© 1999 American Heart Association, Inc.
Thrombosis |
Altered Vascular Injury Responses in Mice Deficient in Protease-Activated Receptor-1
From Drug Discovery, The R.W. Johnson Pharmaceutical Research Institute, Spring House, Pa.
Correspondence to Bruce P. Damiano, Drug Discovery, R.W. Johnson Pharmaceutical Research Institute, Spring House, PA 19477. E-mail bdamiano{at}prius.jnj.com
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Abstract |
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Abstract—Expression of protease-activated receptor-1 (PAR-1), a cell-surface receptor for thrombin, is increased in balloon-injured rat carotid artery and human atherosclerotic tissue. To examine the role of PAR-1 in vascular injury, we compared vascular injury responses in wild-type (WT) and PAR-1–deficient (PAR-1-/-) mice. Arterial injury was induced by inserting a flexible guidewire into the common carotid artery and withdrawing it 6 times with rotation. Bromodeoxyuridine, delivered subcutaneously by osmotic minipump, was used to measure cellular proliferation. Mice were perfusion-fixed at 1, 2, 5, 10, and 14 days after injury. Extensive endothelial damage, mural thrombosis, platelet adherence, and medial smooth muscle cell loss and necrosis were apparent at day 1 in both WT and PAR-1-/- mice. The incidence of thrombosis or platelet deposition in WT and PAR-1-/- mice declined from 100% at day 1 to 25% and 21%, respectively, at 14 days. Endothelial disruption, as assessed by Evan’s blue uptake, was maximum at day 1 and declined by day 14. This apparent endothelial regrowth was similar in WT and PAR-1-/- mice. Significant medial thickening at 14 days after injury was similar in WT (from 22.8±1.7 to 30.7±1.9 µm) and PAR-1-/- (from 23.2±2.1 to 30.5±2.2 µm) mice. Medial area also increased in response to injury but to a lesser extent in PAR-1-/- mice (from 0.0250±0.0044 to 0.0312±0.0047 mm2) than in WT mice (from 0.0266±0.0040 to 0.0398±0.0050 mm2). Neointima was variable and occurred in 6 of 13 WT and 5 of 12 PAR-1-/- mice. However, intimal area tended to be less in PAR-1-/- mice (0.0016±0.0007 mm2) compared with WT mice (0.0082±0.0032 mm2), although this difference did not achieve statistical significance (P=0.06). Cell density was significantly greater in normal carotids from PAR-1-/- (6.4±0.5x103/mm2) compared with WT (4.3±0.8x103/mm2) mice and remained elevated after injury. Vessel and lumen diameters tended to increase in WT mice after injury, whereas vessel diameter was unchanged and lumen diameter actually decreased in PAR-1-/- mice. Cell proliferation in injured carotid arteries was similar in PAR-1-/- and WT mice. These data suggest that PAR-1-/- may play a role in vascular injury responses in this mouse model via possible effects on extracellular matrix regulation.
Key Words: protease-activated receptor • knockout mouse • thrombin • thrombin receptor • cell proliferation
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Introduction |
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Several lines of evidence suggest a significant role for thrombin in vascular injury responses associated with thrombosis, atherosclerosis, and mechanical arterial injury, such as that caused by balloon angioplasty or stent implantation.1 2 3 Concentrations of thrombin at sites of vascular injury are significantly elevated. For example, 5 hours after balloon injury of the rabbit aorta, thrombin activity was 50 fmol · min-1 · cm-2, gradually decreasing to 10 fmol · min-1 · cm-2 at 24 hours after injury.4 These levels were sustained for at least 10 days. In human plasma, thrombin concentrations in the vicinity of a thrombus have been estimated to be as high as 140 nmol/L.5 In addition to evidence for elevated thrombin levels at sites of vascular injury, direct inhibition of thrombin by hirudin has been shown to reduce neointimal thickening induced by balloon angioplasty in normal and hypercholesterolemic rabbits and pigs and in normal rats.6 7 8 9 10 Surprisingly, clinical studies with the direct thrombin inhibitors hirudin11 and hirulog12 and the indirect thrombin inhibitors heparin11 and low-molecular-weight heparin13 have not demonstrated a significant effect on restenosis after angioplasty. However, these thrombin inhibitors were administered during the procedure and for only 3 to 28 days after the procedure. Long-term inhibition of thrombin may be required to produce a significant effect.
Although thrombin’s pivotal role in fibrin polymerization and clot formation contributes to the vascular injury response, activation of specific cell-surface receptors for thrombin may also participate in the vascular injury response. The thrombin receptor, or protease-activated receptor-1 (PAR-1), is a member of a novel family of G protein–coupled receptors activated by proteolytic receptor cleavage.14 15 Receptor activation results in cell signaling and a number of different cellular functions. Within the vasculature, PAR-1 activation causes alteration of vascular tone16 17 18 ; stimulation of vascular smooth muscle proliferation19 ; stimulation of vascular smooth muscle procollagen synthesis20 ; activation of endothelial cells leading to release of nitric oxide,18 endothelin,21 prostaglandins,22 and von Willebrand factor23 ; alteration of adhesion molecule expression24 ; and increased vascular permeability.25 26 Several of these cellular functions, particularly stimulation of vascular smooth muscle proliferation and collagen synthesis, are integral to vascular wound healing. In addition, PAR-1 mRNA and protein expression is upregulated in response to different forms of vascular injury, such as that induced by balloon angioplasty in the rat and baboon27 and in human advanced atherosclerotic plaques.28 29 Thus, elevated thrombin levels at sites of vascular injury can activate the upregulated levels of PAR-1, leading to a number of cellular responses that may participate in the vascular injury response.
Results of recent studies attempting to elucidate the role of PAR-1 in vascular injury responses have been conflicting. In a rabbit arterial injury model, hirudin effectively inhibited neointimal thickening in response to balloon angioplasty, but local administration of PAR-1 antisense oligodeoxynucleotides in vivo did not affect the degree of neointimal thickening in this model.30 In contrast, an antibody to the rat PAR-1 was shown to significantly decrease neointimal formation and PAR-1 mRNA in a rat balloon angioplasty injury model.31 Because PAR-1 does not mediate thrombin-induced platelet aggregation in rats and mice,32 33 the result suggests an important role for a nonplatelet PAR-1 response in vascular injury.
To further elucidate the role of PAR-1 in vascular injury, we evaluated the responses induced by mechanical injury of the carotid artery of mice made deficient in the PAR-1 gene in comparison with age- and weight-matched wild-type (WT) mice.
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Methods |
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PAR-1–Deficient Mice
The gene for the thrombin receptor (PAR-1) was disrupted in mice by homologous recombination as described previously.34 As has been reported previously,34 35 these mice appear phenotypically normal on both gross and histological examinations. However, matings between PAR-1-/- mice occur less frequently and generally produce much smaller litters. The latter is apparently due to partial embryonic lethality at embryonic day 8. Genetically matched WT mice (C57BL/6) were also bred and used for these studies.
Induction of Vascular Injury
All procedures involving the use of animals were performed in
accordance with the Guide for the Care and Use of Laboratory
Animals (1996) and the Animal Care and Use Committee of The R.W.
Johnson Pharmaceutical Research Institute. The left carotid artery was
injured in 95 WT and 95 PAR-1–deficient mice by using a modification
of a previously described procedure.36 The
investigator was unaware of the genotype throughout the
experiment. Mice at least 6 months old and weighing 30 to 35 g
were anesthetized with ketamine/xylazine (80:5 mg/kg
IM). With the use of aseptic techniques, the left carotid artery was
exposed via a midline neck incision, and the carotid bifurcation was
isolated. The external carotid artery was ligated distally, and a
curved, flexible wire (0.45-mm diameter steerable guidewire; Bard) was
introduced into the external carotid artery and advanced to the aortic
arch (
15-mm length). The wire was passed 6 times, with rotation,
along the length of the vessel and then removed. The external carotid
artery was tied off and the incision closed with sutures. Alzet osmotic
minipumps (model 2002, Alza Scientific Products) loaded with
bromodeoxyuridine (BrdU, Sigma Chemical Co) to deliver 25 mg ·
kg-1 · d-1 were
placed subcutaneously via a midback incision. Animals were allowed to
recover in individual cages.
Tissue Collection
At 1, 2, 5, 10, and 14 days after injury, mice were
anesthetized with pentobarbital (100 mg/kg IM), and 50 µL of
5% Evan’s blue solution containing 1000 U/mL heparin was administered
intravenously to demarcate the
deendothelialized area of the carotid artery.
Ten minutes later, the chest was opened, and a 20-gauge needle was
placed in the left ventricle. Mice were perfusion-fixed with 4%
paraformaldehyde in 0.1 mol/L PBS (pH 7.4) at a
constant pressure of 100 mm Hg. The length of blue-stained
carotid artery was measured. The carotid artery was cut transversely at
the center of the blue-stained area. Both carotid halves were fixed in
10% neutral buffered formalin, then placed in PBS, and routinely
processed for paraffin embedding. Four 5-µm-thick sections, 100
µm apart, from the central portion of each vessel half were used for
morphometric analysis. Adjacent sections were processed for
elastin staining or immunohistochemical labeling for BrdU and
PAR-1.
van Gieson Histochemistry
For elastin staining, slides were placed in a solution
containing alcoholic hematoxylin, FeCl3, and
Weigert’s iodine for 10 minutes; rinsed in distilled water; and then
placed into an FeCl3 solution for 3 minutes.
Sections were rinsed in tap water and then in 95% alcohol to remove
the iodine. The slides were then placed into van Gieson’s solution for
3 minutes, rinsed in 95% alcohol, dehydrated, and coverslipped.
Immunohistochemistry
After being dewaxed, the slides were microwaved in Target
solution (Dako), treated with 3%
H2O2 to block
endogenous peroxidase, and incubated with 1N HCl for 30
minutes at 42°C. Slides were then washed in PBS and incubated for 60
minutes with a mouse anti-BrdU biotinylated primary antibody
(anti-BrdU, Zymed) at room temperature. The slides were washed in PBS
and incubated for 30 minutes with peroxidase-conjugated mouse antibody
(avidin horseradish peroxidase–conjugated biotin complex, Vector
Laboratories) at room temperature. After the slides were washed with
PBS, diaminobenzidine tetrahydrochloride (Biomeda) was added as a
chromogen, and the slides were then counterstained with hematoxylin,
dehydrated, and coverslipped. Negative control antibody consisted of
same-species IgG isotype nonimmune serum (Vector Labs).
A rabbit polyclonal antibody to PAR-1, generated by using the carboxyl terminal 10 amino acids of rat PAR-1 (The R.W. Johnson Pharmaceutical Research Institute, La Jolla, Calif), was used to assess the expression of PAR-1 in another set of sections. A biotinylated secondary antibody (goat anti-rabbit), followed by avidin-biotin–horseradish peroxidase complex reagent and 3,3'-diaminobenzidine, was used to detect the primary antibody. Slides were counterstained with hematoxylin.
Morphometry
Morphometric analyses were performed on elastin-stained
tissue sections. For each animal, 8 cross sections from the injured
left carotid and the uninjured right carotid arteries were viewed by
using a Sony CCD video camera attached to a Leitz microscope and
digitized with Image Pro image analysis software (Media
Cybernetics). Representative sections were also
photographed with a conventional camera for presentation in
Figures. For each artery section, the area of the media and any
neointima were measured. A custom program (Phase 3 Imaging
Systems, Glen Mills, Pa) was used with Image Pro to measure the area
and perimeter of the lumen, internal elastic lamina, and external
elastic lamina. Area and average thickness of the media and
neointima were derived from these measurements.
BrdU-labeled and unlabeled nuclei within the media and intima were
counted manually. The investigator performing the analysis was
unaware of the genotype. Statistical analysis consisted
of paired t tests to compare left and right carotid arteries
and unpaired t tests to compare results between
genotypes. Probability values <0.05 were considered
statistically significant.
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Results |
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Responses to Injury at 24 Hours
The wire-injury procedure resulted in significant endothelial denudation and damage or loss of a portion of the medial vascular smooth muscle cells at 24 hours after injury. The latter was indicated by the significant decrease in total nuclei in the injured arteries, expressed as a percent of total nuclei in the corresponding uninjured artery (P<0.001 versus uninjured artery). Acute medial cell loss was comparable in WT and PAR-1-/- mice: 69±9% and 73±4% of the uninjured carotid artery, respectively. Figures 1A
and 1C show examples of uninjured
carotid artery sections from a WT and a
PAR-1-/- mouse, respectively, wherein the
presence of a continuous, normal endothelium in both
can be observed (arrows indicate endothelial nuclei).
Figures 1B and 1D show examples of the vascular injury response
at 24 hours, which consisted of swelling of the media, disruption and
loss of smooth muscle cells, and endothelial damage. In
the particular example from a PAR-1-/- mouse
(Figure 1D), the degree of disruption of vascular smooth muscle
cells and vacuolization were greater than in a WT mouse (Figure 1B). However, when all vessels analyzed at 24 hours were
evaluated, the range of the severity of damage was similar in both
genotypes. The exposed subendothelium had
adherent platelet aggregates visible by light microscopy (Figures 1B and 1D, arrowheads). The degree of platelet deposition
varied between animals, ranging from small platelet aggregates to
extensive multilayered platelet deposition and thrombosis. However,
there were no differences in these characteristics between the 2
genotypes. Fibrin and red blood cells were also sometimes
observed in the media, indicating occasional medial dissection
resulting from the tip of the wire.
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Time Course of Thrombosis and Reendothelialization
At 24 hours after injury, all vessels had some degree of luminal
thrombosis, some of which was occlusive. However, the incidence of
thrombosis (occlusive, nonocclusive, and platelet deposition)
declined from 2 to 14 days after injury (Figure 2, upper panel). The time course of the
incidence of thrombosis was similar in WT and
PAR-1-/- mice. The decline in thrombosis
indicates that mural thrombi were either lysed or consolidated on the
denuded surface of the injured arteries. However, a portion of the
vessels remained completely occluded at day 14. There was no difference
in the rate of occlusive thrombosis in WT and
PAR-1-/- mice at day 14. Animals with occluded
vessels were excluded from morphometric analysis.
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Intravenous Evan’s blue solution administered immediately
before perfusion was used to visualize the proportion of the injured
vessel with disrupted (blue) and intact (white)
endothelium at 1, 2, 5, 10, and 14 days after injury.
Reendothelialization was expressed as the percentage of
the vessel from the aortic arch to the carotid bifurcation with intact
endothelium. At 1 and 2 days after injury, nearly the
entire length of the vessel had disrupted endothelium
(Figure 2
, lower panel). However, from 5 to 14 days after
injury, there was a gradual increase in the percentage of
reendothelialization, reaching a plateau of 80%
reendothelialization. This increase presumably
represents healing and regrowth of the
endothelium over time. The extent or rate of
endothelial regrowth was not different in
PAR-1-/- mice compared with WT mice (Figure 2, lower panel).
Vascular Injury Responses at 14 Days
Figure 3 shows examples of vascular
injury responses in WT mice at 14 days after vascular injury. Left-hand
panels are sections stained for elastin, whereas right-hand panels are
nearly adjacent sections immunolabeled with BrdU. Proliferating cells
that had incorporated BrdU had brown nuclei, whereas nondividing cells
had blue nuclei. Negative control antibody did not produce any
detectable staining (data not shown). Figures 3A and 3B show an
uninjured right carotid artery with normal medial smooth muscle layers,
an intact endothelium, and the absence of BrdU-labeled
nuclei (no cell proliferation). Figures 3C and 3D show a typical
example of medial thickening (arrows) without significant
neointima in an injured carotid artery at 14 days. Medial
thickening was accompanied by a decrease in cell density but an
increase in BrdU-labeled nuclei (arrowheads) in the media. The example
in Figures 3E and 3F are sections from another animal in which
both medial thickening and significant neointimal formation
(arrowheads) were observed. Note the similar decreases in cell density
in the media. BrdU-labeled nuclei were present throughout the
neointima and media.
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Figure 4 shows corresponding examples of
the vascular injury response in PAR-1–deficient mice. Figures 4A and 4B show a normal, uninjured carotid artery that has a
similar appearance to WT arteries. Figures 4C and 4D show an
example of medial thickening with only a thin neointima
(arrowheads). Figures 4E and 4F show an example from another
animal in which medial thickening was accompanied by significant
neointimal formation (arrowheads). In both of these
examples, there was a comparable degree of medial cell loss and an
increase in BrdU-labeled nuclei in the media and
neointima.
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Carotid arteries from both WT and PAR-1–deficient mice were also
subjected to morphometric analysis. The medial areas of the
uninjured right carotid arteries were similar in WT and
PAR-1-/- mice (Table 1, upper portion). The most
consistent aspect of the response to injury at 14 days was an
increase in medial thickness in both WT and
PAR-1-/- arteries. The degree of increase in
thickness was similar in WT and PAR-1-/- mice
(Table 1, upper portion). The medial areas of injured carotid
arteries were also increased in WT and PAR-1-/-
mice, although the degree of increase tended to be less in
PAR-1-/- mice. Thus, increased medial thickness
without a comparable increase in medial area in
PAR-1-/- mice would suggest an inhibition of
compensatory changes in vessel dimensions. Table 1, lower
portion, shows that there was a tendency for vessel and lumen diameters
to increase in response to injury in WT mice. However, vessel diameter
did not change and lumen diameter actually tended to decrease in
response to injury in PAR-1-/- mice. Injury
also induced neointima formation in both groups of mice,
although this response was much less consistent and quite
variable. Nonetheless, intimal area was less in
PAR-1-/- mice, although, this difference
achieved only borderline statistical significance (P=0.06).
Figure 5 summarizes the medial, intimal,
and total area values for individual animals as well as the mean and SE
in the WT and PAR-1-/- groups. Note the wide
range of medial and intimal areas in the WT group and the more narrow
range of areas and lower mean areas in the
PAR-1-/- mice.
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BrdU immunostaining was used to determine the
cumulative number of replicating cells in the vessel wall throughout
the 14 days after injury. There were very few BrdU-labeled cells in the
uninjured vessel, as would be expected. However, the cell density in
normal PAR-1-/- carotid arteries was
significantly greater than in WT carotid arteries (Table 2). The vascular injury procedure caused
significant loss of total nuclei in the media of the injured arteries
compared with the corresponding uninjured artery. This phenomenon was
reflected by the significant decrease in cell density after injury in
both genotypes. The degree of decreased cell density was
comparable in both groups, although cell density remained somewhat
higher in PAR-1-/- mice. The number of
BrdU-labeled cells was greatly increased in the media of injured
arteries. The increase in the percent of BrdU-labeled cells in WT and
PAR-1-/- arteries resulted from significant
increases in cellular proliferation in the media. This increased
proliferative response was similar in both groups. BrdU labeling was
also quantified in the neointima. As might be expected, all
nuclei in the neointima were labeled with BrdU because BrdU
was infused for 14 days. Therefore, there were no differences in the
proportions of BrdU-positive cells between groups. However, the cell
density in the intima of PAR-1–deficient mice was greater than that in
WT mice. In addition, total cell density (media plus
neointima) was elevated in PAR-1–deficient mice compared
with WT mice.
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PAR-1-/- Labeling in Injured Carotid Artery
Figure 6 shows an example of
immunohistochemical labeling of PAR-1 in uninjured and injured mouse
carotid arteries from a WT and a PAR-1-/-
mouse. In WT mice, the PAR-1 label was not apparent in the uninjured
carotid artery (Figure 6B) but was present throughout the
neointima of the injured carotid artery, particularly at
its luminal edge (Figure 6A). Label was absent in both the
uninjured and injured carotid arteries from a
PAR-1-/- mouse (Figures 6C and 6D). This
labeling pattern was confirmed in a total of 2 WT and 2
PAR-1-/- mice.
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Discussion |
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Using mice made deficient in PAR-1, a novel cellular receptor activated by thrombin,14 we investigated the role of PAR-1 in the response to vascular injury induced by mechanical damage to the lumen of the carotid artery. Our results suggest that the absence of PAR-1 alters the vascular injury response in this mouse model. Neointimal thickening was blunted in PAR-1–deficient mice. Although medial thickening in response to injury was comparable in WT and PAR-1–deficient mice, the corresponding increase in total medial area in WT mice tended to be less in PAR-1–deficient mice. In addition, although vessel and lumen diameters tended to increase after injury, vessel diameter was unchanged and lumen diameter actually decreased in PAR-1–deficient mice. Moreover, the reduction in lumen diameter in PAR-1–deficient mice occurred despite the fact that neointimal thickening was less. This profile of vessel wall changes is consistent with a role for PAR-1 in positive remodeling after vascular injury.
The vascular injury response comprises several cellular processes,
including thrombosis, platelet adhesion, cell migration, cell
proliferation, and matrix remodeling. We evaluated and quantified
several components of the injury response in our model. Thrombosis,
either occlusive or nonocclusive, and histological
evidence of platelet deposition were prominent within 24 hours of
injury, as is found after vascular injury in other animal
models37 38 and in humans.39 The incidence of
thrombosis and platelet deposition was not different in WT and
PAR-1–deficient mice. This finding was not unexpected, since
thrombin-induced platelet activation in mice and coagulation in
general do not involve PAR-1.34 35 Early occlusive and
nonocclusive thrombosis resolved over time. The rate of resolution was
not different in WT and PAR-1–deficient mice. The occlusion rate at 14
days was 20% and did not differ in WT and PAR-1–deficient mice.
Therefore, the influence of thrombosis and associated mediators, such
as thrombin and platelet-derived growth factor, was equivalent in
the 2 genotypes and thus, does not explain the observed
differences in vascular injury response. The rate and extent of
endothelial regrowth and the return of barrier function
after injury are other critical determinants of the vascular injury
response. Rapid endothelial regrowth has been shown to
limit neointimal formation.40
Endothelial regrowth, which presumably reflects both
cell migration and proliferation, was unaffected by the absence of
PAR-1, indicating that differences in the rate of
endothelial regrowth do not account for the differences
in vascular injury response. Proliferation plays a critical role in the
vascular injury response and in neointimal thickening.
However, the proportion of proliferating cells in the media, as
assessed by BrdU labeling over the entire 14 days after injury, was not
different in WT and PAR-1–deficient mice. All cells in the
neointima of WT and PAR-1–deficient mice were BrdU
labeled, as would be expected given the continuous 14-day BrdU infusion
that was used. Thus, true proliferation rates in the intima could not
be determined with this BrdU labeling protocol. However, based on
proliferation rates in the media, the absence of PAR-1 does not appear
to affect vascular smooth muscle proliferation, suggesting that the
effects of PAR-1 deficiency on vascular injury responses are not a
result of altered proliferation.
PAR-1 deficiency may also affect tissue remodeling, including cell growth and matrix degradation and synthesis. Normal vessels from PAR-1–deficient mice had a significantly greater smooth muscle cell density compared with that in WT mice. Even though vascular injury decreased cell density in the media of both genotypes, cell density in the PAR-1–deficient mice remained greater than that in WT mice. Cell density in the neointima was also greater in PAR-1–deficient mice. The increased cell density in normal vessels may have resulted from reduced matrix synthesis and secretion during development and/or reduced cell growth rate and volume. Thrombin, acting via PAR-1, has been shown to stimulate collagen synthesis in vascular smooth muscle cells,20 activate matrix metalloproteinases, and increase their synthesis in endothelial cells.41 Thus, thrombin and PAR-1 appear to be involved in matrix remodeling. The greater cell density associated with reduced neointimal area in PAR-1–deficient mice suggests compromised extracellular matrix formation during the injury response. In addition, positive remodeling associated with vascular injury must also involve matrix breakdown, synthesis, and secretion. The tendency for inhibition of positive remodeling in PAR-1–deficient mice after injury may also reflect a deficiency in matrix synthesis and secretion, matrix degradation, and/or alteration in the control of cell growth.
A great deal of evidence implicates thrombin as well as PAR-1 as pivotal mediators of the vascular injury response.1 2 3 Besides the identified functional roles of thrombin and PAR-1 in platelet aggregation, cell proliferation, and matrix synthesis, both thrombin and PAR-1 expression and message appear to be upregulated after vascular injury in animal models27 and in human atherosclerotic vessels.28 29 We have shown that PAR-1 is also upregulated in the mouse carotid artery in response to vascular injury. Inhibition of thrombin’s enzymatic function has been shown to reduce vascular injury response in various animal models.6 7 8 9 10 However, thrombin’s actions involve both pure enzymatic functions as well as activation of its cellular receptor, PAR-1. Therefore, results of studies with thrombin inhibitors do not necessarily prove a role for PAR-1 in vascular injury. The lack of small-molecule PAR-1 antagonists has made it difficult to define a specific role for PAR-1 in vascular injury. Nonetheless, other approaches have been employed to interfere with PAR-1 activation. In a recent study, antisense oligodeoxynucleotide to PAR-1 was used to assess the role of PAR-1 in a rabbit model of vascular injury.30 Local administration of this antisense oligodeoxynucleotide appeared to inhibit expression of PAR-1 mRNA in vivo. However, there was no effect on the degree of intimal thickening. In the same model, hirudin, a direct inhibitor of thrombin, significantly reduced the intimal thickening response.30 Thus, the authors concluded that although thrombin may be involved in the injury response, it did not appear that PAR-1 was involved in this animal model. However, in another study in a rat model, an antibody specific for PAR-1 was shown to inhibit the intimal thickening response at antibody concentrations that effectively inhibited thrombin-induced vascular smooth muscle proliferation in vitro.31 Hirudin was also effective in this model. These results support a role for thrombin and PAR-1 in the vascular injury response in a rat model.
Differences in the apparent participation of PAR-1 in vascular injury responses may clearly result from species and model differences. For example, although PAR-1 mediates thrombin-induced platelet aggregation in humans,14 PAR-1 is not expressed in mouse, rat, and rabbit platelets and thus, does not mediate thrombin-induced platelet aggregation in these species.32 33 The response to thrombin in mouse and rat platelets appears to be mediated by PAR-3 and PAR-4,42 43 2 distinct protease-activated thrombin receptors. Because platelet adhesion, aggregation, and degranulation are important components of the vascular injury process,44 45 these platelet-dependent processes would not be affected in PAR-1–deficient mice. Therefore, our mouse model does not assess the contribution of thrombin-induced platelet aggregation to the vascular injury response. Model-dependent differences in injury responses are likely. Whereas balloon-injury techniques used in rats and rabbits induce a uniform stretch and circumferential denudation, the wire injury does not induce uniform stretch and causes a more focal luminal damage. The resultant injury response may be quite different from the response to balloon angioplasty in rabbits and rats or the process of restenosis in patients with atherosclerosis undergoing balloon angioplasty. Despite these differences, the mouse model does involve several fundamental components of vascular wound repair that are common to this type of injury response in humans. Unfortunately, the focal nature of the lesion, coupled with the relatively thin mouse carotid artery, resulted in great variability, particularly in the incidence of intimal thickening. This variability limited the sensitivity of the model to detect changes in the injury response. Nonetheless, several trends for differences in various aspects of the injury response in PAR-1–deficient mice, taken together, suggest a role for PAR-1 in vascular injury.
Findings in transgenically manipulated mice, in which the gene for a single protein has been deleted, must be interpreted with caution, since cellular and/or physiological changes compensating for the absent protein may occur. Partial embryonic survival in PAR-1–deficient mice may involve such a compensation. In addition, the accentuated cellular response to platelet-derived growth factor in fibroblasts from PAR-1–deficient mice also represents a secondary compensation that may mask the absence of PAR-1.34 The increased vascular smooth muscle cell density in PAR-1–deficient mice, which may be a consequence of the lack of PAR-1 during development, will tend to alter the vascular injury response. Thus, the apparent inhibition of positive remodeling in PAR-1–deficient mice may be a consequence of the higher baseline cell density rather than the absence of PAR-1. Finally, the response to vascular injury involves other important factors besides PAR-1. Many of these factors and pathways are multiply redundant and could compensate for PAR-1’s absence.
In summary, our results suggest that in this mouse model, PAR-1 may play a role in regulation of vascular matrix formation and remodeling associated with vascular injury. Determination of the ultimate significance of PAR-1 in the vascular injury response will require further study, including identification and application of small-molecule PAR-1 antagonists both in animal models and eventually in humans.
Received November 30, 1998; accepted July 8, 1999.
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