© 1999 American Heart Association, Inc.
Rapid Communication |
Blocking Very Late Antigen-4 Integrin Decreases Leukocyte Entry and Fatty Streak Formation in Mice Fed an Atherogenic Diet
From UCLA Departments of Pathology (P.T.S., J.A.B.) and Medicine (P.T.S., M.-L.B., D.K.V., M.C.T., A.J.L., J.A.B.), Los Angeles, Calif; Cytel Corporation (D.S., M.J.E.), San Diego, Calif.
Correspondence to Peggy Shih, Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Center for the Health Sciences, 10833 Le Conte Ave, Los Angeles, CA 90095-1732.
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Abstract—Atherosclerotic lesion development is characterized by the recruitment of leukocytes, principally monocytes, to the vessel wall. Considerable interest has been focused on the adhesion molecule(s) involved in leukocyte/endothelial interactions. The goal of the present study was to determine the role of the very late antigen-4 (VLA-4) integrin/ligand interaction in fatty streak development using murine models. Because
4 null mice are not viable, a peptidomimetic
was used to block VLA-4–mediated leukocyte binding. The ability of a
synthetic peptidomimetic of connecting segment-1 (CS-1 peptide) to
block the recruitment of leukocytes and the accumulation of lipid in
the aortic sinus of either wild-type mice (strain C57BL/6J) or mice
with a low-density lipoprotein null mutation (LDLR-/-) maintained on
an atherogenic diet was assessed. The active (Ac) CS-1 peptide or
scrambled (Sc) CS-1 peptide was delivered subcutaneously into mice
using a mini osmotic pump. Mice were exposed to the peptide for 24 to
36 hours before the onset of the atherogenic diet. In C57BL/6J mice,
leukocyte entry into the aortic sinus, as assessed by en face
preparations, was inhibited by the active peptide (Ac=28±4, Sc=54±6
monocytes/valve; P=0.004). Additionally, frozen sections
stained with Oil Red O were analyzed to assess lipid
accumulation in the aortic sinus. C57BL/6J mice that received the (Ac)
compound demonstrated significantly reduced lesion areas as compared
with mice that received the (Sc) peptide (Ac=4887±4438
µm2, Sc=15 009 ±5619 µm2;
P<0.0001). In a separate study, LDLR-/- mice were
implanted with pumps containing either the (Ac) or (Sc) peptide before
initiation of the atherogenic diet. Because LDLR-/- mice fed a chow
diet displayed small lesions at 14 weeks, the effects of the peptide
seen in these animals represented a change in early lipid
accumulation rather than initiation. By using whole-mount preparations,
the (Ac) but not the (Sc) peptide significantly reduced the area of
lipid accumulation in the aortic sinus, resulting in an approximate
66% decrease. Plasma analysis from all studies revealed
concentrations of peptide to be present at levels previously
determined by in vitro analysis to block adhesion. (Ac) CS-1
peptide, which blocks VLA-4 on the leukocyte surface, is effective in
reducing leukocyte recruitment and lipid accumulation in the aortic
sinus. The present study provides in vivo evidence that the VLA-4
integrin plays an important role in the initiation of the
atherosclerotic lesion and lipid accumulation, and it suggests a
potential therapeutic strategy for this disease.
Key Words: atherosclerosis • monocyte • connecting segment-1 • fibronectin • 4ß1
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Introduction |
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Previous studies have reported that monocyte but not neutrophil adhesion to the vascular endothelium is one of the first steps in the development of the fatty streak.1 2 Recent studies using mice that do not express monocyte activators including macrophage colony-stimulating factor,3 4 monocyte chemoattractant protein-1,5 and the monocyte chemoattractant protein-1 receptor6 have further defined the importance of monocyte recruitment to the endothelium in lesion areas, because all these studies reported significantly decreased lesion formation. Additionally, studies by Boisvert et al7 using an interleukin-8r (the GRO receptor) null mouse model demonstrate the importance of this monocyte activator in lesion formation. Monocytes can adhere to the endothelium by various integrins present on their surface. In vitro studies have demonstrated very late antigen-4 (VLA-4) to be a major ligand mediating firm adhesion of monocytes to the endothelium. Although T lymphocytes are also able to adhere via VLA-4, this interaction may not have a significant effect in fat-fed mice, because several different groups have reported that T lymphocytes did not effect atherosclerosis in fat-fed mice.8 9 10 In spite of the conclusions drawn from these lymphocyte studies, there are indications that lymphocytes may play a role in atherosclerosis under some conditions.8 Considered together, past studies on leukocyte, particularly monocyte, adhesion suggest that VLA-4 may be important in atherogenesis.
Two endothelial ligands for VLA-4 have been described: vascular cell adhesion molecule-1 (VCAM-1) and fibronectin containing the connecting segment-1 (CS-1) region.11 12 13 14 15 Expression of VCAM-1 has been shown to be increased in the aortic endothelium of rabbits given a high-fat diet13 and in the atherosclerotic lesions of certain mouse models of atherosclerosis.16 17 Recently, we have shown that CS-1 is increased in human coronary lesions, and thus it may be an important mediator in monocyte recruitment to the endothelium in vitro.18 The affinity of VLA-4 for VCAM-1 and CS-1 is comparable,19 although VLA-4 recognizes different sequences in VCAM-1 (QIDSPL)20 21 and CS-1 (LDV).14 22 23 Studies by several groups have reported the successful use of a CS-1 peptide in blocking cell-cell interactions in vitro24 and monocyte recruitment in vivo.25 26 Past studies demonstrate the ability of a CS-1 peptidomimetic to block binding of monocytes to VCAM-1 or CS-1.25 26 27 Therefore, we used CS-1 peptide infusion to determine the involvement of VLA-4 in fatty streak formation in an in vivo system.
The present study used C57BL/6J and low-density lipoprotein null mutation (LDLR-/-) mice fed a high-fat, high-cholesterol diet to examine the effect of VLA-4 on the development of early fatty streak lesions. The C57BL/6J mouse is susceptible to atherosclerosis and exhibits slow lesion development on a high-fat, high-cholesterol diet. In contrast, the LDLR-/- mouse developed more extensive lesions in a relatively short time period. Data obtained from both the C57BL/6J and the LDLR-/- mice substantiate an important role for VLA-4 in the development of the early atherosclerotic lesion.
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Materials and Methods |
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In Vitro Studies
Rabbit and human aortic endothelial cells were isolated and cultured as previously described.28 Rabbit leukocytes were prepared using Ficoll gradients and purified by adherence to plastic for 30 minutes. Human monocytes were prepared by a modified Recalde method.29 Preparation of minimally modified low-density lipoprotein (MM-LDL) and monocyte binding studies was performed as previously described.28
Animals and Diets
Female C57BL/6J mice and LDLR-/- mice of a mixed genetic
background (50% C57BL/6J and 50% 129/J) were obtained from Jackson
Laboratories (Bar Harbor, Maine). Age-matched animals were housed
together and fed standard chow (Purina No. 5001) until 
10 weeks of
age. At the onset of the study, the mice were individually housed and
separated into the following groups for the study: (1) chow diet, no
pump, (2) high-fat diet receiving active (Ac) CS-1 peptide, and (3)
high-fat diet receiving scrambled (Sc) CS-1 peptide. Groups receiving
high fat were maintained on an atherogenic diet that consisted of 15%
(wt/wt) fat, 1.25% cholesterol, and 0.5% cholic acid
(Food-Tek, Inc).
Preparation of the CS-1 Native and Scrambled Compounds
Peptides were obtained from Cytel Corporation (San Diego,
Calif). Peptides were coded to perform blinded studies and were
revealed after the completion of the studies. The (Ac) CS-1 compound is
a 3-amino acid peptidomimetic corresponding to the C-terminal portion
of the 25-aa CS-1 sequence, which inhibits VLA-4–mediated cell
binding.26 The (Sc) CS-1 peptide consisted of identical
amino acids; however, the sequence was scrambled.26 The
peptides were diluted with sterile 1x PBS (with Ca and Mg) and loaded
into the reservoir chamber of each mini osmotic pump (Alzet, Alza
Corp). Peptides were delivered at 8 to 15 mg ·
kg-1 · d-1 for 30
days.
Insertion of the Mini Osmotic Pumps
All procedures were carried out under the guidelines of the
Animal Research Committee. Animals were anesthetized using
Aerrane (isoflurane USP, Fort Dalge Animal Health, Fort Dalge,
Iowa). The right lower quadrant of the animal was shaved with clippers
and the skin cleansed with 70% ethanol. A 2-mm transverse incision was
made, and a small subdermal pocket was created using a straight,
long-nosed hemostat. The pump was inserted with the delivery pore
located anterior toward the head of the animal, and the incision was
secured using wound clips. Animals were implanted with the pumps 24 to
36 hours before the initiation of the atherogenic diet to enable the
pump to begin peptide delivery into the bloodstream.
Plasma Lipid Levels and Peptide Analysis
A fasting blood draw was collected from the mice before the
initiation of the study and on the day of their killing. Blood was
collected by previously established methods.17 Total
cholesterol, HDL, and triglyceride levels were
determined by previously described enzymatic methods.30
Additional plasma samples were analyzed for levels of the CS-1
compounds by ELISA immune detection.
Generation of Aortic En Face Preparations
The aortic sinus and en face preparations were isolated and
prepared using a variation on the method reported by Nakashima et
al.16 To determine the effects of the (Ac) CS-1 peptide
and (Sc) CS-1 peptide on leukocyte recruitment to the aortic sinus or
on lipid accumulation, C57BL/6J or LDLR-/- mice fed either chow or a
high-fat diet were killed, and the hearts were quickly perfused with
1x PBS containing 3 U/mL heparin. The heart and the ascending and
descending aortas were removed and the following procedures were
performed.
Leukocyte Identification in C57BL/6J Mice
The heart and ascending aorta were removed and fixed with 100%
acetone. After 24 hours, the heart and aortic samples were rinsed 3
times with 1x PBS, and the tissue and aorta were trimmed until only
the aortic sinus and aortic root remained. Care was taken to avoid
contact with the aortic cusps during the manipulations, and excess fat
and tissue on the back side of the aorta were also removed. The
specimens were placed into a blocking solution of 1x PBS containing
3% BSA (3% APBS) for 1 hour at room temperature followed by rinsing 3
times with PBS and incubation with rat anti-human/mouse Mac-1
(CD11b/CD18) antibody (Boehringer-Mannheim) diluted in 3% APBS
for 16 hours at 4°C. The next day, the tissue was again rinsed 3
times with 1x PBS and blocked for 1 hour at room temperature in a 1x
PBS solution containing normal goat serum. The peroxidase-conjugated
IgG goat anti-rat secondary antibody (Leinco Technologies) was diluted
in 1x PBS/normal goat serum (Dako) solution and incubated for 2 hours
at room temperature. The secondary antibody was recognized using an
amino-9-ethyl carbazole (AEC) kit (Biomeda). The aortic sinus was then
placed onto a microscope slide and mounted using Crystal Mount
(Biomeda). To determine the focal plane used to score the leukocytes,
lipofuscin present on the valve leaflet served as the initial focal
plane (Figure 1
). It has been previously
shown that lipofuscin develops on the flow-exposed surface of the valve
leaflet across from the cusp where lipid, lipoproteins, and
monocytes/macrophages have been detected.31 Once
the level of the lipofuscin had been determined, the plane of focus was
shifted to the area of the aortic sinus. Mac-1+
leukocytes in 5 distinct regions in this area of the aortic valve were
counted (using an eyepiece grid) under a light microscope using x200
magnification.
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Measurement of Lipid Accumulation in the Aortic Sinus From
LDLR-/- Mice
The aortic sinuses were isolated as described above; however,
after removal of adventitial fat, the hearts and attached aortas were
instead fixed with a 4% paraformaldehyde solution
containing 5% sucrose for 24 hours at 4°C. After fixation, the
hearts were thoroughly rinsed 3 times with 1x PBS before rinsing in
70% ethanol for 5 minutes at room temperature. The hearts were stained
with a Sudan IV solution for 6 minutes before destaining with 80%
ethanol to reduce background levels.32 The aortas were
then rinsed with 1x PBS before mounting onto a slide with Crystal
Mount. The area of valve covered by lipid was assessed using the NIH
Image program. Aortic images (21 valves total) were captured by a video
camera mounted to the top of a microscope. Both the lipid-laden regions
and the total area of the valve itself were traced and compared. The
area of valve covered with lipid was expressed as a percentage of the
total area.
Measurement of Lipid Accumulation in Frozen Sections of C57BL/6J
Aortic Sinus
Pumps were inserted into 8 C57BL/6J mice per group 24 hours
before the initiation of the high-fat diet. After 4 weeks of high-fat
feeding, the animals were killed, and the hearts were perfused and
removed to be embedded into OCT compound (Tissue Tek). Hearts
were sectioned and stained with Oil Red O for lipids as previously
described.17 The number of sections that spanned the
aortic sinus of each mouse was determined, and the size of the lesions
present in these sections was measured by previously described
methods.17
Statistical Analysis
Data were analyzed using the Statview 4.5 program. All
P values were calculated using ANOVA and Fisher PLSD
significance test.
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Results |
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In Vitro Effects of the (Ac) CS-1 Peptide
The effect of pretreatment of monocytes with the CS-1 peptidomimetic and subsequent binding to MM-LDL–treated endothelial cells (ECs) was examined. Monocytes, resuspended in 1 mL of medium containing 500 µg/mL CS-1 peptide or no peptide, were incubated for 20 minutes at room temperature. This high level of peptide was used to obtain maximal effects. Monocytes were then added to untreated ECs or to ECs treated for 4 hours with 200 µg/mL of MM-LDL. Treatment with peptide reduced binding both within and across species by 60% to 70% (Figure 2
).
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Active CS-1 Peptide Reduced the Number of Leukocytes Recruited to
the Aortic Sinus of C57BL/6J Mice
To assess the effect of the peptide on lesion initiation as
measured by leukocyte recruitment to the aortic sinus, mini osmotic
pumps containing either the (Ac) CS-1 peptide or (Sc) CS-1 peptide were
implanted subcutaneously into C57BL/6J mice. The pumps were allowed to
begin delivery of the peptides for 24 hours before the mice were
subjected to 4 weeks of high-fat feeding. Leukocytes that were stained
using a Mac-1 (CD18/CD11b) antibody were visualized as red, owing to
AEC (Figure 3A through 3C). A single
aortic cusp and valve from mice on a high-fat diet receiving the (Sc)
CS-1 peptide (Figure 3A) and the (Ac) CS-1 peptide (Figure 3B) are shown at low magnification (x40). At this
magnification, individual Mac-1+ leukocytes
cannot be distinguished, and therefore the accumulated cells appear as
red areas (arrows). At higher magnification, individual cells are
easily identified, as shown in Figure 3C, from animals treated
with the atherogenic diet. Leukocytes were counted in 5 fields from
each valve region. The (Ac) CS-1 peptide significantly reduced by 46%
the number of Mac-1+ leukocytes that adhered to
the aortic sinus as compared with the sinus of mice receiving the (Sc)
CS-1 peptide (Figure 4).
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Active CS-1 Peptide Reduced Lipid Accumulation in the Aortic Sinus
of C57BL/6J Mice as Assessed by Lesion Cross-Sectional Areas
In a separate experiment, the effect of (Ac) CS-1 peptide on
lipid accumulation in C57BL/6J mice was assessed in sections stained
with Oil Red O to detect lipids. Staining was less intense in sections
from mice receiving the (Ac) CS-1 peptide as compared with the mice
receiving the (Sc) CS-1 peptide (Figure 5A and 5B). Lesions from mice receiving
the (Ac) CS-1 peptide were 66% smaller than lesions from mice that
received the (Sc) CS-1 peptide (P=<0.0001; n=8) (Figure 6).
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Active CS-1 Peptide Was Also Effective at Reducing the Amount of
Lipid Accumulation in the Aortic Sinus of LDLR-/- Mice
The ability of the (Ac) CS-1 peptide to decrease the lesion
progression in LDLR-/- mice was determined by the amount of lipid
accumulation in the aortic cusps of LDLR-/- mice after 3 weeks of
high-fat feeding. En face preparations were made from LDLR-/- mice
that were maintained on either a chow or high-fat diet; the latter
group received either the (Ac) CS-1 peptide or (Sc) CS-1 peptide 24 to
36 hours before starting the high-fat diet. Figure 7A shows a single cusp from a mouse
receiving the (Sc) CS-1 peptide. Areas of lipid stained a deep burgundy
color on exposure to Sudan IV. Regions that stained deep burgundy were
able to be visualized before staining under the dissecting microscope
as areas of dense white-yellow accumulations, indicative of large lipid
deposits (data not shown). Figure 7B was taken from a mouse
receiving the (Ac) CS-1 peptide and the high-fat diet. NIH Image
analysis was performed on the aortas, and the amount of lipid
accumulation was expressed as the total area of valve covered with
lipid. The aortas from animals that received the chow diet displayed a
small amount of lipid accumulation (Figure 8). In conclusion, mice receiving the
high-fat diet together with the (Ac) CS-1 peptide had 50% less lipid
accumulation into the aortic sinus area as compared with mice receiving
both the high-fat diet and the (Sc) CS-1 peptide (Figure 8).
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Plasma Lipid and (Ac) CS-1 Peptide Levels
At the end of each experiment, fasting plasma was drawn from the
mice for determination of lipid and peptide levels. Total
cholesterol levels for both the C57BL/6J and LDLR-/- mice
placed on the atherogenic diet were elevated as compared with group
mates on the chow diet (Table).
Differences in the total cholesterol levels from C57BL/6J
mice were not significant between the high-fat groups treated with (Ac)
CS-1 peptide or (Sc) CS-1 peptide. The LDLR-/- mice on the chow diet
exhibited levels of total cholesterol that were
significantly greater than the C57BL/6J mice on a chow diet
(P=<0.0001). This elevated cholesterol level in
the LDLR-/- mice increased even further after high-fat feeding but
also did not differ between peptide groups. Levels of HDL were
found to be dramatically reduced in animals receiving the atherogenic
diet as compared with chow for both strains of mice (Table).
Peptide analysis in the blood demonstrated that the (Ac) CS-1
peptide was present in the bloodstream at concentrations (700±243
ng/mL, 1 µmol/L)26 that have been
previously shown to block monocyte binding in vitro. In summary,
treatment of mice with (Ac) CS-1 peptide did not alter elevated levels
of plasma cholesterol in fat-fed mice. Therefore, the
biological effects of the peptide are likely to be due to reduced
leukocyte recruitment in vivo.
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Discussion |
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The present study uses drug-delivery therapy to investigate the role of VLA-4 in vivo in the initiation and progression of the atherosclerotic lesion. In vivo studies to determine the role of other adhesion molecules in atherosclerosis have used gene targeting. However, this is not an option, because homozygous null mutations of the
4 and VCAM-1 genes have
proven to be lethal.33 34 35 The use of peptides to block
the interaction of leukocytes with the endothelium in
vitro and in sites of inflammation in vivo has previously been reported
by several groups.25 26 The in vitro blocking technique
described in the present study demonstrated the ability of the (Ac)
CS-1 peptide to reduce the adhesion of purified monocytes to either
human or rabbit aortic ECs (Figure 2
). Treatment with the (Ac)
CS-1 peptide reduced binding of monocytes both within and across
species by 60% to 70% (Figure 2). For in vivo studies, Wahl et
al25 induced rheumatoid arthritis in a rat model that was
found to be alleviated by treatment with the CS-1 peptide. In
particular, CS-1 was effective at suppressing both acute and chronic
inflammation, suggesting that the CS-1 peptide could influence both the
initiation and progression of arthritis.25 Additionally, a
CS-1 peptide similar to the compound in the present study has been
reported to reduce the incidence of transplant
atherosclerosis by attenuating intimal lesions in the
coronary arteries of animals.26 Previous studies
using monoclonal antibodies against 4
to investigate allergic asthma and inflammatory bowel disease suggest a
role for 4 in the process of these
inflammatory conditions.36 However, these results should
be interpreted with caution, because some studies report side effects
associated with the administration of an 4
antibody. In the studies described above, peptides were delivered either intravenously or subcutaneously by injection. However, we chose to use a mini osmotic pump implanted subcutaneously into the animal. Different groups have successfully used osmotic pumps to deliver various therapeutic compounds.37 38 Use of mini osmotic pumps allows time-released delivery of the peptides and alleviates the need for daily injections, which can produce inflammation in addition to being labor-intensive. Furthermore, use of the pump reduced the possibility of dose variability from the experimenter or by receiving only a single large dose per day, given that the pump was designed to continuously deliver a set amount of peptide per day.
Monocytes have been shown to be the major VLA-4–containing cell type in atherosclerotic lesions,1 2 and previous studies have shown that neutrophils are not present in lesions.1 Our results demonstrate the ability of the (Ac) CS-1 peptide to reduce the recruitment of Mac-1+ leukocytes in vivo; the majority of these cells most likely are monocytes. The (Ac) CS-1 peptide blocks Mac-1+ leukocyte recruitment to the endothelium by inhibiting VLA-4–mediated binding of leukocytes to their counter-receptors. Because T-cell lymphocytes are also able to adhere via VLA-4, it is possible that the (Ac) CS-1 peptide could inhibit their recruitment. However, studies by 2 separate groups using the apolipoprotein E-/- mouse as an atherosclerosis model independently scored either T-cell lymphocytes10 or macrophages.6 Comparing the 2 studies revealed that apolipoprotein E-/- lesions had a 1:500 lymphocyte to macrophage ratio. Studies by other investigators failed to identify T lymphocytes in mouse lesions at 15 weeks of feeding.17 These studies suggest that monocyte/macrophages represent at least 95% of the leukocytes in mouse lesions. On the basis of these observations, we believe that the majority of leukocytes that stained positively with the Mac-1 antibody and those that are blocked by the (Ac) CS-1 peptide are monocyte/macrophages.
The (Ac) CS-1 peptide was effective at reducing lesion size in both
C57BL/6J and LDLR-/- mouse models. Lipid analysis of frozen
sections from C57BL/6J mice demonstrated that the (Ac) CS-1 peptide
caused a 66% reduction in the size of lesions induced by high-fat
feeding (Figure 6). Although LDLR-/- mice have been reported
not to develop lesions on a chow diet, we and other investigators who
have worked with the LDLR-/- mice have observed these mice to
demonstrate a mild degree of spontaneous lesion development (J.H. Qiao,
MD, oral communication, May 1996). En face sections taken from
LDLR-/- mice demonstrated the presence of baseline lipid accumulation
in mice receiving the chow diet. Mice receiving the (Sc) CS-1 peptide
had a 4-fold increase in lesion size on a high-fat diet as compared
with mice on a chow diet. The (Ac) CS-1 peptide reduced this increase
by 50% (Figure 8). Thus, the peptide blocked the initiation of
lipid accumulation in small lesions. Previous studies have shown that
in the fatty streak, lipids mainly accumulated in macrophage
foam cells whose entry is inhibited by the peptide. Therefore, the
present study suggests that leukocyte VLA-4 binding of CS-1 is
responsible for the inhibition of lesion development.
The present study used 2 different methods to analyze diet-induced atherosclerosis. En face or whole-mount preparations of the aortic sinus were initially used to assess the number of leukocytes bound. The focus of the present study remained on the aortic sinus, because this region has been reported to be the primary site of predilection in several strains of atherosclerosis mouse models including C57BL/6J.17 The same mouse model and technique were also used to determine levels of lipid accumulation. En face preparations have been reported to be less variable with less skewness as compared with traditional histological sections.39 Other advantages to this method include speed of generating samples for analysis. Additionally, en face preparations provide a better orientation of the tissue specimen. However, using this method does not allow for determination of the depth of the lesion. Furthermore, background exogenous tissue may cause difficulty with the analysis by increasing the opacity of the tissue thereby obscuring areas of interest. Therefore, histological sections were also examined to generate a more 3-dimensional image of the lesion. The difference in lesion size was greater in the animals when frozen sections were used, but statistically significant differences were seen with both methods.
The partial inhibition of lesion formation seen in the present study is similar to the effects in other studies targeting single adhesion ligands. Mice with single mutations in CD18, intercellular adhesion molecule-1, and P-selectin exhibited 47%, 63%, and 63% reductions in lesion area, respectively.40 There are several possible explanations for the partial inhibition of leukocytes adhering to the aortic sinus in animals receiving the active CS-1 peptide. The peptide levels used may not be great enough to cause complete saturation of the VLA-4 sites on the surface of leukocytes in vivo. However, previous studies by others showed an IC50 of 0.2 to 0.5 µmol/L in vitro, a concentration that was half of what we observed in the mouse plasma.18 26 Another possibility is that leukocytes may be able to use more than one surface integrin to adhere to the endothelium. Issekutz41 has previously reported that there are 3 leukocyte integrins responsible for in vitro adhesion and in vivo migration to sites of inflammation: Mac-1, lymphocyte function-associated antigen-1 (LFA-1), and VLA-4. Blocking studies demonstrated that, of these 3, LFA-1 and VLA-4 together were mostly responsible for the adhesion and migration of leukocytes. Complete inhibition of monocytes was only achieved after blocking against all 3 integrins.41 Furthermore, Issekutz41 reported that the initiating inflammatory stimulus is able to modify leukocyte integrin use. These studies highlight the importance of VLA-4 in monocyte recruitment in atherosclerosis; however, they cannot exclude the possibility of alternative ligands and/or pathways.
In summary, our data demonstrate the importance of VLA-4 in the
early stages of Mac-1+ leukocyte adhesion and
lipid accumulation that occur in response to a high-fat diet. The
present study has focused on VLA-4–mediated
Mac-1+ leukocyte entry into early fatty streak
lesions and the effect of a CS-1 peptidomimetic on this interaction.
The more recent study reported by Patel et al42
investigated the role of VLA-4 in advanced
atherosclerosis using an
anti-4 antibody. However, the study of Patel
et al used labeled macrophages rather than the natural monocyte
population. The present study together with the study of Patel et
al supports the importance of VLA-4 in regulating leukocyte entry into
both early and advanced lesions where they may contribute to plaque
rupture. Furthermore, our results suggest that VLA-4 peptidomimetics
may be useful in limiting Mac-1+ leukocyte entry
into atherosclerotic lesions. Studies to determine the subunit
recognized by monoclonal antibodies indicate that antibodies against
epitope A of 4 partially block the interaction
between VLA-4 and fibronectin without inhibiting VCAM-1
adhesion.43 This suggests the possibility of generating an
anti-4 peptide that would be specific for
blocking VLA-4–mediated adhesion to CS-1 only.
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Acknowledgments |
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This work was supported by United States Public Health Service grant HL 30568. We thank Drs Elaine Raines and Wolfgang Kaminski for invaluable assistance in en face preparative techniques and YiShou Shi for excellent histological assistance.
Received August 5, 1998; accepted December 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- Gerrity RG. The role of the monocyte in atherogenesis, I: transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181–190.[Abstract]
- Ross R. Cell biology of atherosclerosis. Annu Rev Physiol. 1995;57:791–804.[Medline] [Order article via Infotrieve]
- Qiao JH, Tripathi J, Mishra NK, Cai Y, Tripathi S, Wang XP, Imes S, Fishbein MC, Clinton SK, Libby P, Lusis AJ, Rajavashisth TB. Role of macrophage colony-stimulating factor in atherosclerosis: studies of osteopetrotic mice. Am J Pathol. 1997;150:1687–1699.[Abstract]
-
Smith JD, Trogan E, Ginsberg M, Grigaux C, Tian J,
Miyata M. Decreased atherosclerosis in mice deficient
in both macrophage colony-stimulating factor (op) and
apolipoprotein E. Proc Natl Acad Sci U S A. 1995;92:8264–8268.
[Abstract/Free Full Text] - Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis low density lipoprotein receptor-deficient mice. Mol Cell. 1998;2:275–281.[Medline] [Order article via Infotrieve]
- Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998;394:894–897.[Medline] [Order article via Infotrieve]
- Boisvert WA, Santiago R, Curtiss LK, Terkeltaub RA. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice. J Clin Invest. 1998;101:353–363.[Medline] [Order article via Infotrieve]
-
Dansky HM, Charlton SA, McGee Harper M, Smith
JD. T and B lymphocytes play a minor role in atherosclerotic plaque
formation in the apolipoprotein E-deficient mouse. Proc Natl Acad
Sci U S A. 1997;94:4642–4646.
[Abstract/Free Full Text] - Fyfe AI, Qiao JH, Lusis AJ. Immune-deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet. J Clin Invest. 1994;94:2516–2520.
-
Roselaar SE, Kakkanathu PX, Daugherty A. Lymphocyte
populations in atherosclerotic lesions of apoE-/- and LDL
receptor-/- mice: decreasing density with disease progression.
Arterioscler Thromb Vasc Biol. 1996;16:1013–1018.
[Abstract/Free Full Text] -
Chan BM, Elices MJ, Murphy E, Hemler ME. Adhesion to
vascular cell adhesion molecule 1 and fibronectin. Comparison of
4ß1 (VLA-4) and
4ß7 on the human B
cell line JY. J Biol Chem. 1992;267:8366–8370.
[Abstract/Free Full Text] - Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyj S, Hemler ME, Lobb RR. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell. 1990;60:577–584.[Medline] [Order article via Infotrieve]
-
Li H, Cybulsky MI, Gimbrone MA Jr, Libby P. An
atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable
mononuclear leukocyte adhesion molecule, in rabbit aortic
endothelium. Arterioscler Thromb. 1993;13:197–204.
[Abstract/Free Full Text] -
Guan JL, Hynes RO. Lymphoid cells recognize an
alternatively spliced segment of fibronectin via the integrin receptor
4ß1. Cell. 1990;60:53–61.[Medline]
[Order article via Infotrieve]
-
Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald JA,
Carter WG. Identification and characterization of the T lymphocyte
adhesion receptor for an alternative cell attachment domain (CS-1) in
plasma fibronectin. J Cell Biol. 1989;109:1321–1330.
[Abstract/Free Full Text] -
Nakashima Y, Raines EW, Plump AS, Breslow JL,
Ross R. Upregulation of VCAM-1 and ICAM-1 at
atherosclerosis-prone sites on the
endothelium in the ApoE-deficient mouse.
Arterioscler Thromb Vasc Biol. 1998;18:842–851.
[Abstract/Free Full Text] -
Qiao JH, Xie PZ, Fishbein MC, Kreuzer J, Drake TA,
Demer LL, Lusis AJ. Pathology of atheromatous
lesions in inbred and genetically engineered mice. Genetic
determination of arterial calcification. Arterioscler
Thromb. 1994;14:1480–1497.
[Abstract/Free Full Text] - Shih PT, Elices MJ, Fang ZT, Ugarova TP, Strahl D, Territo MC, Frank JS, Kovach NL, Cabanas C, Berliner JA, Vora DK. MM-LDL induces monocyte adhesion to endothelial CS-1 by activating ß1 integrin. J Clin Invest. In press.
-
Masumoto A, Hemler ME. Multiple activation states of
VLA-4. Mechanistic differences between adhesion to CS1/fibronectin and
to vascular cell adhesion molecule-1. J Biol Chem. 1993;268:228–234.
[Abstract/Free Full Text] -
Vonderheide RH, Tedder TF, Springer TA, Staunton DE.
Residues within a conserved amino acid motif of domains 1 and 4 of
VCAM-1 are required for binding to VLA-4. J Cell Biol. 1994;125:215–222.
[Abstract/Free Full Text] -
Osborn L, Vassallo C, Browning BG, Tizard R, Haskard
DO, Benjamin CD, Dougas I, Kirchhausen T. Arrangement of domains, and
amino acid residues required for binding of vascular cell adhesion
molecule-1 to its counter-receptor VLA-4
(4ß1). J
Cell Biol. 1994;124:601–608.
[Abstract/Free Full Text] -
Komoriya A, Green LJ, Mervic M, Yamada SS, Yamada KM,
Humphries MJ. The minimal essential sequence for a major cell
type-specific adhesion site (CS1) within the alternatively spliced type
III connecting segment domain of fibronectin is leucine-aspartic
acid-valine. J Biol Chem. 1991;266:15075–15079.
[Abstract/Free Full Text] -
Wayner EA, Kovach NL. Activation-dependent recognition
by hematopoietic cells of the LDV sequence in the V region of
fibronectin. J Cell Biol. 1992;116:489–497.
[Abstract/Free Full Text] -
Kumar A, Eckmam JR, Swerlick RA, Wick TM. Phorbol ester
stimulation increases sickle erythrocyte adherence to
endothelium: a novel pathway involving
4ß1 integrin receptors
on sickle reticulocytes and fibronectin. Blood. 1996;88:4348–4358.
[Abstract/Free Full Text] - Wahl SM, Allen JB, Hines KL, Imamichi T, Wahl AM, Furcht LT, McCarthy JB. Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment. J Clin Invest. 1994;94:655–662.
- Molossi S, Elices M, Arrhenius T, Diaz R, Coulber C, Rabinovitch M. Blockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts [see comments]. J Clin Invest. 1995;95:2601–2610.
-
Vanderslice P, Ren K, Revelle JK, Kim DC, Scott D,
Bjercke RJ, Yeh ET, Beck PJ, Kogan TP. A cyclic hexapeptide is a potent
antagonist of 4 integrins.
J Immunol. 1997;158:1710–1718.[Abstract]
-
Kim JA, Territo MC, Wayner E, Carlos TM, Parhami F,
Smith CW, Haberland ME, Fogelman AM, Berliner JA. Partial
characterization of leukocyte binding molecules on
endothelial cells induced by minimally oxidized LDL.
Arterioscler Thromb. 1994;14:427–433.
[Abstract/Free Full Text] - Fogelman AM, Elahi F, Sykes K, Van Lenten BJ, Territo MC, Berliner JA. Modification of the Recalde method for the isolation of human monocytes. J Lipid Res. 1988;29:1243–1247.[Abstract]
- Mehrabian M, Qiao JH, Hyman R, Ruddle D, Laughton C, Lusis AJ. Influence of the apoA-II gene locus on HDL levels and fatty streak development in mice [published erratum appears in Arterioscler Thromb. 1993;13:466]. Arterioscler Thromb. 1993;13:1–10.
-
Mehrabian M, LL Demer, AJ Lusis. Differential
accumulation of intimal monocyte-macrophages relative to
lipoproteins and lipofuscin corresponds to hemodynamic
forces on cardiac valves in mice. Arterioscler Thromb. 1991;11:947–957.
[Abstract/Free Full Text] -
Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D,
Witztum JL. ApoE-deficient mice are a model of lipoprotein oxidation in
atherogenesis: demonstration of oxidation-specific epitopes in lesions
and high titers of autoantibodies to malondialdehyde-lysine in serum.
Arterioscler Thromb. 1994;14:605–616.
[Abstract/Free Full Text] -
Gurtner GC, Davis V, Li H, McCoy MJ, Sharpe A,
Cybulsky MI. Targeted disruption of the murine VCAM1 gene: essential
role of VCAM-1 in chorioallantoic fusion and placentation. Genes
Dev. 1995;9:1–14.
[Abstract/Free Full Text] - Kwee L, Baldwin HS, Shen HM, Stewart CL, Buck C, Buck CA, Labow MA. Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice. Development. 1995;121:489–503.[Abstract]
-
Yang JT, Rayburn H, Hynes RO. Cell adhesion events
mediated by 4 integrins are essential in
placental and cardiac development. Development. 1995;121:549–560.[Abstract]
-
Lobb RR, Hemler ME. The pathophysiologic role of
4 integrins in vivo. J Clin
Invest. 1994;94:1722–1728.
- Leonard JP, Neben TY, Kozitza MK, Quinto CM, Goldman SJ. Constant subcutaneous infusion of rhIL-11 in mice: efficient delivery enhances biological activity. Exp Hematol. 1996;24:270–276.[Medline] [Order article via Infotrieve]
-
Guery JC, Galbiati F, Smiroldo S, Adorini L. Selective
development of T helper (Th) 2 cells induced by continuous
administration of low dose soluble proteins to normal and
ß2-microglobulin-deficient BALB/c mice. J
Exp Med. 1996;183:485–497.
[Abstract/Free Full Text] - Veniant MM, Pierotti V, Newland D, Cham CM, Sanan DA, Walzem RL, Young SG. Susceptibility to atherosclerosis in mice expressing exclusively apolipoprotein B48 or apolipoprotein B100. J Clin Invest. 1997;100:180–188.[Medline] [Order article via Infotrieve]
-
Nageh MF, Sandberg ET, Marotti KR, Lin AH, Melchior EP,
Bullard DC, Beaudet AL. Deficiency of inflammatory cell adhesion
molecules protects against atherosclerosis in mice.
Arterioscler Thromb Vasc Biol. 1997;17:1517–1520.
[Abstract/Free Full Text] -
Issekutz TB. In vivo blood monocyte migration to acute
inflammatory reactions, IL-1, TNF-, IFN-gamma, and C5a
utilizes LFA-1, Mac-1, and VLA-4. The relative importance of each
integrin. J Immunol. 1995;154:6533–6540.[Abstract]
-
Patel SS, Thiagarajan R, Willerson JT, Yeh ETH.
Inhibition of 4 integrin and ICAM-1 markedly
attenuate macrophage homing to atherosclerotic plaques in
ApoE-deficient mice. Circulation. 1998;97:75–81.
[Abstract/Free Full Text] -
Pulido R, Elices MJ, Campanero MR, Osborn L, Schiffer
S, Garcia-Pardo A, Lobb R, Hemler ME, Sanchez-Madrid F. Functional
evidence for three distinct and independently inhibitable adhesion
activities mediated by the human integrin VLA-4. Correlation with
distinct 4 epitopes. J Biol
Chem. 1991;266:10241–10245.
[Abstract/Free Full Text]
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