(Circulation. 2001;103:576.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Plasminogen Activator Inhibitor-1 and Its Cofactor Vitronectin Stabilize Arterial Thrombi After Vascular Injury in Mice
From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, Calif.
Correspondence to David J. Loskutoff, PhD, The Scripps Research Institute, Department of Vascular Biology, VB-3, 10550 N Torrey Pines Rd, La Jolla, CA 92037. E-mail loskutof{at}scripps.edu
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Abstract |
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Background—The origin and contribution of plasminogen activator inhibitor-1 (PAI-1) and its cofactor vitronectin (VN) to arterial thrombosis/thrombolysis in vivo is controversial.
Methods and
Results—Ferric chloride was used to induce
carotid artery injury in 97 wild-type (WT), 84 PAI-1-/-, and 84
VN-/- mice. Complete thrombotic occlusion was observed in 70% of
PAI-1-/- mice versus 92% of WT
(P<0.001) and 87% of
VN-/- (P=0.015) mice. In
vessels that occluded, mean times to occlusion were significantly
longer in PAI-1-/- than in WT or VN-/- mice. The initial
thrombotic response of VN-/- mice was similar to that of WT mice,
but their thrombi were unstable and frequently embolized. As a result,
the patency rate of carotid vessels 30 minutes after injury was as high
in VN-/- mice (36%) as in PAI-1-/- mice (which demonstrate
progressive thrombolysis) and significantly higher than that of WT mice
(12%; P=0.013). Histochemical
and reverse transcription–polymerase chain reaction studies revealed
an early upregulation of PAI-1 mRNA and protein expression in the
thrombus and the vessel wall, which persisted for 
1 week. VN protein
also accumulated after injury, but VN mRNA levels remained low at all
times.
Conclusions—PAI-1 and VN participate in the thrombotic response to arterial injury by preventing premature thrombus dissolution and embolization. The accumulation of PAI-1 in the thrombus/vessel wall after injury may result, at least in part, from local synthesis, whereas the VN protein appears to be derived from plasma.
Key Words: carotid arteries • genes • fibrinolysis • embolism
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Introduction |
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Plasminogen activator inhibitor-1 (PAI-1) is the principal physiological inhibitor of both tissue (tPA) and urokinase (uPA) plasminogen activators and is a key regulator of the fibrinolytic system.1 In humans, up to 90% of circulating PAI-1 is contained within platelet
-granules,
and in vitro studies suggest that it may be responsible, at least in
part, for the resistance of platelet-rich arterial thrombi to
thrombolysis.2 3
Clinical studies correlate elevated circulating levels of PAI-1 with an
increased risk of both arterial and venous
thrombosis,4 and mice lacking
PAI-1 are resistant to venous
thrombosis.5 Despite this,
the contribution of PAI-1 to stabilization of arterial thrombi in vivo
remains controversial. For example, some studies reported that the
thrombotic response to arterial injury was not significantly altered in
PAI-1–deficient mice,6
whereas others demonstrated prolonged times to thrombosis in these
mice.7 8 PAI-1 is a
relatively unstable molecule in solution but is stabilized by binding
to plasma-derived vitronectin
(VN).3 9 Thus, VN
may promote thrombosis by localizing active PAI-1 to sites of vascular
injury. VN also may contribute to thrombosis by binding to platelet
integrins.10 11
However, VN-deficient mice demonstrated a significantly enhanced
thrombotic reaction to arterial
injury,12 suggesting that it
may have an antithrombotic role in vivo. These conflicting results
emphasize the need for further studies to define the exact role of
PAI-1 and VN in thrombus formation and dissolution. In addition to their role in thrombosis and thrombolysis, PAI-1 and VN also influence cell migration and the chronic wound-healing response to vascular injury9 10 through interactions with uPA, the uPA receptor, and integrins.9 13 Although PAI-1 appears to participate in the vascular remodeling process in vivo,10 14 its role in injury models that focus on arterial thrombosis and thrombolysis remains to be established. Furthermore, the origin and expression of VN in the vessel wall after arterial injury have not yet been systematically studied.
In this report, we used the FeCl3 model6 15 in gene-inactivated mice to gain further insight into the role of PAI-1 and VN in the response to arterial injury and thrombosis. Our results indicate that PAI-1 and VN both participate in the early stages of the vascular response to injury by stabilizing the initial thrombus and preventing early fibrinolysis and premature embolization. They suggest that the increased PAI-1 in the organizing thrombus and the vessel wall during the remodeling process reflects local synthesis, whereas the increased VN appears to be derived from plasma.
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Methods |
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Experimental Animals
C57BL/6J wild-type (WT) mice were from Jackson Laboratories (Bar Harbor, Me), PAI-1–deficient (PAI-1-/-) mice were from Dr P. Carmeliet (Leuven, Belgium),5 and vitronectin-deficient (VN-/-) mice were from Dr D. Ginsburg (Ann Arbor, Mich).16 Only animals that were the product of
8 backcrosses to the C57BL/6J
genetic background were used. Genotyping of PAI-1-/- and VN-/-
mice was performed as described
previously.16 17
All animal care and experimental procedures complied with the
Guide for the Care and Use of Laboratory
Animals, US Department of Health and Human Services, and
were approved by the Animal Research Committee of the Scripps Research
Institute.
Carotid Injury and Thrombosis
Mice (6 to 8 weeks old) were subjected to carotid
artery injury with 10% ferric chloride
(FeCl3).6 15
Briefly, animals were anesthetized (Metofane, Schering-Plough), the
left carotid artery was dissected, and then a 0.5x1.0-mm strip of
Whatman No. 1 filter paper soaked in 10% FeCl3
solution was applied to the surface of the adventitia for 3 minutes.
This led to the formation of a 2- to 3-mm-long carotid thrombus
(Figure 1
). Carotid blood flow was monitored with a miniature
ultrasound flow probe (0.5VB, Transonic Systems) interfaced with a
flowmeter (model T106, Transonic Systems) and a computerized data
acquisition program (WinDaq, DATAQ Instruments). At the end of the
30-minute flow-monitoring period, some of the mice were killed with an
overdose of the anesthetic for tissue harvest and processing (see
below). For studies at later times, the surgical wound was sutured, the
anesthesia was discontinued, and the mice were returned to their
cages.
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Tissue Harvest and Processing
At various times after injury, anesthetized animals
were perfusion-fixed with 4% zinc formalin as
described.18 The injured
segment of the carotid artery was then excised such that its proximal,
thinner edge corresponded to the proximal edge of the thrombus, whereas
the distal, thicker edge included the carotid bifurcation. This allowed
orientation of the segments for paraffin embedding. The isolated vessel
segments were immersed in 4% zinc formalin for 4 hours, stored in 70%
ethanol, embedded in paraffin blocks, and sectioned at 5-µm thickness
for histological studies.
For gene expression studies, the animals were gently
perfused with normal saline for 5 to 10 minutes. The thrombosed segment
of the vessel was then removed, together with an 
1-mm-long
noninjured, nonthrombosed margin of the carotid vessel on either side.
The tissues were then snap-frozen in liquid nitrogen, and pairs of
arteries were pooled for preparation of total
RNA.
Histological and Immunohistochemical
Studies
Masson’s trichrome reagent was used to localize
fibrin, collagen, smooth muscle cells (SMCs), and platelets in the
vessel after injury with FeCl3, and Perls’ iron
stain was used to show the distribution of iron. Endothelial cells were
identified with a rabbit anti-human/mouse von Willebrand factor (vWF)
primary antibody (Dako; dilution, 1:100); SMCs were studied with a
monoclonal anti–mouse -actin primary antibody (Boehringer Mannheim;
1:20). Migrating and proliferating SMCs were identified by a polyclonal
antibody to mouse vimentin19
(Santa Cruz; 1:50), and tissue-fixed macrophages were detected with a
rat monoclonal CI:A3-1 antibody (BMA; 1:20). Finally, polyclonal rabbit
anti-mouse antibodies were used to detect PAI-1 and VN. All incubation,
blocking, and antigen unmasking steps were carried out as
described.20 Sections
incubated with nonimmune serum instead of the primary antibody served
as negative controls.
Analysis of Gene Expression
Total RNA was extracted from pooled pairs of mouse
carotid arteries with Ultraspec RNA (Biotecx) and chloroform, and
PAI-1, VN, and ß-actin mRNA levels were determined by reverse
transcription–polymerase chain reaction
(RT-PCR),17 21
with 35 cycles used for VN and ß-actin and 30 cycles for PAI-1 and
ß-actin. Aliquots of the PCR reaction (20 µL) were separated by
electrophoresis through a 1.8% agarose gel, and the relative amount of
the target (PAI-1 or VN) or the control (ß-actin) RNA was determined
densitometrically with a computer-based image analyzer (Alpha Imager
2000, Alpha Innotech). Radiolabeled antisense and sense riboprobes for
murine PAI-1 and VN were prepared, and in situ hybridization was
performed17 21
with the pGEM-3Z vector system (Promega) in the presence of
35S-labeled UTP (>1200 Ci/mmol; Amersham).
Hybridized slides were exposed in the dark as indicated, developed, and
counterstained with hematoxylin and eosin. Parallel sections were
hybridized with a sense probe as a control for nonspecific
hybridization.
Statistical Analysis
Continuous variables are represented by mean
values±SD. Differences between genotypes or time points within the
same genotype were tested by ANOVA followed by the Bonferroni
t test for pairs of means.
Qualitative variables (eg, vascular patency rates at specific time
points) were tested by 
2 and Fisher’s
exact test. All statistical tests were 2-sided, with a value of
P<0.05 indicating statistical
significance.
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Results |
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Role of PAI-1 and VN in Thrombosis and Recanalization After Injury
Carotid artery injury (see Figure 1
) was induced in 97 WT, 84 PAI-1-/-, and 84
VN-/- mice, and hemodynamic
(Figure 2), histological, and gene expression studies were
performed. On induction of injury, rapid and complete thrombotic
occlusion occurred in 92% of WT versus 87% of VN-/- mice but in
only 70% of PAI-1-/- mice
(P<0.001 and
P=0.015 versus WT and VN-/-
animals, respectively). In vessels that occluded
(Figure 2B), the mean time to occlusion was significantly
longer in PAI-1-/- versus WT (11.0±3.0 versus 8.7±1.9 minutes;
P<0.001) or versus VN-/-
(9.2±1.8 minutes; P<0.001)
mice. Although no significant differences could be detected between WT
and VN-/- animals at early times, the patency rate of injured
carotid vessels at 30 minutes after injury
(Figure 2C) was as high in VN-/- as in PAI-/- mice
(36%) and significantly higher than that of WT animals (12%;
P=0.013). Patency was slowly
restored in the arteries of all mice, and by 7 to 21 days, there were
no significant differences between the 3 genotypes. Analysis of flow
profiles revealed that injury of WT mice
(Figure 3A) generally resulted in rapid thrombosis without
subsequent recanalization during the first 30 minutes. In contrast,
30% of the vessels of PAI-1-/- mice failed to thrombose
completely but instead underwent progressive thrombolysis over the
30-minute period
(Figure 3B). In the VN-/- mice that occluded, the initial
thrombotic response appeared to be similar to that of the WT animals,
but the thrombi were unstable and repeatedly embolized
(Figure 3C and 3D), leading to early recanalization and
restoration of flow.
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Morphological Changes in the Carotid Artery
After Injury
Figures 4 and 5 display the histochemical changes that occur
in the carotid arteries of WT mice after injury (similar results were
obtained with the PAI-1-/- and VN-/- mice; not shown). For
example, Masson’s trichrome stain
(Figure 4, left column) revealed the formation of an
occlusive, platelet-rich thrombus within 30 minutes after injury,
progressive accumulation of fibrin in the thrombus (dark red) at 24
hours, and organization of the thrombus at 7 days with disruption of
the internal elastic lamina, migration of cells from the medial layers
to the intima, and increased cellularity with marked thickening of the
adventitia. At 3 weeks, the adventitial reaction to injury had
subsided, and the most prominent finding was the presence of a
multilayered neointima.
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; bottom row). Magnification x1000.
By 30 minutes after injury, vWF was diffusely present in the
platelet-rich thrombus but absent in the intima
(Figure 4, middle column). Endothelial denudation persisted
at least until the end of the first week, with reendothelialization
observed by 3 weeks. Perls’ iron stain
(Figure 4, right column) revealed that iron accumulated at
the interface between the thrombus and the vessel wall at 30 minutes
after injury but then was cleared rapidly during the wound-healing
process.
FeCl3 injury resulted in the loss of
staining for both -actin
(Figure 5, top row) and the cytoskeletal protein vimentin
(Figure 5, middle row) within 30 minutes, and few, if any,
intact cells or cell nuclei could be detected in the media or
adventitia. These changes persisted for 24 hours after injury, but by 7
days, marked repopulation of the vessel wall was observed, with some
-actin–positive cells in the neointima and abundant
vimentin-positive cells throughout the vessel wall. These
round/globular cells were tentatively identified as
proliferating/migrating
SMCs.19 Three weeks after
injury, multiple layers of redifferentiated, flat, -actin–positive
SMCs could be detected in the neointima. Although isolated macrophages
were detected in the adventitia 7 days after injury
(Figure 5, bottom row), most had disappeared again by the end
of the third week. Proliferation rates (proliferating cell nuclear
antigen–stained nuclei; Zymed) in the vessel wall decreased from
13±10% to 7.2±8.1% within 24 hours after injury, then increased
dramatically to 55±27% by the end of the first week
(P<0.001 versus uninjured
vessels), and finally returned to preinjury levels by 3 weeks
(5.1±7.7%). The majority of proliferating cells were dedifferentiated
migrating SMCs (see Reference 1919 ; not
shown).
Changes in PAI-1 and VN Gene Expression in
Injured Vessels
RT-PCR revealed relatively low levels of PAI-1 mRNA in
the carotid artery before and 30 minutes after injury
(Figure 6A and 6B). Increased PAI-1 mRNA was detected 6 hours
after injury, however; it remained relatively high from 1 to 7 days and
then returned to baseline levels by 3 weeks. In contrast, little VN
mRNA was detected in the normal vessel, and it did not increase at any
time after injury
(Figure 6C and 6D). In situ hybridization
(Figure 7) revealed that in uninjured vessels and at very
early times (30 minutes) after injury, PAI-1 mRNA could be detected
only within isolated cells (-actin–positive; not shown) of the
media and adventitia. By 6 hours after injury, some intact endothelial
cells (vWF-positive; not shown) and SMCs also began to express PAI-1
mRNA. At 7 days, a strong multifocal signal for PAI-1 mRNA (blue color)
was detected in the organizing thrombus and in all layers of the vessel
wall
(Figures 7 and 8). This pattern was similar to the
distribution of PAI-1 antigen
(Figure 8). The PAI-1 signal was localized predominantly to
proliferating/migrating
SMCs19 in the media and
adventitia. Three weeks after injury, the intensity of the PAI-1 signal
decreased in accordance with the PCR findings.
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Although VN gene expression could not be detected by in situ
hybridization in the carotid wall before (not shown) or after
(Figure 7) injury, VN antigen was readily detected in the
thrombus by 30 minutes after injury (not shown) and in the vessel wall
by 6 hours. It remained in the organizing thrombus and in all layers of
the vessel wall throughout the wound-healing process
(Figure 8). Its diffuse immunosignal, however, was distinctly
different from the multifocal signal for PAI-1 mRNA and
protein.
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Discussion |
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Recent studies6 7 8 12 yield contradictory results with regard to the thrombotic response of PAI-1– and VN-deficient mice to vascular injury and thus raise questions about the true effects of PAI-1 and VN on the balance between thrombosis and fibrinolysis in vivo. The data summarized in this report address these inconsistencies. They demonstrate that PAI-1 and VN are both essential for preventing early fibrinolysis and premature thrombus embolization. For example, arterial injury with FeCl3 led to thrombotic occlusion in a significantly lower proportion of PAI-1-/- (70%) compared with WT (92%) mice (Figure 2
). Moreover, the mean time to thrombosis in vessels
that occluded was significantly longer in PAI-1-/- animals, and
>30% of the PAI-1-/- vessels were patent within 30 minutes
(versus 12% in WT mice). Our results thus confirm and extend the
findings of previous studies that reported enhanced endogenous
thrombolysis in the absence of
PAI-1.7 8 22
VN-deficient mice also exhibited an attenuated thrombotic
response to injury
(Figures 2 and 3). In this case, the proportion of vessels
that initially thrombosed after injury and the mean time to thrombotic
occlusion did not differ significantly versus WT mice
(Figure 2B). A large proportion of the thrombi were unstable,
however, and frequently embolized
(Figure 3C and 3D), leading to a high patency rate 30 minutes
after injury
(Figure 2C). This instability may reflect the absence of the
stabilizing effect of VN on PAI-1 at the site of arterial
injury3 23 or
impaired hemostatic function because of a defect in platelet
aggregation caused by VN
deficiency.10 11
Further studies are needed to clarify this issue.
Our results indicating instability of arterial thrombi in
VN-/- mice differ from those of recent
studies12 showing shortened
times to thrombosis in these mice (ie, 
3.0 minutes). The mouse
strains and protocols used to induce arterial injury in the 2 studies
were similar, suggesting that the differences might be due to
differences in the anesthetics used. To test this hypothesis, we
induced carotid artery injury in WT and VN-/- mice after
anesthetizing the animals with either 120 mg/kg pentobarbital
IP12 or inhaled
methoxyflurane according to our protocol. Baseline carotid blood flow
was lower in both mouse genotypes under pentobarbital compared with
methoxyflurane anesthesia (0.8±0.3 versus 1.5±0.3 mL/min;
P=0.002), and the time to
thrombotic occlusion after injury was significantly shorter (6.7±0.6
versus 9.5±2.3 minutes;
P=0.007). None of the mice,
however, whether anesthetized with metofane or pentobarbital, developed
occlusive carotid thrombi 3.0 minutes after injury. It is possible
that the carotid vessels were handled more vigorously in the earlier
study, and that this, together with the lower flow rate induced by
pentobarbital, contributed to the shortened times to
occlusion.
The mechanisms underlying the formation of
platelet-rich arterial thrombi after application of
FeCl3 to the adventitia of the mouse vessel are
largely unknown. Iron may injure endothelial
cells24 and/or promote
platelet activation and
thrombosis.25 Whether
changes in plasma components, in local gene expression, or in specific
proteins and cells also contribute to the vascular remodeling process
in this model is similarly unknown. These issues also were addressed in
this study. We found that FeCl3 accumulates at
the interface between the thrombus and the vessel wall
(Figure 4), resulting in endothelial denudation and loss of
medial SMCs within 30 minutes. The intima, media, and adventitia
appeared to be acellular at early times after injury (ie, 30 minutes,
24 hours). By 7 days, however, there was a peak in cellular
proliferation
(Figures 4 and 5), particularly in the adventitia, and the
beginning of cell migration into the media and neointima. The vast
majority of the proliferating and migrating cells exhibited
characteristics of dedifferentiated SMCs (so-called synthetic
phenotype).19 By 3 weeks,
reendothelialization, repopulation of the media, and formation of a
multilayered, -actin–positive neointima had occurred, and the
cellular proliferation rate in the vessel wall had returned to
preinjury levels. Thus, the temporal response of cells to
FeCl3 injury in the mouse resembles that
observed in the electrical and the rose bengal injury
models.18 26
Cellular proliferation and migration occur earlier after mechanical
carotid injury,27 probably
because of the absence of severe damage to the medial cells in that
model.
Although PAI-1 gene expression was very low in the wall of
uninjured mouse carotid vessels, it increased significantly early (6
hours) after injury
(Figure 6), consistent with previous reports in the
rat28 and the
rabbit.29 In situ
hybridization studies showed that at 6 hours, local PAI-1 synthesis was
confined to surviving endothelial cells and SMCs in the injured
vascular segment
(Figure 7). By 7 days, however, dedifferentiated
proliferating/migrating SMCs in the organizing thrombus and throughout
the vessel wall appeared to synthesize PAI-1
(Figures 7 and 8). PAI-1 gene expression returned to
background levels when arterial remodeling was complete (by 3 weeks).
The spatial and temporal changes in PAI-1 mRNA and protein expression
observed after injury suggest that the inhibitor may play a role in the
regulation of cell migration and neointima formation in
vivo.30 It remains to be
determined, however, whether the extent or the rate of restenosis in
this model differs significantly between wild-type and PAI-1–deficient
mice. Accelerated neointima formation in PAI-1-/- mice was reported
after electrical injury,14
suggesting that PAI-1 may inhibit, or at least retard, excessive
thickening of the vessel wall and narrowing of the lumen during the
remodeling process. The effects may be different in human
atherothrombosis, however, and in models of experimental injury that
induce a marked thrombotic response in the vessel (ie, the
FeCl3 model). In such instances, earlier and
more complete dissolution of the thrombus in the absence of PAI-1 may
remove an important stimulus for ongoing cell proliferation and
migration and thus actually reduce neointimal thickening.
Finally, our observations revealed an abundance of VN antigen in the thrombus and the vessel wall over the 3-week period after FeCl3-induced injury. The diffuse distribution pattern of VN antigen, the presence of very low levels of VN mRNA, and the absence of upregulation of VN gene expression in the thrombus and vessel wall after injury, however, suggest that the VN protein was derived from plasma and not synthesized locally. This hypothesis is consistent with other reports20 21 31 (see, however, a differing view32 ). As in the case of PAI-1, further studies are necessary to confirm the role of VN in vascular remodeling in vivo. In particular, the possibility that the lack of VN may affect the rate and/or extent of neointima formation after arterial injury with FeCl3 in the mouse needs to be tested.
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Acknowledgments |
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This work was supported by a grant from Boehringer Ingelheim to Dr Konstantinides, a grant from the Deutsche Forschungsgemeinschaft (German Research Association, DFG Scha 808/1-1) to Dr Schäfer, and NIH grants HL-31950 and HL-47819 to Dr Loskutoff. The authors gratefully acknowledge B. Fay and D. Ginsburg (University of Michigan, Ann Arbor, Mich) for helping us to establish the FeCl3 model.
Received May 2, 2000; revision received July 18, 2000; accepted August 4, 2000.
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