(Circulation. 1997;96:4333-4342.)
© 1997 American Heart Association, Inc.
Articles |
Remodeling and Neointimal Formation in the Carotid Artery of Normal and P-Selectin–Deficient Mice
From Cardiovascular Pharmacology, Pharmacia and Upjohn, Inc, Kalamazoo, Mich, and Department of Surgery, Maine Medical Center Research Institute, South Portland (V.L.). Dr. Kumar is currently at Preclinical R & D Genetics Institute, Andover, Me.
Correspondence to Ronald J. Shebuski, PhD, Pharmacia and Upjohn, Inc, 301 Henrietta St, Kalamazoo, MI 49007. E-mail akumar{at}genetics.com
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Background Inflammatory reactions such as leukocyte activation with platelet adherence and release of inflammatory mediators occur after percutaneous transluminal coronary angioplasty and may play a role in restenosis. Vascular remodeling with neointimal formation was studied in normal C57Bl/J6 and P-selectin–deficient mice.
Methods and Results The left common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, left carotids and contralateral controls were snap-frozen. Computer-aided morphometry was performed to measure ratios of neointimal to medial area (NI/M) in 10 sections per animal as a measure of the thickness of the neointimal lesion. For normal mice, NI/M was 1.13±0.2 (n=20), whereas NI/M was reduced by 76% to 0.27±0.1 (n=19) in P-selectin knockout mice. Vascular constriction (as measured by the length of external elastic lamina) was the same in both groups, but the circumference of the lumen in knockout mice was 26% larger. Also, normal and P-selectin–deficient mice were killed at 3 and 7 days after ligation (n=6 for each group per time point). Histological staining and immunostaining for CD45 showed no inflammatory cell presence in P-selectin knockout mice. However, in normal mice, leukocyte infiltration was observed in the adventitia, media, and developing neointima. Also, P-selectin immunostaining was observed in media and developing neointima of normal mice.
Conclusions These data suggest that P-selectin is involved in processes leading to cell migration and proliferation associated with vascular remodeling, presumably by mediating leukocyte recruitment and the interaction between platelets and leukocytes.
Key Words: restenosis • immunohistochemistry • remodeling • cells
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Introduction |
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Restenosis is one of the major limitations of PTCA and can be thought of as a combination of neointimal formation and arterial remodeling in response to balloon injury. Although intimal hyperplasia has been described as the "pathological hallmark of restenosis after angioplasty,"1 it may not be the primary mechanism by which restenosis occurs. Vascular remodeling has a significant impact on chronic lumen area2,3 and may be responsible for 50% to 90% of late luminal area loss.4–7
Remodeling is an adaptive process that occurs in response to chronic changes in hemodynamic conditions8,9 and involves changes in many processes, such as cell growth, cell death, cell migration, and changes in extracellular matrix composition, that lead to a compensatory adjustment in vessel diameter and lumen area. In the context of restenosis after balloon angioplasty, vascular remodeling refers to loss of lumen area by a combination of reduction in vessel diameter and neointimal thickening. The exact mechanism of arterial remodeling remains to be fully understood and may involve growth factors, vasoactive agents, and matrix modulators. Endothelial cells play an important role in sensing changes in mechanical and biochemical forces9; however, the time to endothelial regrowth after angioplasty in humans is unknown. The blood vessel essentially is thought to remodel itself in response to long-term changes in flow, such that the lumen area is modified to maintain a predetermined level of shear stress.10
P-selectin is a membrane glycoprotein contained within
platelet 
-granules11,12 and Weibel-Palade
bodies of endothelial cells13,14
that is rapidly mobilized to the plasma membrane on cell activation and
granule secretion. It is a member of the selectin family, which also
includes E-selectin and L-selectin. Selectins have been implicated in
mediating transient interactions between endothelial
cells and leukocytes in what is known as leukocyte "rolling,"
generally believed to be the prerequisite for firm
adhesion.15,16 In addition, P-selectin has also
been reported to mediate adherence of activated platelets
to monocytes and neutrophils17,18 via its
carbohydrate ligands sialyl LewisX and P-selectin
glycoprotein ligand-1.19
Previous studies suggest that inflammatory reactions and platelet accumulation occur after PTCA. Activation of granulocytes20,21 and neutrophils22 after coronary angioplasty in humans provides evidence that these cells may be important in the restenotic process. Marmur et al1 reported that thrombin is generated in human coronary arteries after PTCA. Thrombin-activated platelets adhere to monocytes and neutrophils via P-selectin.17,18 Recently, Mickelson et al23 reported that leukocyte activation with platelet adherence occurs after coronary angioplasty and that the magnitude of leukocyte activation and platelet adherence was higher in patients experiencing late clinical events. In addition, higher plasma P-selectin levels have been reported in patients with restenosis within 6 months of PTCA.24
Platelet-leukocyte interaction is of considerable pathophysiological interest because it not only targets both cell types to appropriate sites of inflammation and/or hemostasis but also causes functional alteration in these cells. For example, neutrophil-platelet interaction has been associated with neutrophil activation, adherence, and aggregation.25,26 Metabolic cooperation, such as the transcellular metabolism of lipid intermediates from one cell type by the other,27,28 may be important in inflammation, thrombogenesis, and wound healing. P-selectin–mediated binding to monocytes may also induce tissue factor expression29,30 and hence play a role in thrombogenesis.31
We recently established a model for studying vascular remodeling in the mouse.32 Interruption of flow caused by ligating the left common carotid artery just proximal to the carotid bifurcation caused an 80% reduction in lumen area by a combination of intimal hyperplasia together with decreased vessel diameter. This model has the potential to identify molecules contributing to the remodeling process in vivo by studying animals carrying targeted disruption of genes or expressing transgenes. In this study, neointimal lesion formation in normal C57Bl/J6 mice and P-selectin knockout mice was compared to investigate the role of P-selectin in the restenotic process.
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Methods |
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All animals used in this study were handled in compliance with the Guide for the Care and Use of Laboratory Animals, 1996, a publication of the National Research Council, National Academy Press, Washington, DC. All experiments were performed in an American Association for Accreditation of Laboratory Animal Care–approved laboratory according to protocols that were reviewed and approved by the Institutional Animal Care and Use Committee.
Animal Procedures
Mouse Carotid Ligation Model
Adult male C57Bl/J6 mice (n=20) and P-selectin–deficient mice
(n=20) were used in the carotid ligation model. P-selectin knockout
mice, generated on the C57Bl/J6 strain, were provided by Transgenics,
Pharmacia & Upjohn, Inc. Briefly, the animals were anesthetized
with a solution of ketamine (80 mg/kg body wt; Fort Dodge
Laboratories, Inc) and xylazine (5 mg/kg; Lloyd Laboratories) injected
intraperitoneally. The left common carotid artery
was exposed through a small midline incision in the neck. The artery
was completely ligated just proximal to the carotid bifurcation to
disrupt blood flow. The animals were allowed to recover for 4 weeks.
All animals except one P-selectin knockout mouse recovered. The animals
were then reanesthetized as described above, and 50 µL of a
5% solution of Evans blue dye (Sigma Chemical Co) in Ringer's
solution (Baxter Healthcare Corp) was injected into the renal vein and
allowed to circulate for 1 minute. For each animal, after perfusion
fixation under physiological pressure with
formaldehyde solution (10% vol/vol in aqueous phosphate buffer) as
previously described,33 a 5-mm segment of the
left carotid just proximal to the suture and a similar segment of the
right contralateral control artery were excised.
Perfusion-fixed artery segments were placed (ligated end down for left carotid segments) in Tissue-Tek O.C.T. embedding medium (Miles Inc), snap-frozen at -160°C in liquid nitrogen–cooled isopentane (Baxter Scientific), and stored at -84°C. Fresh-frozen samples were sectioned on a Leitz cryostat and placed on Fro-Pen–coated (Zymed Laboratories Inc) ProbeOn slides (Fisher Scientific) for immunohistochemical analysis and Fro-Pen–coated microscope slides for histological staining. Normal C57Bl/J6 and P-selectin knockout mice were also killed at days 3 and 7 (6 of each at both time points) after carotid artery ligation and tissue samples were analyzed for possible early involvement of inflammatory cells.
Bleeding Time Measurements
Determinations of bleeding times for 25 each of the C57 Bl/J6
and P-selectin knockout mice were made as previously
described.34,35 Briefly, conscious mice were held
in a restrainer, and a distal 2-mm segment of the tail was transected
with a disposable surgical blade. The tail was quickly immersed in a
100-mL beaker of 0.9% isotonic saline at 37°C. Bleeding time was
measured from the moment the tip of the tail was severed until bleeding
ceased completely and there was no rebleeding within 30 seconds, up to
a maximum observation period of 10 minutes. Those animals for which
bleeding did not cease in this period were assigned a bleeding time of
10 minutes.
Complete Blood Counts
Normal C57Bl/J6 and P-selectin–deficient mice were
anesthetized as above (n=10 each), and blood samples were drawn
by cardiac puncture into EDTA-containing Microtainer tubes
(Becton-Dickinson). These samples were analyzed by Clinical
Research, Pharmacia & Upjohn, Inc, and complete blood counts and
differential leukocyte counts were reported. Briefly, complete blood
and differential leukocyte counts were performed in a Bayer Technicon
H-1 loaded with species-specific software. This apparatus
uses fixation followed by flow-cytometric light scattering to determine
parameters such as red cell count, size, hemoglobin
content, and platelet count and volume in a laser-based optics
channel. The peroxidase method was used for leukocyte counts after red
cell lysis and fixing of white blood cells and intracellular enzymes.
Differential leukocyte counts were performed after cytochemical
staining at the sites of peroxidase activity. The final counts were
obtained by a combination of light scatter, peroxidase staining, and
nuclear complexity. Blood smears were examined to check cell
morphology. The automated Technicon system was frequently calibrated by
manual hemocytometric counting after red cell lysis.
Immunohistochemistry
P-Selectin Immunostaining
Representative cryo-step sections (50 µm
apart/30 per animal) from normal and P-selectin knockout mice at 3, 7,
and 28 days after ligation were immunohistochemically assessed for
P-selectin antigen expression with an indirect immunoenzymatic
ultrastreptavidin detection method (Signet Laboratories). Briefly,
tissue cryosections were postfixed in cold 10% formalin (4°C) for 10
minutes. Endogenous peroxidase activity was blocked by
0.3% (vol/vol) H2O2 in
methanol for 10 minutes. An avidin/biotin blocking kit (Vector Labs,
Inc.) was also applied to block nonspecific binding, followed by
preincubation in 5% normal goat serum for 20 minutes. The primary
polyclonal P-selectin antibody (PharMingen) or an appropriate nonimmune
rabbit IgG control antibody (Sigma ImmunoChemical) was applied for 30
minutes, followed by application of a 1:2 diluent of biotinylated
secondary antibody for 20 minutes. An ultrastreptavidin horseradish
peroxidase–conjugated labeling complex was applied for 20 minutes.
P-selectin antigen expression was visualized with a DAB chromogen
substrate. Sections were counterstained with Mayer's hematoxylin
(Sigma Chemical Co) and mounted with Crystal Mount (Fisher Biotech) for
light microscopic analysis. Confirmation of method specificity
was achieved by PBS buffer substitution of primary and secondary
antibodies or staining with DAB substrate only.
CD45 Immunostaining
To further characterize involvement of inflammatory leukocytes
at days 3 and 7 after ligation, CD45 immunohistochemistry was conducted
on 30 adjacent cryostat step sections. CD45-positive cells were
immunolocalized by incubation with a rat monoclonal antibody against
CD45 (PharMingen) (1.25 µg/mL) overnight (4°C) followed by
application of a biotinylated mouse anti-rat secondary antibody
(Jackson ImmunoResearch) (5 µg/mL) for 30 minutes. Detection of CD45
was completed with a DAB chromogen substrate that produces a brown cell
surface stain on CD45 positive cells.
Histopathology
Adjacent cryostat step sections (50 µm apart) were also
stained with H&E for morphometric and light microscopic evaluation of
3- and 7-day (n=6 for both groups per time point) and 4-week (n=20 for
normal and n=19 for knockout mice) lesion formation after ligation. To
better identify infiltrating inflammatory cells, additional 3- and
7-day samples were embedded in paraffin, and step sections were stained
with H&E to achieve superior morphology.
Morphometry
The extent of neointimal proliferation was
quantified by measuring the area (µm2) of the
neointima and media for 10 H&E–stained cross sections of
each left carotid artery (from 20 normal and 19 P-selectin knockout
mice). Typically, 30 to 40 H&E–stained sections, 50 µm apart,
were obtained from each animal. To select the 10 sections to be
measured, the sections from an animal were divided into five nearly
equal groups. All sections were examined under a Jenaval
photomicroscope (The Microscope Company), and two sections were picked
from each group. This was to ensure that the entire length of the
arterial sample could be used in the analysis,
especially because the thickness of the lesion varied along the length
of the artery. Also, all sections were within 5 mm of the ligation
site, because only a 5-mm segment had been excised in the first place.
The Optimas analysis software (Bioscan, Inc) and a Microcomp
image analysis system (The Microscope Company) was used to
measure areas enclosed by the EEL, IEL, and the vessel lumen. The
medial area was calculated by subtracting the area defined by the IEL
from the area defined by the EEL. The neointimal area was
determined by subtracting lumen area from the area defined by the IEL.
In addition, the length of the EEL (in micrometers) was
determined in both groups as a measure of the vascular constriction.
The circumference of the IEL and lumen (in micrometers)
were also measured. These comparisons were made on the basis of length
measurements, which are less sensitive to changes due to collapse of
sections during processing.
Data Analysis
Data from morphometric analyses were reported as an
average NI/M ratio and circumference of EEL and lumen (in
micrometers) for each animal, which were measures of the
thickness of the neointimal lesion, vascular constriction,
and lumen area, respectively. Values were averaged for the two groups
of mice: normal C57Bl/J6 and P-selectin knockout. Bleeding time was
reported in minutes. All data are reported as mean±SEM for the two
groups of mice. Hypothesis testing on the means of the two groups was
performed with unpaired Student's t tests, and probability
values were reported. Statistical significance was judged at
P
.05.
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Comparison of Lesion Formation in Normal C57Bl/J6 and P-Selectin–Deficient Mice
For normal mice 4 weeks after ligation, the average NI/M ratio calculated for the ligated left carotid arteries by measurement of 10 histological sections per animal was 1.129±0.15 (n=20, Fig 1a
). Intimal hyperplasia was maximal
closest to the ligation site and decreased in thickness in the
direction of the aortic arch. As also shown in Fig 1a, for
P-selectin–deficient mice, NI/M was 0.270±0.07 (n=19). Compared with
normal mice, neointimal thickness in P-selectin knockout
mice was decreased by 76% (P<.001). We established earlier
that in this model, lumen area is reduced by a combination of
neointimal formation and reduction in vessel
area.32 Fig 1b shows that there was no difference
in the average circumference of EEL in normal and knockout mice. Hence,
both groups exhibit a similar degree of vascular constriction in
response to carotid artery ligation. The length of the IEL was the same
in both groups too (data not shown), suggesting that medial area was
not different. The circumference of the lumen in P-selectin knockout
mice was 26% larger than that of normal mice (P=.003, Fig 1b).
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Fig 2a shows a
histological section from the right control artery of a
normal C57Bl/J6 mouse. Fig 2b is a representative
section from the left ligated artery of the same mouse as in Fig 2a. As
is clear from Fig 2a and 2b, there is neointimal formation
in response to interruption of flow in this model in normal C57Bl/J6
mice. Similarly, Fig 2c is a typical right control artery from a
P-selectin knockout mouse. Fig 2d is a section from the left artery of
the same knockout mouse as in Fig 2c. Fig 2c and 2d, along with Fig 1
, shows that neointimal formation is clearly reduced in
response to flow interruption in P-selectin knockout mice.
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Role of Inflammatory Cells in Lesion Formation
H&E–stained paraffin-embedded carotid sections from normal and
P-selectin knockout mice at 3 and 7 days after ligation (n=6 for each
group at each time point) are shown in Fig 3. Evidence of inflammatory leukocyte
recruitment was visible in normal mouse sections, around the lumen and
in the adventitia, at day 3 (Fig 3a). By day 7, inflammatory cells were
in the developing neointima, media, and adventitia (Fig 3c). In P-selectin knockout mice, no inflammatory cells were observed
either at day 3 (Fig 3b) or day 7 (Fig 3d) after ligation. To better
identify inflammatory leukocytes, immunostaining for
the common leukocyte antigen CD45 was performed on frozen sections from
normal and P-selectin knockout mice 3 and 7 days after ligation.
CD45-positive leukocytes, indicated by a brown surface stain in Fig 4, were observed in the same pattern as
that described for Fig 3 above. Again, P-selectin knockout mice had no
evidence of inflammatory cell recruitment either at day 3 (Fig 4b) or
at day 7 (Fig 4d).
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P-selectin immunostaining on frozen sections taken from
animals killed 3 and 7 days after ligation is shown in Fig 5 for one each of the normal and
P-selectin knockout mice. Fig 5a shows a section from a normal mouse at
day 3. Some P-selectin staining (indicated in brown) is visible around
the lumen, probably derived from endothelial cells and
adhering platelets. Staining is absent in the P-selectin knockout
mouse section, as shown in Fig 5b. By day 7, P-selectin
immunostain for the normal mouse was more intense and
extended not only around the lumen but also into the developing
neointima and media (Fig 5c). Again, there was no staining
in the P-selectin knockout mouse at day 7, as shown in Fig 5d.
P-selectin immunostaining for normal animals was absent
at 4 weeks after ligation (data not shown).
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Bleeding Time Measurements
The average time for bleeding to cease after the tip of the tail
was severed in normal mice was 2.33±0.6 minutes (n=25); for P-selectin
knockout mice (n=25), it was 2.06±0.38 minutes. Hence, bleeding times
for normal and knockout mice were not different (P=.70).
Bleeding time measurements were made according to the same protocol as
Subramaniam et al35; however, unlike the findings
in the present work, these investigators reported a 40% increase
in the bleeding time for P-selectin knockout mice compared with normal
mice. This might be related to the fact that in their study,
Subramaniam et al used P-selectin knockout mice on a 129Sv/C57BL
background, whereas in the present study, knockout mice were C57BL
and were confirmed homozygous, as were the normal animals.
Complete Blood Counts
Complete blood counts and differential leukocyte counts were
performed on blood samples from normal C57Bl/J6 and
P-selectin–deficient mice (n=10 each). A twofold to threefold increase
in basal neutrophil counts in P-selectin–deficient animals compared
with normal wild-type animals has been reported
previously.36 Also, no difference in the total
peripheral leukocyte and platelet counts was reported
in either group of animals. In this study, however, total leukocyte
count in P-selectin knockout mice was found to be 49% higher than in
normal mice (4.81±0.5x103 versus
3.22±0.7x103/µL; n=10, P=.05). The
percentage of leukocytes that were neutrophils, lymphocytes, and
monocytes was the same in both cases (32%, 62%, and 1%,
respectively). Therefore, total counts of neutrophils, lymphocytes, and
monocytes were nearly 50% higher in the P-selectin knockout mice than
in normal mice, probably as a result of increased half-life and/or
reduced margination. Also, the platelet count in
P-selectin–deficient mice was found to be 18% lower than in normal
mice (1084.6±20.8x103 versus
1317.2±103.3x103/µL; P=.04).
Again, these differences from previous reports in blood counts may be a
result of differences in the genetic background of the mice
studied.
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Discussion |
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Coronary restenosis remains a complex pathophysiological problem that continues to defy preventive pharmacological therapy. Processes such as smooth muscle cell migration and proliferation, extracellular matrix synthesis, and mural thrombosis, among others, are posited to contribute to restenosis after PTCA. However, a variety of agents targeting these processes have been largely unsuccessful in clinical trials despite positive effects in animal models of neointimal proliferation. In some of the more promising studies, neointimal hyperplasia in rabbit carotid arteries after balloon injury was reduced by the administration of a small peptide antagonist, GpenGRGDSPCA, which blocked the
Vß3 integrin,
implicated in smooth muscle cell migration.37
Also, a monoclonal antibody directed against the platelet GP
IIb/IIIa (IIbß3)
receptor reduced clinical restenosis, as assessed by composite
end-point analysis (death, myocardial infarction, or repeat
revascularization).38 This
was, however, associated with serious bleeding complications. Although the use of animal models is not without limitations, it has contributed immensely to the understanding of the process of restenosis. It has now become possible to effectively define appropriate molecular targets by studying animals (usually mice) carrying targeted disruption of genes (knockout mice) or expressing transgenes (transgenic mice). We developed a murine model of vascular remodeling in which a disruption of the flow field was created in the left common carotid artery.32 During the 4 weeks that these animals were allowed to recover, these altered shear stress conditions caused the vessel to "remodel" and shrink in luminal area because of reduction in vessel diameter and neointima formation. The proliferative response of smooth muscle cells may also be stimulated by the increase in arterial wall tension39–41 that occurs proximal to the ligating suture. In this work, this model was used in normal C57Bl/J6 and P-selectin knockout mice to investigate the role of P-selectin in the restenotic process.
The NI/M was reduced by 76% in P-selectin knockout mice compared with
normal mice, and the circumference of the lumen was found to be 26%
larger (Fig 1). Fig 2 shows the dramatic difference in lesion thickness
between the two groups. Clearly, P-selectin is involved in the
processes leading to cell proliferation associated with vascular
remodeling, much like that which occurs after balloon injury. These
data also strongly suggest the early involvement of inflammatory cells
in mediating this effect, because leukocytes were observed in the
adventitia, media, and developing neointima of normal mouse
carotid sections at 3 and 7 days after ligation, but not in P-selectin
knockout mice. In other murine models, such as thioglycollate-induced
neutrophil influx into the peritoneal cavity,42 a
1- to 2-hour lag in neutrophil recruitment after thioglycollate
injection was observed. The recovery of neutrophil recruitment at later
time points (>2 hours) was attributed to E-selectin. In the
present study, normal C57Bl/J6 and P-selectin–deficient mice were
examined 3, 7, 14 (data not shown), and 28 days after carotid ligation.
The complete absence of inflammatory cell infiltrate in knockout mouse
carotid sections at 3 and 7 days is presented (Figs 3 and 4).
Inflammatory cells were not detected within the arterial
wall at 14 or 28 days in either group (data not shown). The lesion
thickness at 28 days, however, was significantly reduced in P-selectin
knockout mice, as shown in Figs 1 and 2. It appears that E-selectin is
most likely not involved in this injury model. Although the
endothelial cells remain intact, the changes in the
local environment on ligation may not be sufficient to induce
E-selectin expression, which requires robust cytokine or LPS
stimulation over a period of 4 to 6 hours.43
E-selectin immunostaining was not performed in this
work, but endothelial cells were found to stain
positive for P-selectin. In addition, P-selectin
immunostaining in the media and developing
neointima reveals that platelets may have a role in
contributing to smooth muscle cell migration and proliferation.
In this model, secondary effects associated with no net flow in the left common carotid artery together with turbulence just proximal to the ligating suture may lead to activation of platelets and leukocytes. Both platelets and leukocytes may gain access to the media at sites at which the integrity of the endothelial monolayer is compromised, in the region of turbulence proximal to the ligating suture. Alternatively, platelets and leukocytes adhering to the endothelium may simply be covered by migratory, hyperplastic vascular smooth muscle cells. However, because these processes could just as easily occur in the P-selectin knockout mice, but did not, a third possibility seems more likely. The transmigration of leukocytes into the vessel wall has been extensively characterized.16 Also, the adherence of platelets via P-selectin to its carbohydrate ligands on monocytes and neutrophils has been well documented.17,18 Therefore, it is possible that platelets may be transported to the media through heterotypic aggregation with migratory leukocytes mediated by P-selectin. This would create a more direct spatial association between vascular smooth muscle cells and platelet-derived mitogenic and chemotactic factors, thus enhancing neointimal formation. Conversely, absence of P-selectin may have a two-pronged effect in P-selectin knockout mice. First, the process of leukocyte recruitment is impaired in these mice36 because of the absence of P-selectin, which mediates leukocyte rolling on activated endothelium. In addition, heterotypic aggregation between platelets and leukocytes mediated by P-selectin is not likely to occur in P-selectin–deficient mice. This would explain why inflammatory cells were not observed in P-selectin knockout mouse carotid arteries at 3 and 7 days after ligation, even though it was not surprising to find little or no P-selectin immunostaining in P-selectin–deficient mouse sections. However, P-selectin immunostaining in normal mice, especially in the media at day 7, which is probably platelet derived, suggests that in this model, platelets migrate into the vessel wall either directly or via P-selectin–mediated adherence to leukocytes, potentially influencing smooth muscle cell migration and proliferation.
A recent report suggested a mechanism for platelet-dependent lymphocyte recruitment to high endothelial venules in which activated platelets bound to peripheral node addressin on the endothelium in L-selectin–deficient mice can capture circulating lymphocytes through high-density expression of P-selectin.44 Adherent platelets at sites of vascular damage may recruit circulating neutrophils through P-selectin–mediated adhesion and ß2-integrin–dependent transmigration.45 Interactions such as these are probably responsible for the colocalization of platelets and neutrophils in acute inflammation,46 myocardial infarction,47 and atherosclerosis.48,49 In addition, platelet-leukocyte adherence has been reported in patients undergoing cardiopulmonary bypass50 and after coronary angioplasty,23 the magnitude of leukocyte activation and platelet adherence being higher in patients experiencing late clinical events. Diacovo et al44 suggested that platelets may have the capacity to deliver leukocytes to vascular beds that may not express selectins or selectin ligands but do have receptors for other platelet adhesion molecules. These investigators also report a "dynamic interplay between activated platelets and vascular endothelium" that is characterized by the occurrence of reversible but continuous interactions between endothelial cells and deposited platelets. In this scenario, it is tempting to speculate that migratory leukocytes may also have the capacity to deliver platelets to the media via a P-selectin–dependent interaction in which platelet-derived growth factors and chemotactic factors may influence smooth muscle cell migration and proliferation leading to neointimal formation in such pathological situations as restenosis.
Previously described animal models of atherogenesis have also reported neointimal thickening accompanied by leukocyte infiltration.51,52 In the present model, vascular constriction with accompanying neointimal proliferation was studied, because it relates to loss of lumen area in restenosis after angioplasty. No endothelial denudation or thrombus formation was observed. The results obtained suggest early involvement of leukocytes and implicate P-selectin in this process. There is evidence in the literature that agents that antagonize selectins/P-selectin help protect the host from damage occurring during inflammation,53 myocardial infarction, and reperfusion injury.54–57 In addition, antibody inhibition of P-selectin blocked leukocyte accumulation and fibrin deposition within Dacron grafts.31 Also, pretreatment with an anti–P-selectin antibody accelerated streptokinase-induced thrombolysis in a primate model of arterial thrombosis.58 Recently, it was reported that in a rabbit balloon injury model, intimal hyperplasia was reduced by blocking selectins with a sialyl LewisX analogue.59 These data, together with the results from the present study, suggest that P-selectin may be an important therapeutic target for cardiovascular disorders.
P-selectin–deficient mice were found to be grossly normal and fertile. P-selectin immunostaining on blood smears was performed to confirm that these mice in fact did not express platelet P-selectin (data not shown). In addition, the same P-selectin knockout mice were also supplied to another group, which showed that there was no histamine- or LPS-induced expression of P-selectin in the tissues of these mice compared with the wild-type (C57Bl) mice.60 In the present work, tail bleeding times for P-selectin knockout mice were not found to be significantly different from those for normal C57Bl/J6 mice, unlike previously reported data.35 This indicates that P-selectin–deficient mice do not suffer from abnormal intravascular coagulation activity, despite an 18% reduction in platelet count. These mice did exhibit moderate leukocytosis, probably due to reduced margination and/or increased half-life of the cells. It therefore appears that P-selectin antagonism will be appropriate and effective as a therapeutic strategy and will probably not be associated with bleeding complications.
This work provides compelling evidence for a role of P-selectin in a mouse model of arterial remodeling and neointimal formation. This may have implications in the treatment of restenosis, especially because it is now recognized that remodeling has a significant impact on chronic lumen area and is largely responsible for late lumen area loss. Administration of various types of P-selectin inhibitors, such as neutralizing antibodies and competing oligosaccharide ligands, have thus far been successful in animal models for various related pathological conditions, as outlined above. Given the limitations of currently available preventive therapy for restenosis, P-selectin antagonism may be a highly attractive strategy for further clinical investigation.
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Acknowledgments |
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The authors wish to acknowledge Timothy P. Boyle and Susan L. Kuiper of Transgenics (Unit 7241), Pharmacia and Upjohn, Inc, for providing P-selectin knockout mice and Clinical Research (Unit 7262) for analyzing blood samples. The expert advice of Carol W. Johnson (Unit 7227) on immunohistochemistry is gratefully acknowledged. The completion of these experiments was also greatly facilitated by the assistance of the staff of Laboratory Animal Medicine and Care (Unit 7273), Pharmacia and Upjohn, Inc.
Received July 10, 1997; revision received September 5, 1997; accepted September 11, 1997.
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References |
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H. Weiler, V. Lindner, B. Kerlin, B. H. Isermann, S. B. Hendrickson, B. C. Cooley, D. A. Meh, M. W. Mosesson, N. W. Shworak, M. J. Post, et al. Characterization of a Mouse Model for Thrombomodulin Deficiency Arterioscler Thromb Vasc Biol, September 1, 2001; 21(9): 1531 - 1537. [Abstract] [Full Text] [PDF]
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K. Wang, Z. Zhou, X. Zhou, K. Tarakji, E. J. Topol, and A. M. Lincoff Prevention of intimal hyperplasia with recombinant soluble P-selectin glycoprotein ligand-immunoglobulin in the porcine coronary artery balloon injury model J. Am. Coll. Cardiol., August 1, 2001; 38(2): 577 - 582. [Abstract] [Full Text] [PDF]
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S. S. Smyth, E. D. Reis, W. Zhang, J. T. Fallon, R. E. Gordon, and B. S. Coller {beta}3-Integrin-Deficient Mice but Not P-Selectin-Deficient Mice Develop Intimal Hyperplasia After Vascular Injury : Correlation With Leukocyte Recruitment to Adherent Platelets 1 Hour After Injury Circulation, May 22, 2001; 103(20): 2501 - 2507. [Abstract] [Full Text] [PDF]
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J. A. McPherson, K. G. Barringhaus, G. G. Bishop, J. M. Sanders, J. M. Rieger, S. E. Hesselbacher, L. W. Gimple, E. R. Powers, T. Macdonald, G. Sullivan, et al. Adenosine A2A Receptor Stimulation Reduces Inflammation and Neointimal Growth in a Murine Carotid Ligation Model Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 791 - 796. [Abstract] [Full Text] [PDF]
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S. R. Steinhubl, S. G. Ellis, K. Wolski, A. M. Lincoff, and E. J. Topol Ticlopidine Pretreatment Before Coronary Stenting Is Associated With Sustained Decrease in Adverse Cardiac Events : Data From the Evaluation of Platelet IIb/IIIa Inhibitor for Stenting (EPISTENT) Trial Circulation, March 13, 2001; 103(10): 1403 - 1409. [Abstract] [Full Text] [PDF]
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A. M. Miller, A. R. McPhaden, R. M. Wadsworth, and C. L. Wainwright Inhibition by leukocyte depletion of neointima formation after balloon angioplasty in a rabbit model of restenosis Cardiovasc Res, March 1, 2001; 49(4): 838 - 850. [Abstract] [Full Text] [PDF]
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J.-G. Bienvenu, J.-F. Tanguay, J.-F. Theoret, A. Kumar, R. G. Schaub, and Y. Merhi Recombinant Soluble P-Selectin Glycoprotein Ligand-1-Ig Reduces Restenosis Through Inhibition of Platelet-Neutrophil Adhesion After Double Angioplasty in Swine Circulation, February 27, 2001; 103(8): 1128 - 1134. [Abstract] [Full Text] [PDF]
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D. Manka, R. G. Collins, K. Ley, A. L. Beaudet, and I. J. Sarembock Absence of P-Selectin, but Not Intercellular Adhesion Molecule-1, Attenuates Neointimal Growth After Arterial Injury in Apolipoprotein E-Deficient Mice Circulation, February 20, 2001; 103(7): 1000 - 1005. [Abstract] [Full Text] [PDF]
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T. Kawasaki, M. Dewerchin, H. R. Lijnen, I. Vreys, J. Vermylen, and M. F. Hoylaerts Mouse Carotid Artery Ligation Induces Platelet-Leukocyte-Dependent Luminal Fibrin, Required for Neointima Development Circ. Res., February 2, 2001; 88(2): 159 - 166. [Abstract] [Full Text] [PDF]
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S. Kawashima, T. Yamashita, M. Ozaki, Y. Ohashi, H. Azumi, N. Inoue, K.-i. Hirata, Y. Hayashi, H. Itoh, and M. Yokoyama Endothelial NO Synthase Overexpression Inhibits Lesion Formation in Mouse Model of Vascular Remodeling Arterioscler Thromb Vasc Biol, February 1, 2001; 21(2): 201 - 207. [Abstract] [Full Text] [PDF]
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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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K. Yogo, H. Shimokawa, H. Funakoshi, T. Kandabashi, K. Miyata, S. Okamoto, K. Egashira, P. Huang, T. Akaike, and A. Takeshita Different Vasculoprotective Roles of NO Synthase Isoforms in Vascular Lesion Formation in Mice Arterioscler Thromb Vasc Biol, November 1, 2000; 20 (11): e96 - e100. [Abstract] [Full Text] [PDF]
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 F Kee, C Morrison, A E Evans, E McCrum, D McMaster, J Dallongeville, V Nicaud, O Poirier, F Cambien, and G F BAXTER Polymorphisms of the P-selectin gene and risk of myocardial infarction in men and women in the ECTIM extension study Heart, November 1, 2000; 84(5): 548 - 552. [Abstract] [Full Text]
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J. E. Rectenwald, L. L. Moldawer, T. S. Huber, J. M. Seeger, and C. K. Ozaki Direct Evidence for Cytokine Involvement in Neointimal Hyperplasia Circulation, October 3, 2000; 102(14): 1697 - 1702. [Abstract] [Full Text] [PDF]
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S.-i. Hayashi, N. Watanabe, K. Nakazawa, J. Suzuki, K. Tsushima, T. Tamatani, S. Sakamoto, and M. Isobe Roles of P-Selectin in Inflammation, Neointimal Formation, and Vascular Remodeling in Balloon-Injured Rat Carotid Arteries Circulation, October 3, 2000; 102(14): 1710 - 1717. [Abstract] [Full Text] [PDF]
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C. Emanueli, M. B. Salis, J. Chao, L. Chao, J. Agata, K.-F. Lin, A. Munao, S. Straino, A. Minasi, M. C. Capogrossi, et al. Adenovirus-Mediated Human Tissue Kallikrein Gene Delivery Inhibits Neointima Formation Induced by Interruption of Blood Flow in Mice Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1459 - 1466. [Abstract] [Full Text] [PDF]
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K. J. Harmon, L. L. Couper, and V. Lindner Strain-Dependent Vascular Remodeling Phenotypes in Inbred Mice Am. J. Pathol., May 1, 2000; 156(5): 1741 - 1748. [Abstract] [Full Text] [PDF]
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B. Chandrasekar and J.-F. Tanguay Platelets and restenosis J. Am. Coll. Cardiol., March 1, 2000; 35(3): 555 - 562. [Abstract] [Full Text] [PDF]
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P. M. H. Schiffers, D. Henrion, C. M. Boulanger, E. Colucci-Guyon, F. Langa-Vuves, H. van Essen, G. E. Fazzi, B. I. Levy, and J. G. R. De Mey Altered Flow-Induced Arterial Remodeling in Vimentin-Deficient Mice Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 611 - 616. [Abstract] [Full Text] [PDF]
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K.-Y. Chyu, P. Dimayuga, J. Zhu, J. Nilsson, S. Kaul, P. K. Shah, and B. Cercek Decreased Neointimal Thickening After Arterial Wall Injury in Inducible Nitric Oxide Synthase Knockout Mice Circ. Res., December 3, 1999; 85(12): 1192 - 1198. [Abstract] [Full Text] [PDF]
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A. Kumar, M. P. Villani, U. K. Patel, J. C. Keith Jr, and R. G. Schaub Recombinant Soluble Form of PSGL-1 Accelerates Thrombolysis and Prevents Reocclusion in a Porcine Model Circulation, March 16, 1999; 99(10): 1363 - 1369. [Abstract] [Full Text] [PDF]
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S. R. Bryant, R. J. Bjercke, D. A. Erichsen, A. Rege, and V. Lindner Vascular Remodeling in Response to Altered Blood Flow Is Mediated by Fibroblast Growth Factor-2 Circ. Res., February 19, 1999; 84(3): 323 - 328. [Abstract] [Full Text] [PDF]
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A. J. Palazzo, S. P. Jones, D. C. Anderson, D. N. Granger, and D. J. Lefer Coronary endothelial P-selectin in pathogenesis of myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, November 1, 1998; 275(5): H1865 - H1872. [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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V. Fuster, M. Poon, and J. T. Willerson Learning From the Transgenic Mouse : Endothelium, Adhesive Molecules, and Neointimal Formation Circulation, January 13, 1998; 97(1): 16 - 18. [Full Text] [PDF]
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