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Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
1Correspondence: Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail: einar.eriksson{at}fyfa.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 subunit
(n=5) had no effect on rolling flux (107±25%,
P=0.782) or rolling velocity (89±6.1%,
P=0.147), despite endothelial expression of vascular
cell adhesion molecule 1 (VCAM-1). Leukocytes interacting with
atherosclerotic endothelium were predominantly neutrophils, because
treatment with antineutrophil serum decreased rolling and neutrophil
counts in peripheral blood to the same extent. In conclusion, we
present the first direct observations of atherosclerosis in
vivo. We show that transient dynamics of leukocyte-endothelium
interactions are important regulators of arterial leukocyte recruitment
and that leukocyte rolling in atherosclerosis is critically dependent
on the endothelial selectins. This experimental technique and the data
presented introduce a novel perspective for the study of
pathophysiological events involved in large-vessel disease.—Eriksson,
E. E., Xie, X., Werr, J., Thoren, P., Lindbom, L. Direct viewing
of atherosclerosis in vivo: plaque invasion
by leukocytes is initiated by the endothelial selectins.
Key Words: intravital • atherosclerosis • inflammation • selectin • neutrophils
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An important event in atherogenesis and in the destabilization of
developed plaque is the recruitment of leukocytes to the arterial wall
(1
, 2)
. The process of leukocyte recruitment in
atherosclerosis is believed to be mediated by sequential steps of
leukocyte-endothelium interactions similar to those occurring in
postcapillary venules in inflammation. In venules, such interactions
are mediated by various cell adhesion molecules (CAMs) on leukocytes
and on endothelial cells (3)
. Initial leukocyte capture
and rolling along the endothelium are mediated primarily by the
selectin family of CAMs and their counterreceptors (4
, 5)
,
whereas firm adhesion to the endothelium is mediated primarily by
integrin receptors on leukocytes interacting with their endothelial
ligands (6)
. In contrast to the relatively well known
mechanisms mediating these processes in inflammation, the cellular and
molecular events involved in leukocyte recruitment in atherosclerosis
remain largely unclear. Special interest has focused on vascular cell
adhesion molecule 1 (VCAM-1), because it is up-regulated on arterial
endothelium after atherogenic stimuli (7
, 8)
and because
it preferentially mediates recruitment of mononuclear leukocytes such
as monocytes and T lymphocytes (9)
, subtypes of leukocytes
that are present in atherosclerotic lesions (10
, 11)
.
However, studies indicate roles in atherosclerosis for other CAMs as
well (9
, 12
13
14)
.
In the present study, we used a novel intravital microscopic technique
(15)
to study the dynamics of leukocyte recruitment in the
aorta of atherosclerotic mice in vivo. We found that
leukocyte tethering, rolling, and firm adhesion occurred and could be
observed in atherosclerosis, whereas leukocyte-endothelium interactions
were virtually absent in healthy mice of similar age. Interactions on
atherosclerotic endothelium were transient, and the efficiency of
rolling leukocytes to initiate firm adhesion was low. All rolling
interactions depended critically on the endothelial selectins. These
data unravel specific features of leukocyte-endothelium interactions in
atherosclerosis previously not accessible to experimental
investigation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental procedure
Mice were anesthetized by inhalation of 2% isoflurane in 40%
O2, and catheters were placed in the left carotid
artery and in the left jugular vein. Blood pressure was measured
through the carotid catheter connected to a pressure transducer and a
Grass amplifier. An intravenous (i.v.) infusion of bicarbonate-buffered
glucose (0.4 ml/h) was used to maintain normal acid-base balance.
Rectal temperature was kept at 37°C with a heating pad and an
infrared heat lamp. The exposed tissue was superfused with a
thermostated (37°C) bicarbonate-buffered saline solution equilibrated
with 5% CO2 in nitrogen to maintain
physiological pH or was covered with a physiologically buffered
hyaluronic acid solution (Healon, 14 mg/ml, Pharmacia-Upjohn, Uppsala,
Sweden). All parameters were recorded via computer using Grass Polyview
software and stored for later analysis. Serial blood samples (10 µl)
were taken through the carotid catheter and analyzed for white blood
cells (WBCs) in a Bürker chamber.
Surgical procedure
The abdomen was opened through a midline incision and the
intestines were retracted and kept moist during the experiment. The
aorta was exposed and, without direct manipulation of the vessel, was
separated from the vena cava for a distance of 2–3 mm immediately
inferior of the renal arteries. The mouse was placed under the
microscope, and an ultrasonic flow probe connected to a flowmeter
(Transonic T-106 flowmeter, 0.7 V flow probe, sample rate: 200/s) was
placed around the artery. Direct intravital microscopic observations
were performed on the abdominal aorta at least 4–5 mm downstream of
the flow probe.
Intravital microscopy
Microscopic observations were made by using an intravital
microscope (Leitz Biomed, Wetzlar, Germany) with a water immersion
objective (Leitz SW 25x). Epi-illumination fluorescence microscopy
(Leitz Ploem-o-pac, filter block M2 illuminated by a cooled infrared
filtered lamp [Osram HBO 200W/4]) was started 2 min after labeling of
circulating leukocytes with an i.v. injection of rhodamine 6G (0.3
mg/ml, 0.67 mg/kg). Images were televised and recorded on videotape by
using a light-sensitive Panasonic WV-1900 video camera.
Analysis of in vivo experiments
In the aorta and in the iliac arteries, rolling and adhering
leukocytes were clearly visualized on the anterior half of the vessels
facing the objective. However, on severe atherosclerotic lesions
visualization was difficult because of the increased thickness of the
arterial wall. Rolling leukocyte flux was determined as the number of
rolling leukocytes passing a reference line perpendicular to blood
flow. Leukocyte rolling velocity was determined as the mean velocity of
individual rolling leukocytes. Leukocyte-endothelium contact time and
leukocyte rolling distance were determined as the respective time and
distance individual leukocytes were in contact with, and rolled along,
the endothelium. Adhesion efficiency was determined as the percentage
of rolling leukocytes that went from rolling to firm adhesion.
Scanning electron microscopy
Animals were perfusion fixed through the left ventricle with
2.5% glutaraldehyde in phosphate buffer (20 min, 100 mmHg) with
outflow through severed jugular veins. The aorta was excised, dissected
free from perivascular tissue, opened longitudinally, and attached en
face on glass slides (Superfrost Plus Gold) using superglue. The
specimens were then dehydrated in increasing concentrations of ethanol
and Freon 113 and finally through critical point dehydration with
CO2. After gold sputter coating, the aortas were
examined in a scanning electron microscope (Philips SEM
515).
Immunofluorescence microscopy
Mice were perfusion fixed through the left ventricle with 1%
paraformaldehyde in phosphate buffer (10 min, 100 mmHg). The aorta was
excised, dissected free from perivascular tissue, kept in fixative for
1–2 h at 4°C, opened longitudinally, cut in 1-cm segments, and
washed in PBS followed by blockage of unspecific binding with 10%
rabbit serum in PBS for 30 min at 4°C. The vessels were kept
overnight in primary antibody or irrelevant isotype-matched control
antibody (10 µg/ml), after which they were washed, incubated with
fluorescein isothiocyanate (FITC)-conjugated rabbit
F(ab')2 anti-rat immunoglobulin G (IgG) (dilution
1:50), washed, mounted en face on glass slides, and viewed in a laser
scanning confocal microscope (Insight Plus, Meridian Instruments,
Okemos, MI) under normal transmitted and laser-emitted fluorescent
light.
Antibodies and reagents
The antibodies used in vivo in this study were monoclonal
antibody (mAb) RB40.34 against mouse P-selectin (30 µg/mouse;
Pharmingen, San Diego, CA), mAb 9A9 against mouse E-selectin (30
µg/mouse; a kind gift from B. A. Wolitzky, Hoffmann-La Roche,
Nutley, NJ), mAb R1–2 against mouse integrin
4 subunit (150 µg/mouse; Pharmingen), and
the isotype-matched control mAb R3–34 (60 µg/mouse; Pharmingen). All
antibodies used in vivo were previously shown to be specific for and
block the function of their respective CAMs. Antibodies used for
immunofluorescence microscopy were mAb RB40.34, mAb R3–34, and mAb
MK2.7 against mouse VCAM-1 (Serotec, Oxford, U.K.). FITC-conjugated
rabbit F(ab')2 anti-rat IgG came from Serotec.
Rabbit anti-mouse antineutrophil serum (ANS; 25 µl/mouse) came from
Inter-Cell Technologies, Hopewell, NJ. Rhodamine 6G came from Sigma
Chemical, St. Louis, MO.
Statistical analysis
The data represent the mean ± SE of
measurements obtained in the indicated number of experiments.
Statistical analysis was performed using t test, the
Mann-Whitney rank sum test, the paired t test, and the
Wilcoxon signed rank test for paired samples. Statistical significance
was set at P < 0.05. In figures, *, **, and *** denote
differences from control values by significance levels of
P < 0.05, P < 0.01, and
P < 0.001, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leukocyte-endothelium interactions occur and can be observed in
atherosclerosis in vivo
In 5- to 15-month-old control C57BL/6 mice (n=24),
leukocyte-endothelium interactions were absent except in four mice in
which minimal rolling of leukocytes was observed in the aortic
bifurcation. In atherosclerotic mice, leukocyte-endothelium
interactions were not observed in animals that did not show signs of
arterial lesions. However, when lesions were present, leukocyte rolling
was observed in 45 of 64 mice (70%, a similar percentage in both
atherosclerotic strains). The average number of rolling leukocytes in
atherosclerotic mice was 5.8 ± 1.8 and 14 ± 3.2 cells/min
in ApoE0 (n=13) and
ApoE0LDLR0
(n=51) animals, respectively (P=0.117, range:
0–92 cells/min, Fig. 1
). Stereoscopic microscopic, intravital microscopic, and scanning
electron microscopic appearances of an atherosclerotic plaque where
leukocyte rolling was observed are shown in Fig. 2A
B
C
.
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In early atherosclerotic lesions, leukocyte-endothelium interactions were detected on the entire injured area; in severe lesions, however, interactions were typically observed in the periphery of plaque, primarily downstream and along the sides of lesions. On severe plaque, most rolling leukocytes were also captured in the periphery of those lesions. Thus, it seems that both tethering and rolling of leukocytes in developed atherosclerosis occur preferentially at the border between the plaque and the parts of the arterial wall less affected by lesion formation. In areas of the aorta distant from plaque and unaffected by lesions, interactions were rare. However, in a few mice such interactions were observed, possibly reflecting early lesion development.
Leukocyte rolling in atherosclerosis does not occur in clusters
and is not mediated by platelets adherent to the endothelium
Leukocytes rolling along the endothelium in atherosclerotic
arteries were observed as single rolling cells. Clusters of leukocytes
and platelets, similar to what have previously been described in
systems in which platelets are important in leukocyte recruitment
(16
, 17)
, were not detected. To further investigate the
potential role of platelets in leukocyte-endothelium interactions in
atherosclerosis, scanning electron microscopy was used on arteries in
which leukocyte rolling had previously been observed in vivo (Fig. 2C
). Atherosclerotic plaques were observed as elevated areas
in the arterial wall and the endothelial lining was in each specimen
intact (n=4). Adherent leukocytes were found on and in the
periphery of plaque. Platelet adhesion was rare.
Leukocyte-endothelium interactions in atherosclerosis are transient
and leukocyte adhesion efficiency is low
The characteristics of leukocyte-endothelium interactions in
atherosclerosis were investigated and compared with previous data from
cytokine-treated venules (18)
and cytokine-treated mouse
aorta (15)
. Data are shown in Table 1
. The typical cumulative rolling distance as a function of time is shown
in Fig. 3
. Leukocyte rolling in atherosclerosis occurred in small areas in the
periphery of atherosclerotic lesions, resulting in a short leukocyte
rolling distance. This should be compared with a fivefold longer
leukocyte rolling distance previously observed in cytokine-stimulated
venules. The difference in leukocyte-endothelium contact time between
atherosclerosis and cytokine-treated venules was even more pronounced,
because of a large difference in leukocyte rolling velocity. The
differences in the characteristics of leukocyte-endothelium
interactions between atherosclerosis and inflamed venules is likely
partly due to differences in the local hemodynamics, inasmuch as wall
shear rate is higher in arteries than in venular systems. This result
is supported by previous data demonstrating that wall shear rate
strongly influences leukocyte rolling in various vessel types (4
, 15
, 19)
. In accordance with the transient dynamics of
interactions, adhesion efficiency of rolling leukocytes in
atherosclerosis was found to be low compared with that in venules.
During a total time of 90 min of intravital observations, 1267
leukocytes were observed rolling on atherosclerotic endothelium. Among
these 1267 leukocytes, only 4 went from rolling to firm adhesion
(0.3%); in contrast, 91% of leukocytes rolling in inflamed venules
eventually adhere. In cytokine-treated aorta, leukocyte rolling
velocity was quite similar to that in atherosclerosis. However, rolling
distance and leukocyte-endothelium contact time were greater,
reflecting a more widespread expression of CAMs compared with that in
atherosclerotic endothelium. Likewise, the adhesion efficiency of
rolling leukocytes was higher in cytokine-treated aorta at a magnitude
that correlated with the differences in rolling distance and contact
time. This result may indicate that the higher adhesion efficiency in
cytokine-treated aorta than in atherosclerosis was due to the longer
time that leukocytes were in contact with the endothelium and,
accordingly, a longer exposure to chemoattractants present in the
arterial wall. Thus, transit time for leukocytes rolling along the
endothelium may be an important parameter influencing the induction of
firm leukocyte adhesion in arteries and atherosclerosis, as previously
shown in venules (20)
. This may be a critical factor in
limiting leukocyte recruitment in atherogenesis.
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Leukocyte rolling in atherosclerosis depends on the endothelial
selectins
The molecular basis for leukocyte rolling in atherosclerotic mice
was studied by using function-blocking antibodies against various CAMs.
Antibodies were given as i.v. injections in 60–70 µl of PBS. Blood
flow in the aorta was stable during the experiments. Antibody
experiments were primarily performed in
ApoE0LDLR0 mice fed the WD
for 5–6 months. At this time, the progression of atherosclerosis in
the abdominal aorta below the renal arteries was moderate, and lesions
were visible in 30 of 44 mice (68%). Additional experiments with
selectin-blocking antibodies were performed in
ApoE0 mice at various ages (5–18 months) in
which atherosclerotic lesion formation ranged from minimal progression
up to almost complete coverage of the abdominal aorta by plaques.
Notably, the adhesion molecules involved in leukocyte rolling were the
same in all stages of lesions and in all age groups of both strains;
therefore, data for selectin-blocking antibodies presented in
Fig. 4
were pooled. This approach revealed that leukocyte rolling in
atherosclerosis was abolished by an antibody blocking the function of
P-selectin (P<0.001, ApoE0 mice
n=4, ApoE0LDLR0
mice n=4, pooled data are shown in Fig. 4A
). In
addition, rolling leukocyte flux was significantly decreased after
function inhibition of E-selectin (ApoE0:
53±8.9% of the rolling flux before antibody treatment,
n=3, P<0.05;
ApoE0LDLR0: 49±15%,
n=7, P<0.05; all mice: 51±9.9%,
P<0.01). In contrast, no effect was seen after treatment
with an antibody against the integrin 4
subunit (ApoE0LDLR0:
107±25%, n=5, P=0.782) or an isotype-matched
control antibody
(ApoE0LDLR0: 108 ±
6.7%, n=5, P=0.277). Furthermore, function
inhibition of P-selectin after treatment with antibodies blocking the
function of E-selectin (n=6) or integrin
4 (n=5) again almost abolished
leukocyte rolling (P<0.001). To further investigate the
importance of P-selectin in leukocyte rolling in atherosclerosis, the
time of observation after treatment with function-blocking antibodies
against this adhesion molecule was extended. No leukocytes interacted
with the endothelium during 130 min of observation after function
inhibition of P-selectin alone (n=8). Moreover, during 65
min of observation after simultaneous blockage of P- and E-selectin
(n=6), only one (1)
leukocyte interacted
transiently (<0.5 s) with the aortic endothelium. Furthermore, in four
of five mice treated with an antibody against the integrin
4, additional function inhibition of
P-selectin abolished interactions for a total time of 45 min,
regardless of the result that the integrin 4
antibody was without obvious effect. However, in one
ApoE0LDLR0 mouse treated
with the antibody against integrin 4, function
inhibition of P-selectin was not sufficient to abrogate
leukocyte-endothelium interactions, and a rolling flux of 0.67 cell/min
remained after antibody treatment. These remaining interactions were
abolished by function inhibition of E-selectin.
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The qualitative appearance of leukocyte-endothelium interactions after
antibody treatment was also investigated. Leukocyte-endothelium contact
time was decreased by function inhibition of E-selectin
(ApoE0: 42±13% of mean contact time in
individual mice before antibody treatment, P<0.05;
ApoE0LDLR0: 69±9.0%,
P<0.05; all mice: 60±8.2, P<0.01, Fig. 4B
) but not integrin 4
(ApoE0LDLR0: 96±23%,
P=0.879) or control antibodies
(ApoE0LDLR0: 108±11%,
P=0.545). Leukocyte rolling distance was not affected
by any of these treatments (data not shown). However, leukocyte rolling
velocity was increased by function inhibition of E-selectin. Mean
rolling velocity increased to 162 ± 18%
(P<0.01; ApoE0: 194±42%,
P=0.157;
ApoE0LDLR0:
147±16%, P<0.05) of the velocity before treatment
based on calculations of mean velocity in individual mice (153±6.9%,
by comparing the velocity of individual rolling leukocytes,
P<0.001, Fig. 4C
). No effect on leukocyte
rolling velocity was observed either after treatment with integrin
4 (mean velocity: 89±6.1%,
P=0.147; individual leukocytes: 88±5.1%,
P=0.139; n=5) or control antibodies (mean
velocity: 100±17%, P=0.980; individual leukocytes:
105±9.9%, P=0.662; n=5). It is important to
note that the E-selectin antibody (9A9) proved not to influence
P-selectin-dependent interactions and E-selectin-independent
interactions inasmuch as treatment with 9A9 antibody did not alter
rolling flux or rolling velocity either in trauma-induced rolling in
the mouse cremaster muscle (rolling flux: 108±21% of rolling flux
before antibody treatment, P=0.502; rolling velocity:
107±12, P=0.542; n=3) or in the aorta of C57BL/6
mice when rolling was rapidly up-regulated by topical application of
irritant (rolling flux: 95±2.5%, P=0.184; rolling
velocity: 85±8.1%, P=0.198; n=3). Taken
together, the data indicate that capture and rolling of leukocytes in
atherosclerosis depend almost totally on the endothelial selectins.
Neutrophils roll along atherosclerotic endothelium in vivo
Because P- and E-selectin are known to predominantly mediate
rolling of neutrophils in inflamed venules (21)
, it was of
interest to investigate whether neutrophils could contribute to
selectin-dependent leukocyte rolling in atherosclerosis as well. In
atherosclerotic mice in which leukocyte rolling was observed
(n=5), ANS was administered acutely through an i.v.
injection. Results are shown in Fig. 5
. ANS reduced the systemic granulocyte count and mononuclear cell count
to 21 ± 3.7% and 61 ± 11% of the counts before ANS
treatment, respectively. Leukocyte rolling decreased at the same time
to 18 ± 5.3%, suggesting that a majority of the leukocytes
rolling along atherosclerotic endothelium were neutrophils.
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Expression of CAMs on atherosclerotic endothelium
To further characterize the CAMs responsible for leukocyte rolling
in atherosclerosis, we used immunofluorescence microscopy to analyze
atherosclerotic aortas in which leukocyte rolling had been observed
in vivo. Of three experiments, strong expression of
P-selectin was found in two aortas and weak expression was found in one
aorta at these specific sites (Fig. 6A
). In areas where no leukocyte rolling had been observed,
staining was negative. Positive staining for VCAM-1 was regularly found
in the periphery of plaque, as described previously (22)
(Fig. 6B
), whereas no positive staining was detected using
an isotype-matched control antibody. Because leukocyte rolling and
P-selectin expression were found mainly in the periphery of lesions,
P-selectin seems to colocalize with the typical expression pattern of
VCAM-1. As in previous studies (22)
, we were not able to
safely stain E-selectin on arterial endothelium, possibly because of
limited expression of this CAM. This result agrees with previous data
from the microcirculation where E-selectin has eluded staining in
arterioles treated with mouse tumor necrosis factor (TNF-)
(23)
despite a clear-cut effect of this CAM in arteriolar
leukocyte-endothelium interactions in vivo
(19)
. Nonetheless, in vivo data (this paper and
refs 13
and 14
) strongly indicate that E-selectin is expressed and
plays an important role in leukocyte recruitment in atherogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies investigating the mechanisms of leukocyte recruitment
in atherosclerosis have detected different CAMs expressed on arterial
endothelium and have revealed the presence of adherent and
transmigrated leukocytes. However, it has not been clearly shown
whether the multistep pathway responsible for leukocyte recruitment in
tissue inflammation is also critical in atherosclerosis. Thus, the
specific roles for different CAMs found on arterial endothelium in
arterial leukocyte recruitment remain unclear. In this study, we found
that leukocyte rolling and firm adhesion occur in various stages of
atherosclerosis, thus demonstrating that leukocyte recruitment on
atherosclerotic lesions takes place in a multistep fashion. Lesions in
which interactions were investigated ranged from early lesions to
developed fibrous plaque. On developed lesions, leukocyte-endothelium
interactions were observed primarily in the periphery of plaque,
corresponding to preferential CAM expression in these areas (this paper
and ref 22
).
Leukocyte-endothelium interactions in atherosclerosis are transient compared with those occurring in venules at sites of inflammation. Leukocyte-endothelium contact time and leukocyte rolling distance are lower and leukocyte rolling velocity is higher than those in venules. These parameters are likely influenced by hemodynamic conditions, with high shear stressing the molecular bonds of leukocyte-endothelium interactions in arteries. Conceivably, these transient interactions in atherosclerosis and the consequent brief exposure of rolling leukocytes to chemoattractants present in plaque restrict the efficiency of leukocytes rolling along atherosclerotic endothelium to initiate firm adhesion. However, because atherosclerosis in ApoE0 and ApoE0LDLR0 mice is a process that progresses over months and years, the conspicuously low number of leukocytes that firmly adhere is likely sufficient for lesion development. In fact, the low adhesion efficiency may be a rate-limiting step in the progress of atherogenesis, and small changes in the absolute number of recruited leukocytes may lead to significant changes in the development of lesions. Accordingly, the levels of chemoattractants present in the arterial wall, potentially influencing adhesion efficiency, may be important in regulation of recruitment of leukocytes and hence in the progression of the atherosclerotic disease.
Initial attachment and rolling of leukocytes on atherosclerotic
endothelium depend critically on P-selectin. The importance of
P-selectin in arterial leukocyte-endothelium interactions is supported
by data from the cytokine-treated mouse aorta where leukocyte rolling
was dramatically decreased, although not abolished, by P-selectin
blockage (15)
. In addition to P-selectin, E-selectin
influences rolling interactions, inasmuch as function inhibition of
this CAM increases rolling velocity and decreases leukocyte-endothelium
contact time. This result indicates that E-selectin stabilizes and
slows down leukocyte rolling in atherosclerosis and thereby, as
previously shown in venules (20)
, increases the tendency
for firm adhesion. Evidently, the endothelial selectins mediate
virtually all leukocyte rolling in atherosclerosis, and because no
leukocytes firmly adhered without initial rolling along the
endothelium, the endothelial selectins seem to play critical roles in
leukocyte recruitment in atherosclerosis. In addition, in
vivo experiments and scanning electron microscopy clearly show
that leukocyte-endothelium interactions in atherosclerosis occur on an
intact endothelial lining and that there is no obvious role for
platelets in this process. Thus, the molecular mechanisms responsible
for leukocyte rolling in atherosclerosis are similar to the mechanisms
previously shown in cytokine-challenged mice (15)
and
suggest that cytokines present in atherosclerosis (24
, 25)
are important in inducing expression of CAMs and thereby in the
recruitment of leukocytes.
In contrast to previous studies supporting a role for VCAM-1 in
leukocyte recruitment in atherosclerosis, our data suggest that the
integrin 4/VCAM-1 pathway may not mediate
initial attachment or rolling of leukocytes regardless of expression of
VCAM-1 on arterial endothelium. However, this pathway may still
influence rolling of certain leukocyte subclasses, such as monocytes
and T lymphocytes, which cannot be discriminated from other subclasses
in intravital microscopy experiments in which intravascular labeling of
leukocytes is used. Roles for integrin 4 and
VCAM-1 in leukocyte rolling in atherosclerosis are supported by
findings that these CAMs can influence rolling and adhesion in carotid
arteries from ApoE0 mice perfused with monocytic
cell lines (26)
. Moreover, integrin
4 or VCAM-1 could be important in firm
adhesion and transmigration.
Results from the present study strongly indicate that tethering and
rolling of all types of leukocytes interacting with atherosclerotic
endothelium depend critically on the endothelial selectins. These roles
for the endothelial selectins explain previous findings of delayed
lesion development in atherosclerotic mice lacking P-selectin,
E-selectin, or P- and E-selectin in combination (13
, 14)
.
In other vascular systems, such as venules (21)
and
cytokine-treated arterioles (19)
and arteries (E. E.
Eriksson et al., unpublished data), the endothelial selectins primarily
mediate interactions with neutrophils. Interestingly, we found that
this is most likely also true in atherosclerosis, a fact that could
explain the lack of effect of function inhibition of the integrin
4, a CAM that is predominantly expressed on
mononuclear leukocytes (27)
. However, in the literature,
neutrophils are rarely mentioned in the context of atherosclerosis.
Nonetheless, because leukocyte-endothelium interactions alone may
influence functions of both leukocytes and endothelial cells by
signaling via CAMs (28
29
30)
, we predict that neutrophils
could play roles in atherogenesis despite the fact that they may not be
a prominent histological feature of atherosclerotic lesions. Moreover,
a few studies suggest that small numbers of neutrophils,
neutrophil-specific substances, and chemoattractants with the ability
to enhance recruitment of neutrophils may be found in atherosclerotic
lesions (31
32
33)
. Given the limited life span of
neutrophils in inflamed tissue (34)
, even small numbers of
neutrophils detected in histological sections may reflect significant
invasion of these cells in plaque in vivo. Thus, even though
the present study does not specifically identify which CAMs mediate
recruitment of monocytes and T lymphocytes into atherosclerotic
lesions, these first dynamic studies of leukocytes interacting with
atherosclerotic endothelium indicate that leukocyte subclasses other
than mononuclear cells may be involved in the pathophysiology of this
disease. Although roles for such leukocytes in atherogenesis are at
this stage hypothetical, the issue of transiently interacting
leukocytes in atherosclerosis clearly requires further investigation.
In summary, we have performed, for the first time, direct observations of leukocyte recruitment in atherosclerosis in vivo. Using this technique, we establish that aspects of the dynamic cellular mechanisms in atherosclerosis can be observed and investigated in an in vivo situation. The data reveal that leukocyte-endothelium interactions in atherosclerosis are transient compared with those occurring in tissue inflammation, thereby limiting the capability of leukocytes to firmly adhere and be recruited to atherosclerotic lesions. Moreover, we show that initial attachment and rolling of leukocytes in atherosclerosis are critically dependent on the endothelial selectins, results that together with other data also raise questions about the potential involvement of leukocytes other than monocytes and T lymphocytes in this disease. This new technique and the data presented reveal the feasibility of dynamic observations of atherosclerosis in vivo. What is more, new opportunities for investigating the acute and chronic events of large-vessel disease such as balloon injury, plaque rupture, and vascular thrombosis now evolve.
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ACKNOWLEDGMENTS |
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Received for publication August 28, 2000.
Revision received December 11, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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