© 1996 American Heart Association, Inc.
Articles |
Migration of Arterial Wall Cells
Expression of Plasminogen Activators and Inhibitors in Injured Rat Arteries
From the Department of Pathology (M.A.R., C.I.), University of Washington, Seattle, and the Maine Medical Center Research Institute (V.L.), South Portland, Me.
Correspondence to Michael A. Reidy, PhD, University of Washington/Department of Pathology, Vascular Biology/Box 357335, Seattle, WA 98195-7335.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract The expression of plasminogen activators and inhibitors was examined in denuded arteries. Within 5 days, smooth muscle cells (SMCs) on the luminal surface expressed the mRNA for tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), the receptor for UPA (UPAR), and plasminogen activator inhibitor type-1 (PAI-1). Similar results were seen after 8 days. Six weeks later, only TPA mRNA was still expressed by SMCs on the luminal surface. En face casein zymograms revealed a net fibrinolytic activity in areas covered with luminal SMCs. Reverse zymography showed no antifibrinolytic activity in these zones. Quiescent endothelial cells did not express TPA, UPA, UPAR, or PAI-1 mRNA. Regenerating endothelium at the wound edge strongly expressed TPA, UPA, and UPAR, as well as PAI-1. UPA and UPAR expression was highly restricted to cells at the wound edge and was not present elsewhere. En face zymography showed no plasmin activity in endothelialized areas, and reverse zymography showed a net antifibrinolytic activity in endothelialized zones. These results suggest that plasminogen activator and inhibitor expression correlates with the migration of both SMCs and endothelial cells into an arterial wound.
Key Words: arterial injury • plasminogen activators • smooth muscle cells • endothelium
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Plasminogen activators have been shown to be important in the regeneration of large-vessel endothelium and in angiogenesis.1 2 3 In vitro endothelial cells at wound edges express both UPA and TPA. These same cells exhibit lytic activity as assessed by casein zymograms.3 4 The plasminogen activators convert plasma-derived inactive plasminogen to active plasmin, which can degrade a variety of matrix proteins and activate metalloproteinases,5 and activation of these enzymes is associated with the ability of cells to migrate through the extracellular matrix.6 7
Plasminogen activator expression is not restricted to the endothelium, and the invasion of many cancer cells is linked to the presence of plasminogen activators.6 7 8 9 These cells have also been found to express UPAR, which binds UPA and allows the activation of plasminogen at sites where it is protected from PAIs.10 11 Nonmalignant cells also express UPAR, and recently this receptor has been shown to play an important role in endothelial cell migration.4
In a previous report, we showed that the SMCs of injured arteries express both TPA and UPA within days after injury and that plasmin activity is significantly increased at this time. Furthermore, the migration of SMCs into the arterial intima was inhibited by a plasmin inhibitor.12 These data have led us to propose that, like endothelial cells, the medial SMCs need to express plasminogen activators to migrate into the intima. The aim of this article, therefore, was to document the expression of both plasminogen activators and PAIs (PAI-1 and PAI-2) in both regenerating endothelium and migrating SMCs by in situ hybridization in arteries injured with a balloon catheter. We also wished to correlate the expression of UPA with the expression of its receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Arterial Injury Model
All animal studies were approved by the animal care committee of the University of Washington. Forty-eight male Sprague-Dawley rats (400 g, 3 to 4 months old) (Bantin & Kingman, Edmonds, Wash) were used in all the experiments. All surgical procedures were carried out with the animals under general anesthesia by intraperitoneal injection of xylazine (2.2 mg/kg, AnaSed, Lloyd Laboratories) and ketamine (50 mg/kg body wt, Ketaset, Aveco Co, Inc). For time points of 5 days and later, the left carotid artery and the aorta were denuded completely with a 2F balloon catheter (Fig 1
).
This results in endothelial outgrowth from intercostal
arteries. For studying early time points of wounded
endothelium up to 3 days, the aortic
endothelium was only partially denuded by passing an
uninflated balloon catheter along the aorta (Fig 1). SMCs were
studied
in normal and balloon-injured carotid arteries. This allowed us to
study SMCs in cross-sectional (embedded in paraffin, 5 µm thick)
and en face preparations during neointimal formation as
well as in chronically denuded vessels.
Deendothelialized segments of arteries were
identified by intravenous injection of Evans blue (0.3 mL
of 5% solution in saline) 10 minutes before death. For in situ
hybridization, all animals were perfusion fixed with phosphate (0.1
mol/L, pH 7.4)-buffered 4% paraformaldehyde. Four or
more rats were studied per time point (between 2 hours and 6 weeks
after injury). Vessels from the same animal were used for incubation
with the sense and antisense probes of TPA, UPA, UPAR, and PAI-1. A
total of 153 specimens were analyzed, of which 35 were
incubated with sense probes.
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In Situ Hybridization
Arteries were prepared for en face in
situ hybridization
according to our published protocol.13
Arterial segments were cut open longitudinally, and the
tissue was pinned out flat on
polytetrafluoroethylene cards (luminal side
facing up). Incubation with proteinase K (1 µg/mL, 37°C,
Boehringer Mannheim) was 15 minutes, followed by
prehybridization for 2 hours with 0.3 mol/L NaCl, 20 mmol/L Tris (pH
7.5), 5 mmol/L EDTA, 1x Denhardt's solution, 10% dextran sulfate,
10
mmol/L dithiothreitol, and 50% formamide. T3, Sp6, and T7 polymerases
(Promega) were used to generate corresponding sense and antisense
strands of [35S]UTP-labeled riboprobes from linearized
cDNA. An equal number of counts of sense or antisense probe was applied
to the specimens. After hybridization (at 55°C overnight), the
specimens were washed with 2x SSC (1x SSC contains 150 mmol/L NaCl
and 15 mmol/L sodium citrate, pH 7.0), 10 mmol/L
ß-mercaptoethanol, and 1 mmol/L EDTA (twice for 10 minutes each),
treated with RNase A (20 µg/mL, 30 minutes at 37°C, Sigma Chemical
Co), and washed in 2x SSC (as above). This procedure was followed by a
high-stringency wash at 55°C for 2 hours (0.1x SSC, 10 mmol/L
ß-mercaptoethanol, and 1 mmol/L EDTA). Subsequent steps followed
the protocol described previously.14 The Häutchen
procedure for en face preparations15 was carried out after
the probe hybridization. All slides were coated with
autoradiographic emulsion (Kodak, NTB2), exposed for 3
weeks, and then developed (Kodak, D-19). Sections and Häutchen
preparations were observed under the light microscope using
dark-field, bright-field, and a combination of epiluminescence
and bright-field illumination (reflective light). Shown are the
representative results of three or four independent
runs of in situ hybridization carried out in parallel with antisense
and the corresponding sense probes.
cDNA Probes Used for In Situ Hybridization
Rat cDNAs for TPA
and UPA (both 
350 bp) were kindly provided
by Dr Jay L. Degen (Cincinnati, Ohio). A rat cDNA for PAI-1 was
received from Dr Gelehrter (Ann Arbor, Mich).16 A 1.35-kb
Pst I–Kpn I fragment of PAI-1 was subcloned into
pBluescript SK+ (Stratagene) for in situ hybridization. A rat PAI-2
cDNA was kindly provided by Dr Grundmann (Behring Werke, Marburg,
Germany). Rat UPA receptor was cloned by reverse
transcription–polymerase chain reaction using RNA isolated from
rat SMCs. RNA was reverse transcribed using random hexamer primers, and
cDNA was amplified with rat UPAR-specific primers based on the
published sequence.17 The 5' primer (position 123) was
CTGCCTGGTGGAGGAGTG, and the 3' primer (position 513) was
CTGCCACAGCCTTTGGTG. These primers amplified a 400-bp sequence of coding
region that is present in all splice variants of the UPAR. The
identity of the sequence was verified using multiple restriction
digests and sequencing.
En Face Zymography
Plasminogen (human plasma, Sigma), 1 mg
protein/mL
in 50 mmol/L Tris (pH 8.1), and urokinase (high molecular weight,
Calbiochem) in 50 mmol/L Tris (pH 8.1) were aliquoted and kept at
-80°C. Nonfat dry milk (8% in 0.1 mol/L Tris, pH 8.1) and agar
(2.5% in distilled water) were boiled for 20 minutes, cooled,
centrifuged, and used immediately. Glass slides, Pasteur
pipettes, and glass beakers were all warmed in a 50°C oven as were
the above solutions, with the exception of plasminogen and
urokinase. Balloon-injured or unmanipulated arteries were perfused
with cold lactated Ringer's solution in situ, removed from the
animals, and placed on ice until the gels had been prepared.
The gel was prepared in a warmed glass beaker in the following proportions: milk 25%, 1 mol/L Tris 10%, dH2O 15%, and agar 50%, with the addition of 40 µg of plasminogen per milliliter of gel. This mixture was poured, avoiding bubble formation, onto warmed glass slides and spread quickly with the side of a Pasteur pipette to form a uniform opaque layer. Slides were removed from the 50°C oven and allowed to cool at room temperature. Segments of opened arteries were laid flat onto the gel surface, with the lumen side in contact with the gel. Slides were then incubated in a humid chamber at 37°C for 3 to 5 hours. Consistent results were obtained from all 16 independent experiments, and representative examples are shown.
For reverse zymography, the gel was made as above but with the addition of urokinase (0.8 U/mL of gel). These gels were incubated at 37°C for 4 to 6 hours and visualized under dark-field illumination. The data shown reflect consistent and representative examples of eight independent experiments.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Gene expression by SMCs was examined in carotid arteries, because these vessels, in contrast to the aorta, do not completely reendothelialize after endothelial denudation. This allowed us to examine SMCs at times when they are known to be replicating and migrating (ie, 5 to 8 days after balloon catheter injury) and at a time when these cells have returned to quiescence in the chronically denuded vessel (6 weeks after injury).18 19 We were unable to study SMCs of uninjured arteries with the en face technique, since only the luminal cell layer, which in uninjured arteries is the intact endothelium, can be studied by this technique. Endothelial cells were studied in the rat aorta at various times after total or partial denudation, ranging from 2 hours to 6 weeks (Fig 1
).
En Face In Situ Hybridization
SMCs
Expression
of TPA was not detectable in cross sections from normal
animals (Fig 2a), but strong expression of TPA was seen
in SMCs on the luminal surface of carotid arteries after balloon
injury. The earliest time examined was 5 days after injury, but a
similar result was obtained after 8 days (Fig 2b). Six weeks
after
injury, TPA was still expressed by these cells (Fig 2c),
although the
vast majority of these luminal SMCs were no longer replicating.
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UPA was
not expressed in uninjured arteries (Fig 3a);
however, many luminal SMCs at 5 and 8 days after injury showed
expression of UPA mRNA (Fig 3b). At later times after injury (6
weeks),
only a very few luminal SMCs were positive for UPA mRNA (Fig
3c). The
expression of UPAR was similar to that of UPA in that no expression was
detectable in normal carotid arteries (Fig 4a), and 5
and 8 days after injury, large numbers of luminal SMCs expressed this
transcript (Fig 4b). By 6 weeks, no mRNA for UPAR was detected
in
luminal SMCs (Fig 4c). The hybridization signal seen with the
UPAR
probe was the weakest among all the genes studied, which may explain
our inability to detect this transcript by Northern blot
analysis of total RNA extracted from the entire vessel wall
(data not shown).
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The expression of both PAI-1 and PAI-2 was also
examined in SMCs from
unmanipulated arteries and at various times after injury. PAI-1 mRNA
was not detected in normal vessels (Fig 5a). The
majority of the luminal SMCs at 5 and 8 days expressed PAI-1 (Fig
5b),
but at 6 weeks after injury only a few cells were still positive (Fig
5c). PAI-2 mRNA was not detected in any artery.
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Endothelial Cells
The endothelial monolayers were
examined in the
unmanipulated rat aorta and at various times after injury. Since
defined zones of the aorta were denuded, we were able to examine the
regenerating cells within hours after injury. Control quiescent
endothelial cells did not express TPA (Fig 6a), but within 24
hours, those cells adjacent to the
wound edge showed strong expression (Fig 6b). At both 5 and 8
days
after injury, TPA was expressed at the leading edge of the
endothelial wound and 20 cells back from the wound
edge (Fig 6c). TPA mRNA, however, was not observed in other
areas of
the endothelium, nor was it detected in the regenerated
endothelium of arteries 6 weeks after balloon injury
(Fig 6d). Expression of TPA mRNA was also seen in some
inflammatory
cells adhering to the endothelium (data not shown).
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The mRNA for UPA was
detected in the regenerating
endothelium after 24 hours (Fig 7b) but
was tightly restricted to a few rows of endothelium
directly adjacent to the leading edge. A similar pattern of expression
was observed in the endothelium at 5 and 8 days after
injury (Fig 7c). No UPA expression was seen in the
endothelium 6 weeks after injury (Fig 7d) or in
endothelial cells from control uninjured arteries (Fig 7a).
Adhering inflammatory cells also frequently showed expression of
UPA mRNA (not shown).
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No expression of UPAR mRNA was found in uninjured
aortic
endothelium (Fig 8a), but this
transcript was observed within 24 hours in the leading edge cells.
Expression was restricted to a few rows adjacent to the leading edge at
both 5 and 8 days after injury (Fig 8b). At later times, when
endothelium had stopped regenerating (6 weeks), no
expression was detected (Fig 8c).
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No expression of PAI-1
mRNA was detectable in normal
endothelium (Fig 9a). A marked increase
in PAI-1 expression occurred as early as 2 hours after injury in those
cells adjacent to the leading edge (Fig 9b). Replicating
endothelium at the leading edge at 8 days showed high
levels of PAI-1 mRNA that extended several cell rows into the monolayer
(Fig 9c). Regenerated endothelium at 6 weeks again
showed no detectable levels of PAI-1 expression (Fig 9d).
Expression of
PAI-2 mRNA was not detected at any time in endothelial
cells.
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En Face Zymography
To determine the net balance of the
fibrinolytic activities on the
luminal surface of these rat arteries, en face zymography was used.
With this technique, arteries were cut open longitudinally and placed
(luminal side down) on slides coated with substrate gels containing
casein and plasminogen. Lysis of this substrate was
indicative of net plasmin activity. In injured arteries, at both 1 and
6 weeks the zones covered by luminal SMCs caused marked lysis of the
gel, whereas those areas covered by new endothelium
showed no lysis (Fig 10A and 10C). Control
uninjured
arteries did not lyse the plasminogen gel.
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The antifibrinolytic
activity of the arteries was measured by reverse
zymography. A known amount of urokinase was added to the substrate gel
that would initiate lysis. Inhibition of lysis, presumably by the
action of PAIs, was visualized as a light undigested zone in contrast
to the dark lytic zone seen throughout the remainder of the gel (Fig
10B and 10D). At 1 and 6 weeks after injury,
those denuded areas with
luminal SMCs and no overlying endothelium showed no
inhibition of lysis. Inhibition of gel lysis was observed in those
zones repopulated by new endothelial cells (Fig 10D)
and was also observed in control arteries, which had a confluent
endothelium (Fig 10B and 10D). The resolution
of this
procedure, however, did not allow us to make any judgments on the lytic
activity of those endothelial cells at the leading
edge.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Plasminogen activators, important in the generation of plasmin, have now been shown to be important mediators of cell migration. This is true for metastatic cells, for invading cells during embryonic development, and in wound healing.4 6 7 8 9 20 More recent data show that the expression of UPAR is also important in this process.11 21 The presence of UPAR on the migrating cell and the expression of UPA either by the same cell or by adjacent cells provide an environment in which the latent UPA can bind to its receptor and be activated at a site where it is protected from inactivation by plasmin inhibitors, which are often present in excess concentrations.4 12 The cellular localization of UPAR is also important, since this receptor and its ligand are concentrated in the region of the cell where movement occurs and where, presumably, lysis of the matrix and cell attachment is required.11 22 23 24 25 The present study was focused on the role of plasminogen activators and inhibitors as well as UPAR in the movement of both SMCs and endothelial cells into an arterial wound.
SMC Migration and UPA/UPAR Expression
The presence of
plasminogen activators in
endothelial cells in vitro has been well
documented,3 26 27 28 29
but there are few data with respect to
migrating SMCs, especially in vivo. In part, this is because
measurement of SMC migration is difficult; furthermore, it has been
unclear what role SMC migration plays in the repair of
arterial wounds. We have recently shown that both TPA and
UPA are expressed in rat carotid arteries at early times after
arterial denudation, and an overall increase in plasmin
activity has been detected.12 The upregulation of
plasminogen activators was associated with the
onset of SMC migration into the intima, and inhibition of SMC migration
was linked with a decrease in plasminogen
activators and plasmin generation.12 This
association of plasminogen activators with the
ability of SMCs to migrate to the intima was strengthened by an
experiment by Jackson et al12 in which plasmin
inhibitors blocked SMC migration. In that study, the
plasmin inhibitor tranexamic acid was given to rats
immediately before and after balloon injury, and the number of SMCs
that had migrated into the intima was significantly reduced. In
contrast to our in vivo data, Grainger and
coworkers30 31
have reported that plasmin may have inhibitory effects on
cultured SMCs via its ability to activate TGF-ß1, which acts
as an inhibitor of SMC proliferation in vitro. It is
questionable, however, whether these in vitro data are relevant to the
rat balloon injury model, since infusion of TGF-ß1 into rats after
balloon injury caused an increase in SMC replication.32
Furthermore, using the same model, a neutralizing antibody against
TGF-ß1 inhibited intimal lesion formation.33
In the present study, we examined the expression of plasminogen activators and inhibitors by the luminal cells present in injured arteries using en face in situ hybridization.13 We chose to examine arteries at times when migration and intimal lesion growth are known to occur.18 19 The en face technique was used because it is sensitive and can detect low-abundance mRNA and because it allowed us to sample all those cells on the luminal surface of arteries that were actively migrating. Our results show that those SMCs that migrated onto the luminal surface of the injured arteries express both TPA and UPA. This result agrees with our data obtained from Northern blots in an earlier experiment,12 although in that study we were not able to identify the location of the plasminogen activator–expressing cells.
An interesting further aspect of the present study was the
apparent
coordinated expression of UPA and UPAR. Inactive prourokinase binds to
the UPAR, where it can be activated. The presence of UPA and
its receptor on the same cell serves to regulate cell surface
proteolytic activity and focalizes plasmin generation at a site where
it is protected from the plasma inhibitor
2-antiplasmin.34 35 UPAR has been
shown to
be expressed by invasive cancer cells,36 although these
same cells do not necessarily have to express UPA while maintaining
their metastatic property. Indeed, it now appears that cancer cells
frequently express UPAR while the surrounding stromal cells synthesize
UPA.36 With regard to the vessel wall, expression of UPAR
was recently reported in macrophages and SMCs in
atherosclerotic lesions of rabbit and human arteries.37
Our data show that within days after injury, when SMCs are known to be
migrating into the intima, these cells express both UPA and UPAR,
although it is unclear if the same cell expresses the ligand and the
receptor. The fact that both UPA and UPAR are coordinately expressed
suggests that the same factors may trigger their synthesis. In the
injured artery, both basic FGF-2 and platelet-derived growth
factor significantly increase TPA and UPA activities,38
and both these factors would be present in the injured
artery.8 39 40 Mignatti et
al22 have shown
that FGF-2 also induces UPAR in vascular endothelial
cells. One possibility, therefore, is that after balloon catheter
injury, released FGF-2 from traumatized SMCs is the stimulus for UPA
and UPAR expression. This may explain why UPA and UPAR were noted only
at 5 and 8 days after injury but not after 6 weeks. Thus, the absence
of UPA and UPAR at this later time may be explained by the fact that
there is no FGF-2 in the extracellular pool of these
arteries.41 42
The present study shows that SMCs express PAI-1 at the time when migration occurs; therefore, both plasmin inhibitors and activators are present in these arteries. The finding of PAI-1 expression does not necessarily mean that the inhibitor is dominant, since PAI-1 can exist in an inactive form43 and the relative amounts of active inhibitor and active plasminogen activators cannot be determined by in situ techniques. A more realistic evaluation of the balance of plasminogen factors on the surface of these arteries perhaps may be obtained from the result with en face zymograms. Presumably, in these assays we are detecting the fibrinolytic activity of the arterial surface cells, although it is possible that factors could diffuse to the luminal surface from deep within the arterial wall. The data obtained with this technique suggest that plasmin generation is favored in areas with no endothelium and where SMCs form the luminal surface. Thus, the cells that have migrated from the media and are present in the intima would have the ability to digest a variety of matrix extracellular proteins either by direct lysis by plasmin or indirectly via activation of matrix metalloproteinases.5 In contrast to our findings, Schneiderman et al44 reported that diseased human arteries show an increase in PAI-1 expression and suggested that this may relate to the severity of the atherosclerotic lesions. The rat arteries in the present study are markedly different from human atherosclerotic lesions in that there is neither ulceration nor calcification. Furthermore, the rat lesions in the present study contain mostly SMCs, which may well be different from the mesenchymal-appearing intimal cells that dominate human lesions.
One interesting observation is that at early and late times after injury, SMCs still express TPA. We believe that TPA is also important in SMC migration, but the fact that it is expressed at times when intimal SMCs have long stopped migrating (6 weeks) may suggest an alternative action. TPA is known to be synthesized by endothelial cells, and this activity is normally associated with fibrinolysis.45 One possible function for SMC TPA is to maintain a nonthrombogenic surface. As we have noted in previous work,19 46 47 the surface of these still-denuded arteries is surprisingly nonthrombogenic and devoid of fibrin. Therefore, this property may be attributed to the active fibrinolytic activity of the luminal SMCs. It should be noted, however, that intimal SMCs can also synthesize nitric oxide and prostacyclin, which will undoubtedly contribute to the nonthrombogenicity of this surface.48 49
Endothelial Cell Proliferation and Protease
Expression
The pattern of TPA and UPA expression by the
endothelium in vivo is very similar to data from an
earlier study by Pepper et al3 in which
plasminogen activators, especially UPA, were
found to be expressed at the leading edge of the wounded
endothelial monolayers in vitro. Later work by this
group showed that the endothelial cells at the leading
edge bound 125I-labeled UPA and expressed
UPAR.4 Our data would confirm these findings, since UPA,
UPAR, and TPA were all expressed by endothelial cells
at the wound edge and only at times when endothelial
cells were replicating. Furthermore, when endothelial
cell replication stopped, UPA, UPAR, and TPA were not expressed. These
data would strongly support the hypothesis that plasmin and
plasminogen activators are necessary for in
vivo endothelial cell proliferation over a denuded
surface.
In light of the above data, it was interesting to note that both the control and regenerated endothelium in these arteries did not lyse the casein gel, and antifibrinolytic activity was observed using reverse zymograms. Contrary to normal endothelium, those endothelial cells at the leading edge strongly expressed UPA, UPAR, and TPA, as well as PAI-1 and thus might be expected to possess a net caseinolytic activity. In the gel overlay assay, however, we were not able to discriminate the leading edge endothelial cells from the abutting SMCs, which also express UPA and TPA; therefore, we cannot accurately comment on the fibrinolytic activity of endothelium at these interfaces. Endothelial cells have been shown to express PAI-1,50 and PAI-1 activity has been detected by reverse zymography in confluent endothelial cells in vitro.3 In fact, in vivo studies have shown PAI-1 expression in endothelial cells in both diseased and healthy arteries.43 50 51 Therefore, it would seem that endothelial cells in vivo normally have net antifibrinolytic activity corresponding to undetectable levels of UPA and TPA expression. The balance of proteolytic factors with their inhibitors is regulated in favor of fibrinolysis at sites with active endothelial regeneration where UPA, UPAR, and TPA are expressed.
In summary, a marked expression of UPA, UPAR, TPA, and PAI-1 was observed by in situ hybridization in arterial SMCs that had recently migrated to the intimal surface after balloon injury. After 6 weeks, however, only TPA was still expressed by these cells. En face zymography of these denuded arteries showed lysis of casein gels and no antifibrinolytic activity on reverse zymograms. These data would suggest that plasminogen activators, especially UPA and UPAR, are strongly associated with the migration of SMCs from the media into the intima. Those endothelial cells bordering arterial wounds (5 and 8 days) express UPA, UPAR, PAI-1, and TPA. In particular, UPA, TPA, and UPAR were restricted to those cells adjacent to the wound edge. At a later time after injury (6 weeks) and in normal endothelium, no expression of the plasminogen activator was observed. Zymography revealed no lytic activity associated with confluent endothelium, although a marked antifibrinolytic activity was noted in these same endothelialized areas.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-41103 and HL-03174 and by a Grant-in-Aid from the American Heart Association, with funds contributed in part by the American Heart Association, Alaska Affiliate, Inc. The authors thank Dr Degen for the TPA and UPA probes, Dr Gelehrter for PAI-1, and Dr Grundmann for the PAI-2 probe. The technical assistance of Veronica Poppa is greatly appreciated.
Received September 6, 1995; accepted November 28, 1995.
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