Rapid Communications

Blocking Very Late Antigen-4 Integrin Decreases Leukocyte

Entry and Fatty Streak Formation in Mice Fed
an Atherogenic Diet
Peggy T. Shih, Marie-Luise Brennan, Devendra K. Vora, Mary C. Territo, Dana Strahl,
Mariano J. Elices, Aldons J. Lusis, Judith A. Berliner

AbstractAtherosclerotic 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 a4 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 (LDLR2/2) maintained on an atherogenic
Downloaded from http://circres.ahajournals.org/ by guest on August 2, 2017

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 (Ac52864, Sc55466 monocytes/valve; P50.004). 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
(Ac5488764438 mm2, Sc515 009 65619 mm2; P,0.0001). In a separate study, LDLR2/2 mice were implanted with
pumps containing either the (Ac) or (Sc) peptide before initiation of the atherogenic diet. Because LDLR2/2 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. (Circ Res. 1999;84:345351.)
Key Words: atherosclerosis n monocyte n connecting segment-1 n fibronectin n a4b1

P revious 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 chemoattrac-
tant 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 endothe-
lium. 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 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

Received August 5, 1998; accepted December 4, 1998.

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.
1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org

345
 346 Blocking VLA-4 Reduces Fatty Streak Development
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.1115 Ex-
pression 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 atheroscle-
rosis.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 se-
quences 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
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monocytes to VCAM-1 or CS-1.2527 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 lipopro-
tein null mutation (LDLR2/2) mice fed a high-fat, high-cho-
lesterol diet to examine the effect of VLA-4 on the develop-
ment of early fatty streak lesions. The C57BL/6J mouse is
susceptible to atherosclerosis and exhibits slow lesion devel-
opment on a high-fat, high-cholesterol diet. In contrast, the
LDLR2/2 mouse developed more extensive lesions in a
relatively short time period. Data obtained from both the
C57BL/6J and the LDLR2/2 mice substantiate an important
role for VLA-4 in the development of the early atheroscle-
rotic lesion.
Materials and Methods
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 LDLR2/2 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 athero-
genic 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 por-
tion 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 13 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 z kg21 z d21 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 de-
scribed 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 LDLR2/2 mice fed either chow or a high-fat diet were
killed, and the hearts were quickly perfused with 13 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 13 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 13 PBS contain-
ing 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 4C. The next day, the tissue was again
rinsed 3 times with 13 PBS and blocked for 1 hour at room
temperature in a 13 PBS solution containing normal goat serum.
The peroxidase-conjugated IgG goat anti-rat secondary antibody
(Leinco Technologies) was diluted in 13 PBS/normal goat serum
(Dako) solution and incubated for 2 hours at room temperature. The
secondary antibody was recognized using an amino-9-ethyl carba-
zole (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 detect-
ed.31 Once the level of the lipofuscin had been determined, the plane
of focus was shifted to the area of the aortic sinus. Mac-11
leukocytes in 5 distinct regions in this area of the aortic valve were
counted (using an eyepiece grid) under a light microscope using
3200 magnification.

Measurement of Lipid Accumulation in the Aortic Sinus
From LDLR2/2 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 4C. After fixation, the hearts were thoroughly rinsed
3 times with 13 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 back-
ground levels.32 The aortas were then rinsed with 13 PBS before
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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.
Results
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-LDLtreated
endothelial cells (ECs) was examined. Monocytes, resus-
pended in 1 mL of medium containing 500 mg/mL CS-1
peptide or no peptide, were incubated for 20 minutes at room
temperature. This high level of peptide was used to obtain
Shih et al February 19, 1999 347
Figure 1. Schematic diagram of the aortic sinus
where leukocytes are scored. Lipofuscin forms on
the valve leaflet across from where monocytes/mac-
rophages accumulate. For frozen sections, the cusp
region is round (A). In en face preparations, the valve
leaflet is flattened against the back of the aortic sinus
(B). Identifying the lipofuscin allowed for orientation of
the plane of focus. Mac-11 leukocytes were identi-
fied and counted in the plane of the aortic sinus
found below that of the lipofuscin.
maximal effects. Monocytes were then added to untreated
ECs or to ECs treated for 4 hours with 200 mg/mL of
MM-LDL. Treatment with peptide reduced binding both
within and across species by 60% to 70% (Figure 2).
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 (340). At this magnifi-
cation, individual Mac-11 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-11 leukocytes that
adhered to the aortic sinus as compared with the sinus of mice
receiving the (Sc) CS-1 peptide (Figure 4).
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
Figure 2. (Ac) CS-1 peptide reduces mono-
cyte adhesion in vitro. Monocytes isolated
from either humans or rabbits were pretreated
with the (Ac) CS-1 peptidomimetic before
adhesion to MM-LDLtreated ECs. Monocytes
were resuspended in 1 mL of medium con-
taining 500 mg/mL CS-1 peptide or no pep-
tide and then incubated for 20 minutes at
room temperature. Monocytes were then
added to untreated ECs or to ECs treated for
4 hours with 200 mg/mL of MM-LDL at 37C.
Treatment with the peptide reduced binding
both within and across species by 60% to
70%. *P,0.001; n512. Data are represented
as mean6SD.
 348 Blocking VLA-4 Reduces Fatty Streak Development
Figure 3. Representative aortic cusp from a mouse receiving
either (Ac) CS-1 peptide or (Sc) CS-1 peptide was stained for
leukocytes using a Mac-1 (CD11b/CD18) antibody. Mini osmotic
pumps containing either the (Ac) CS-1 peptide or (Sc) CS-1
peptide were implanted subcutaneously into the backs of
C57BL/6J mice. Mice were then subjected to an atherogenic
diet for 4 weeks. The aortic sinus was isolated, fixed with 100%
acetone, and then stained with Mac-1 (CD11b/CD18) for the
presence of leukocytes. The Mac-1 antibody was visualized
using AEC; therefore, the positively stained cells appear as red.
Mac-11 leukocytes can be seen on the aortic sinus of mice
receiving the (Sc) CS-1 peptide (arrow) (A). Mice receiving the
(Ac) CS-1 peptide also contain Mac-11 leukocytes but are more
difficult to visualize at this magnification (arrow) (B) (magnifica-
tion 340). With higher magnification of the aortic sinus from a
mouse receiving the (Sc) CS-1 peptide, Mac-11 leukocytes
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appear as red (C) (magnification 3200).
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
(P5,0.0001; n58) (Figure 6).
Active CS-1 Peptide Was Also Effective at
Reducing the Amount of Lipid Accumulation in
the Aortic Sinus of LDLR2/2 Mice
The ability of the (Ac) CS-1 peptide to decrease the lesion
progression in LDLR2/2 mice was determined by the
amount of lipid accumulation in the aortic cusps of
LDLR2/2 mice after 3 weeks of high-fat feeding. En face
preparations were made from LDLR2/2 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
Figure 4. (Ac) CS-1 peptide significantly reduces the amount of
Mac-11 leukocytes bound to the aortic sinus of C57BL/6J mice
maintained on an atherogenic diet. The aortic valve from each
mouse was divided into 5 fields, and Mac-11 leukocytes were
counted in each field. The (Ac) CS-1 peptide significantly
reduced the levels of leukocytes bound to the aortic sinus of
mice receiving the atherogenic diet. *P50.004; n515 total num-
ber of valves for (Ac) CS-1 peptide and (Sc) CS-1 peptide. Data
are represented as mean6SD.
Figure 5. Frozen sections from C57BL./6J mice stained with Oil
Red O. Aortic sections from C57BL/6J mice that received either
the (Ac) CS-1 peptide or (Sc) CS-1 peptide were stained for Oil
Red O. Mice receiving the (Sc) CS-1 peptide exhibited concen-
trated lipid accumulation within the aortic sinus (A). The lipid
accumulation from mice receiving the (Ac) CS-1 peptide stained
with less intensity (B) (magnification 3100).
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 receiv-
ing 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).
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
LDLR2/2 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 LDLR2/2
mice on the chow diet exhibited levels of total cholesterol that
were significantly greater than the C57BL/6J mice on a chow
diet (P5,0.0001). This elevated cholesterol level in the
LDLR2/2 mice increased even further after high-fat feeding
but also did not differ between peptide groups. Levels of
Figure 6. (Ac) CS-1 peptide significantly reduced the amount of
lipid accumulated in the aortic sinus of C57BL/6J mice receiving
an atherogenic diet. The area of lipid accumulation was calcu-
lated using an eyepiece counting grid. The lesion area of mice
receiving the (Ac) CS-1 peptide was '2-fold less than that from
mice receiving the (Sc) CS-1 peptide. *P,0.0001; n58 animals
for either (Ac) CS-1 peptide or (Sc) CS-1 peptide. Data are rep-
resented as mean6SD.

Figure 7. Aortic sinus of mice receiving either (Ac) CS-1 peptide
or (Sc) CS-1 peptide was stained for lipids using Sudan IV.
LDLR2/2 mice were used to determine the effect of the pep-
tides on lipids. The aortic sinuses of these mice were isolated in
the same manner as that outlined above for the C57BL/6J mice.
However, for this experiment, the en face preparation was fixed
with a 4% paraformaldehyde/5% sucrose solution before stain-
ing with Sudan IV. Mice receiving the (Sc) CS-1 peptide demon-
strated regions of lipid accumulation that appear as deep bur-
gundy (arrows) (A). Mice receiving the (Ac) CS-1 peptide also
contained some amount of lipid accumulation (arrow) (B) (mag-
nification 3400).
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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 (7006243 ng/mL,
'1 mmol/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 biolog-
ical effects of the peptide are likely to be due to reduced
leukocyte recruitment in vivo.
Discussion
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 a4 and VCAM-1 genes
Figure 8. (Ac) CS-1 peptide significantly reduced the amount of
lipid accumulated in the aortic sinus of LDLR2/2 mice receiving
an atherogenic diet. After the en face preparations had been
stained and mounted onto slides, the valves were analyzed
using the NIH Image program. The total area of each valve was
measured and standardized. The region of valve containing lipid
was outlined and the area measured. The area covered with
lipid was expressed as a percentage of the total area of the
valve. The (Ac) CS-1 peptide was able to significantly reduce
the amount of lipid that accumulated in the valves of the mice.
*P50.02; n521 values for (Ac) CS-1 peptide and n520 for (Sc)
CS-1 peptide. Data are represented as mean6SD.
Shih et al February 19, 1999 349
Plasma Lipid Levels
Total Cholesterol HDL LDL1VLDL
C57BL/6J
Chow 7168 6467 763
(Ac) CS-1 117617 2565 92619
(Sc) CS-1 119628 2769 92627
LDLR 2/2
Chow 3566102 102623 2546105
(Ac) CS-1 1991646 30617 1961640
(Sc) CS-1 18206222 2167 17996224
Values are expressed in mg/dL.
have proven to be lethal.3335 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 progres-
sion 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 a4 to investigate
allergic asthma and inflammatory bowel disease suggest a
role for a4 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 a4 antibody.
In the studies described above, peptides were delivered
either intravenously or subcutaneously by injection. How-
ever, we chose to use a mini osmotic pump implanted
subcutaneously into the animal. Different groups have suc-
cessfully 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 experi-
menter 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-11 leukocytes in
vivo; the majority of these cells most likely are monocytes.
The (Ac) CS-1 peptide blocks Mac-11 leukocyte recruitment
to the endothelium by inhibiting VLA-4 mediated binding of
 350 Blocking VLA-4 Reduces Fatty Streak Development
leukocytes to their counter-receptors. Because T-cell lympho-
cytes 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 E2/2
mouse as an atherosclerosis model independently scored
either T-cell lymphocytes10 or macrophages.6 Comparing the
2 studies revealed that apolipoprotein E2/2 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 leuko-
cytes 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 LDLR2/2 mouse models. Lipid
analysis of frozen sections from C57BL/6J mice demon-
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strated that the (Ac) CS-1 peptide caused a 66% reduction in
the size of lesions induced by high-fat feeding (Figure 6).
Although LDLR2/2 mice have been reported not to develop
lesions on a chow diet, we and other investigators who have
worked with the LDLR2/2 mice have observed these mice
to demonstrate a mild degree of spontaneous lesion develop-
ment (J.H. Qiao, MD, oral communication, May 1996). En
face sections taken from LDLR2/2 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 inhib-
ited 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 prepara-
tions 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 histo-
logical sections.39 Other advantages to this method include speed
of generating samples for analysis. Additionally, en face prepa-
rations 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. There-
fore, 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, intercel-
lular 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 leuko-
cytes 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 mmol/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 respon-
sible 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-11 leukocyte adhesion and lipid
accumulation that occur in response to a high-fat diet. The
present study has focused on VLA-4mediated Mac-11 leuko-
cyte 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-a4 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-11 leukocyte entry into atherosclerotic lesions.
Studies to determine the subunit recognized by monoclonal
antibodies indicate that antibodies against epitope A of a4
partially block the interaction between VLA-4 and fibronectin
without inhibiting VCAM-1 adhesion.43 This suggests the pos-
sibility of generating an anti-a4 peptide that would be specific for
blocking VLA-4mediated adhesion to CS-1 only.
Acknowledgments
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.
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 Blocking Very Late Antigen-4 Integrin Decreases Leukocyte Entry and Fatty Streak
Formation in Mice Fed an Atherogenic Diet
Peggy T. Shih, Marie-Luise Brennan, Devendra K. Vora, Mary C. Territo, Dana Strahl, Mariano
J. Elices, Aldons J. Lusis and Judith A. Berliner
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Circ Res. 1999;84:345-351

doi: 10.1161/01.RES.84.3.345
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