049 - 1059. [Abstract] [Full Text] [PDF]
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N. A. Giese, M. M. H. Marijianowski, O. McCook, A. Hancock, V. Ramakrishnan, L. J. Fretto, C. Chen, A. B. Kelly, J. A. Koziol, J. N. Wilcox, et al. The Role of Alpha and Beta Platelet-Derived Growth Factor Receptor in the Vascular Response to Injury in Nonhuman Primates Arterioscler Thromb Vasc Biol, April 1, 1999; 19(4): 900 - 909. [Abstract] [Full Text] [PDF]
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A. A.-R. Higazi, K. Bdeir, E. Hiss, S. Arad, A. Kuo, I. Barghouti, and D. B. Cines Lysis of Plasma Clots by Urokinase-Soluble Urokinase Receptor Complexes Blood, September 15, 1998; 92(6): 2075 - 2083. [Abstract] [Full Text] [PDF]
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H. R. Lijnen, B. Van Hoef, F. Lupu, L. Moons, P. Carmeliet, and D. Collen Function of the Plasminogen/Plasmin and Matrix Metalloproteinase Systems After Vascular Injury in Mice With Targeted Inactivation of Fibrinolytic System Genes Arterioscler Thromb Vasc Biol, July 1, 1998; 18(7): 1035 - 1045. [Abstract] [Full Text] [PDF]
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P. Carmeliet, L. Moons, and D. Collen Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis Cardiovasc Res, July 1, 1998; 39(1): 8 - 33. [Abstract] [Full Text] [PDF]
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M.-L. Bochaton-Piallat, G. Gabbiani, and M. S. Pepper Plasminogen Activator Expression in Rat Arterial Smooth Muscle Cells Depends on Their Phenotype and Is Modulated by Cytokines Circ. Res., June 1, 1998; 82(10): 1086 - 1093. [Abstract] [Full Text] [PDF]
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H.R. Lijnen, J. Silence, B. Van Hoef, and D. Collen Stromelysin-1 (MMP-3)-Independent Gelatinase Expression and Activation in Mice Blood, March 15, 1998; 91(6): 2045 - 2053. [Abstract] [Full Text] [PDF]
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 P. Carmeliet, L. Moons, M. Dewerchin, S. Rosenberg, J.-M. Herbert, F. Lupu, and D. Collen Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice J. Cell Biol., January 12, 1998; 140(1): 233 - 245. [Abstract] [Full Text] [PDF]
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P. Carmeliet, L. Moons, R. Lijnen, S. Janssens, F. Lupu, D. Collen, and R. D. Gerard Inhibitory Role of Plasminogen Activator Inhibitor-1 in Arterial Wound Healing and Neointima Formation : A Gene Targeting and Gene Transfer Study in Mice Circulation, November 4, 1997; 96(9): 3180 - 3191. [Abstract] [Full Text]
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S. Mukhina, V. Stepanova, D. Traktouev, A. Poliakov, R. Beabealashvilly, Y. Gursky, M. Minashkin, A. Shevelev, and V. Tkachuk The Chemotactic Action of Urokinase on Smooth Muscle Cells Is Dependent on Its Kringle Domain. CHARACTERIZATION OF INTERACTIONS AND CONTRIBUTION TO CHEMOTAXIS J. Biol. Chem., May 26, 2000; 275(22): 16450 - 16458. [Abstract] [Full Text] [PDF]
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Y. Zhu, H. Bujo, H. Yamazaki, S. Hirayama, T. Kanaki, K. Takahashi, M. Shibasaki, W. J. Schneider, and Y. Saito Enhanced Expression of the LDL Receptor Family Member LR11 Increases Migration of Smooth Muscle Cells In Vitro Circulation, April 16, 2002; 105(15): 1830 - 1836. [Abstract] [Full Text] [PDF]
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