© 1999 American Heart Association, Inc.
Original Contribution |
Patterns of Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 Expression in Rabbit and Mouse Atherosclerotic Lesions and at Sites Predisposed to Lesion Formation
From the Department of Laboratory Medicine and Pathobiology (K.I., L.H., M.I., M.I.C.), University of Toronto and Toronto General Hospital Research Institute, Ontario, Canada, and Vascular Research Division, Department of Pathology (H.L., M.D., B.D.M.), Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Myron I. Cybulsky, MD, Department of Laboratory Medicine and Pathobiology, Toronto General Hospital, 200 Elizabeth St, CCRW 1-855, Toronto, Ontario M5G 2C4, Canada. E-mail myron.cybulsky{at}utoronto.ca
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Abstract |
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Abstract—The recruitment of mononuclear leukocytes and formation of intimal macrophage-rich lesions at specific sites of the arterial tree are key events in atherogenesis. Inducible endothelial cell adhesion molecules may participate in this process. In aortas of normal chow-fed wild-type mice and rabbits, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), but not E-selectin, were expressed by endothelial cells in regions predisposed to atherosclerotic lesion formation. En face confocal microscopy of the mouse ascending aorta and proximal arch demonstrated that VCAM-1 expression was increased on the endothelial cell surface in lesion-prone areas. ICAM-1 expression extended into areas protected from lesion formation. Hypercholesterolemia induced atherosclerotic lesion formation in rabbits, LDL receptor and apolipoprotein E knockout mice, and Northern blot analysis demonstrated increased steady-state mRNA levels of VCAM-1 and ICAM-1, but not of E-selectin. Immunohistochemical staining revealed that VCAM-1 and ICAM-1 were expressed predominantly by endothelium in early lesions and by intimal cells in more advanced lesions. In early and advanced lesions, staining was most intense in endothelial cells at and adjacent to lesion borders. ICAM-1 staining extended into the uninvolved aorta. These expression patterns were highly reproducible in both species. The only difference was that VCAM-1 expression in endothelium over the central portions of lesions was found frequently in rabbits and rarely in mice. The expression of VCAM-1 by arterial endothelium in normal animals may represent a pathogenic mechanism or a phenotypic marker of predisposition to atherogenesis.
Key Words: atherosclerosis • endothelium • vascular cell adhesion molecule-1 • intercellular adhesion molecule-1 • expression pattern
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Introduction |
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Atherosclerosis is a disease process in which blood leukocytes and medial smooth muscle cells migrate into the arterial intima and are organized to form lesions with characteristic morphological features.1 2 Adhesion molecules expressed by endothelium and intimal cells may have pathogenic functions during this process. They may contribute to lesion initiation by participating in the recruitment of blood monocytes and lymphocytes to the intima in the earliest stages of atherogenesis and to lesion expansion at the periphery of lesions.3 Adhesion molecule expression within lesions may influence the organization of cells and promote cytokine/growth factor production and migration of medial smooth muscle cells into the intima, as well as influence cell replication.1
In a variety of species ranging from pigeons to humans, leukocyte accumulation and atherosclerotic lesion formation occur reproducibly at specific sites in the arterial tree, such as the lesser curvature of the aortic arch or adjacent to arterial branches.4 5 6 Leukocyte composition in lesions is highly regulated, consisting of monocytes and lymphocytes, but not polymorphonuclear leukocytes. These features suggest that local events contribute to leukocyte recruitment during atherogenesis. Potential mechanisms include hemodynamic factors, production of chemokines specific for mononuclear leukocytes, and distinct patterns of local adhesion molecule expression.
Our focus was on the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin, because their expression on vascular endothelium and other cell types is regulated by induction of transcription7 and they may be potential targets for therapy. Other endothelial cell adhesion molecules may also be relevant to atherogenesis. For example, P-selectin expression on the endothelial surface can be upregulated rapidly by translocation from cytoplasmic granules. In contrast, ICAM-2 is expressed constitutively by most endothelial cells and, therefore, is not likely to contribute to the topographic pattern of lesion formation.
Our goal was to evaluate and compare inducible adhesion molecule expression in normal and hypercholesterolemic mice and rabbits. This may provide valuable clues to similarities or differences in mechanisms of lesion formation. Atherosclerotic lesion formation has been studied extensively in rabbits, and mice have been used in many recent studies. Through genetic manipulation, mice provide a unique opportunity to assess the roles of adhesion molecules in atherosclerotic lesion formation. Expression patterns of adhesion molecules will provide insights into potential functions at specific stages of lesion formation and will aid in the interpretation of data from genetically altered animals.
Two knockout models, apolipoprotein E (ApoE-/-) and LDL receptor (LDLR-/-) mice, have been particularly popular for studies of atherogenesis.8 9 10 Both develop marked hypercholesterolemia and lesions throughout the aorta. Lesions in ApoE-/- and LDLR-/- mice have morphological features closely resembling human atherosclerosis,11 12 13 which suggests that similar pathogenic mechanisms may be involved. LDLR-/- mice fed a normal chow diet have only a 2-fold elevation in plasma cholesterol and do not develop lesions.10 When fed a 1.25% cholesterol diet (including 7.5% cocoa butter, 7.5% casein, and 0.5% cholic acid), these mice develop marked hypercholesterolemia and extensive lesions throughout the aorta.13 ApoE-/- mice develop hypercholesterolemia and atherosclerotic lesions when fed a normal chow mouse diet; however, if they are fed a Western-type diet (0.15% cholesterol, 21% fat) lesions develop more rapidly.8 11 Each model has advantages and disadvantages. An appealing feature of LDLR-/- mice is that the atherogenic process is initiated with the start of cholesterol feeding, whereas the onset of this process is more difficult to pinpoint in the ApoE-/- model.
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Materials and Methods |
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Animals and Diets
Animals were maintained in accordance with guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (DHEW publication No. [NIH] FS-23) and the Canadian Council on Animal Care. Male and female LDLR-/- mice (mixed 129-C57BL/6 strain) and ApoE-/- mice (crossed 6 times into the C57BL/6 strain) were purchased from The Jackson Laboratory (Bar Harbor, ME). In LDLR-/- mice, hypercholesterolemia was induced by feeding a 1.25% cholesterol diet containing 0.5% cholic acid10 for 4, 5, 8, 10, 12, 20, or 24 weeks (4 to 6 mice per group). Control groups of LDLR-/- mice and ApoE-/- mice received standard mouse laboratory chow. For en face experiments, C57BL/6 and LDLR-/- mice (crossed 10 times into the C57BL/6 strain) were purchased from The Jackson Laboratory. At 2 to 4 months of age, they were fed standard chow or a cholate-free AIN-76A semipurified diet containing high fat (40% of energy intake) and 1.25% cholesterol (diet D12108, Research Diets, Inc).14
Pasturella-free New Zealand White male rabbits weighing 2.0 to 3.0 kg were purchased from Millbrook Farms (Amherst, Mass). Rabbits were assigned to 1 of 2 dietary groups: standard rabbit chow (Purina) or a diet with 0.3% cholesterol and 9% partially hydrogenated coconut oil (Research Diets Inc). Food and water were provided ad libitum. Groups of rabbits were maintained on standard chow for at least 2 weeks and on cholesterol chow for 3, 6, or 9 weeks (5 rabbits per group). Watanabe heritable hyperlipidemic rabbits 10 weeks to 1 year of age were purchased from the NIH (Bethesda, MD) and were fed standard chow.
Systemic Activation of Endothelium
Mice received an intraperitoneal injection
of 100 µg Escherichia coli 055:B5 endotoxin
(lipopolysaccharide [LPS], Sigma). Rabbits were injected
intravenously with 50 µg of LPS. Animals were euthanized
4 hours after LPS injection.
Immunohistochemistry on Aortic Cross Sections
The arterial tree of mice anesthetized with
ethyl ether was perfused at 100 mm Hg via the left ventricle with
20 mL PBS (pH 7.0), followed by 25 mL 2%
paraformaldehyde (4°C). Unfixed aortas were harvested
from rabbits euthanized by intravenous sodium pentobarbital
overdose. Aortas were removed attached to the heart and placed into
ice-chilled PBS, and adipose tissue was removed in situ. Segments of
aorta were immersed in OCT compound, snap frozen in liquid
nitrogen–cooled 2-methylbutane, and stored at -80°C.
Serial aortic cross sections (5 to 6 µm) were cut on a cryostat and placed on tissue section adhesive–coated slides (Vectabond, Vector Laboratories). Rabbit sections were air dried, whereas mouse sections were incubated at 45°C overnight to firmly adhere tissue to the slide. Slides were fixed for 5 minutes in acetone at -20°C and incubated with primary antibodies for 1 hour at 22°C (rabbit) or overnight at 4°C (mouse). Subsequent steps included the following: biotinylated polyclonal secondary antibodies, blocking endogenous peroxidase activity with 0.3% hydrogen peroxide in PBS (20 minutes), avidin-biotin-peroxidase complexes (ABC Elite kit, Vector Laboratories), 3-amino-9-ethylcarbazole, Gill's hematoxylin counterstain, and glycerol gelatin mount (Sigma).
Primary antibodies included the following: monoclonal rat anti-mouse VCAM-1 (M/K-2.7, IgG1, American Type Culture Collection), ICAM-1 (YN1/1.7.4, IgG2b, American Type Culture Collection), and CD31-platelet/endothelial cell adhesion molecule-1 (IgG2a, PharMingen Inc), and mouse anti-rabbit VCAM-1 (Rb1/9, IgG1), ICAM-1 (Rb2/3, IgG1), E-selectin (14G2, IgG1, Hoffman-La Roche Inc), and macrophages (RAM 11, IgG1, Dako). Rabbit endothelial cells were identified with cross-reacting antibodies to human von Willebrand factor (polyclonal goat, Atlantic Antibodies) or human CD31 (JC/70A, mouse IgG1 monoclonal antibody, Dako). Negative controls were nonimmune rat IgG and E1/C15, a mouse IgG1 monoclonal antibody that did not bind to rabbit tissues.
Northern Blotting
Aortas were pooled from 4 mice per group, and total RNA was
obtained by homogenization in guanidinium
thiocyanate and cesium chloride
ultracentrifugation.15 mRNA was isolated
by oligo(dT) cellulose chromatography.15
Rabbit aortas were divided into segments. Total cellular RNA was
extracted with an acid-guanidinium thiocyanate-phenol-chloroform
mixture16 from the portions of the ascending aorta and
portions of arch and descending thoracic aorta. Immunohistochemistry
was performed on segments containing the brachiocephalic and fourth
pair of intercostal artery ostia, and oil red O staining17
on abdominal aortas and thoracic segments with the fifth pair of
intercostal artery ostia.
RNA was electrophoresed through formaldehyde-containing 1% agarose
gels (45 V, overnight), capillary transferred to nylon membranes
(Bio-Trans[+], ICN),15 and hybridized overnight
at 65°C18 with
[
-32P]dCTP-labeled cDNA probes (random
sequence decanucleotide primer kit, Amersham Corp). Washed
membranes were exposed at -80°C to XAR-5 film (Kodak) with enhancing
screens (Dupont), and autoradiographs were analyzed by
densitometry.
Rabbit VCAM-1, ICAM-1, and E-selectin cDNAs were constructed by hybridizing an LPS-activated rabbit endothelial cell cDNA library, and rabbit MCP-1 cDNA was produced by reverse transcriptase–polymerase chain reaction. Murine VCAM-1 cDNA was obtained from Biogen Inc and ß-actin from Ambion Inc. Probes were generated by restriction enzyme digestion and purified by agarose gel electrophoresis and glass beads.15
Mapping of Sites Predisposed and Resistant to
Atherosclerotic Lesion Formation in Proximal Aortas of Mice
The arterial tree of
halothane-anesthetized mice was perfused with PBS, pH 7.4 (5
minutes), and then 2% paraformaldehyde (20 minutes) at
100 mm Hg via the left ventricle. The ascending aorta and arch
were harvested, and adipose tissue was dissected while immersed in cold
PBS. Regions predisposed and resistant to atherosclerotic
lesion formation were determined by oil red O staining in
LDLR-/- mice fed a semipurified, 1.25%
cholesterol, cholate-free diet14 for 4 or 12
weeks (8 mice/group). The ascending aorta and arch were opened in a
highly reproducible manner as outlined in Figure 6
, allowing the
tissue to lie flat on a microscope slide. En face oil red O-stained
aortas were photographed and scanned into a computer. Using image
analysis software (C-imaging) and 3 anatomic landmarks, each
aorta was transformed to a standard size and overlaid with a grid.
Squares in the grid were scored for presence or absence of oil red O
staining, and probability maps for lesion development were
constructed.
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En Face Analysis of Endothelial Cell
Surface VCAM-1 Expression in Normal Mice
Immunostaining for
endothelial cell surface VCAM-1 was carried out in 2-
to 4-month-old C57BL/6 and LDLR-/- mice fed
standard chow. After perfusion fixation and dissection, the ascending
aorta and proximal arch segment were incubated with 20% donkey serum
(15 minutes) and then overnight on a rotator with M/K-2.7 hybridoma
supernatant (4°C). The secondary antibody was Cy-3–labeled donkey
anti-rat IgG (1:200 dilution, 30 minutes, 22°C). Aortas were
washed 3 times with PBS between incubations, stained with green nucleic
acid stain (1:100 dilution, 30 minutes, Sytox, Molecular
Probes), opened as above, and mounted on slides using mounting medium
(Vectashield, Vector Laboratories). The distal arch served as the
negative control and was incubated with nonimmune rat IgG (10 µg/mL)
instead of M/K-2.7. Images of the endothelial cell
monolayer were obtained using a Bio-Rad MRC-600 confocal microscope
equipped with a krypton/argon laser and a 60x 1.4–numerical aperture
objective (Nikon). For each mouse, images were obtained from regions
with high and low probabilities (HPs and LPs, respectively) for lesion
development (3 or 4 images per region) and from the negative control
using the same confocal settings. The HP and LP regions were located
using a simple mathematical equation and 3 anatomic landmarks as
reference points. Fluorescence in the Cy-3 channel (excitation,
568 nm; emission, >585 nm) was quantified using the confocal software
frequency histogram function. For each mouse, the negative control was
used to establish a pixel intensity that eliminated 99% of the
background signal. Background fluorescence was then subtracted
by applying this threshold to all HP and LP images, and the percentage
pixels with remaining signal and the average signal intensity were
determined for each image. The data showed a normal distribution, and
statistical differences were evaluated with an unpaired t
test. Detection of only cell surface VCAM-1 was verified by
immunostaining with a goat polyclonal antibody to
ß-catenin (Santa Cruz Biotechnology). ß-Catenin is a cytoplasmic
protein and was not detected unless cells were
permeabilized before staining with 0.2% Triton
X-100.
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Results |
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Aortic VCAM-1 and ICAM-1 Expression Is Upregulated by Hypercholesterolemia
Northern blot analysis was used to quantify adhesion molecule expression in aortas of normal or hypercholesterolemic rabbits and mice (Figure 1
). VCAM-1 and ICAM-1 steady-state mRNA
was increased 2- to 3-fold (normalized to ß-actin) in
LDLR-/- mice fed a cholesterol diet
for 12 weeks and in 34-week-old ApoE-/- mice
fed a chow diet (Figure 1a and Table 1). Similar increases in VCAM-1 and
ICAM-1 expression (2.7- and 10-fold, respectively, normalized to
ethidium bromide–stained 28S rRNA) were observed in rabbits fed a
cholesterol diet for 9 weeks (Figure 1b and Table 2). In rabbits, the magnitude of
VCAM-1, but not ICAM-1, expression correlated (r=0.99) with
the extent of surface area involved by oil red O–stained lesions in
segments of descending thoracic and abdominal aorta from individual
rabbits (Table 2). In mice, 2 VCAM-1 transcripts were detected
(Figure 1a). The upper band (below 28S rRNA) is the 7
Ig–like domain form, and the lower (below 18S rRNA) band is an
alternatively spliced 3 Ig–like form that is unique to
rodents.19 20 Two VCAM-1 mRNA transcripts were also
detected in rabbit aortas (Figure 1b). The upper band may be an
alternatively polyadenylated or spliced form of VCAM-1. We
identified an 8 Ig–like domain form of VCAM-1 in LPS-activated
rabbit endothelium (data not shown). The additional
Ig-like domain was unique to rabbits, was located between the 7th and
transmembrane domains, and was homologous to nonfunctional domains.
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E-selectin expression was not detected in aortas of mice (not
shown) and rabbits (Figure 1b
), yet E-selectin mRNA was readily
detected in lungs of LPS-treated rabbits and mice, which indicates that
the sensitivity of Northern blotting did not account for the absence of
detectable transcripts in the aorta.
VCAM-1 and ICAM-1 Are Expressed Predominantly by
Endothelial Cells in Early Atherosclerotic Lesions and
by Intimal Cells in More Advanced Lesions
In early lesions composed predominantly of
macrophage foam cells, expression of VCAM-1 and ICAM-1, but not
of E-selectin, was detected by immunohistochemistry predominantly in
endothelium. In more advanced lesions, expression was
most abundant in intimal cells (Figure 2), which consist mostly of
macrophages with variable numbers of smooth muscle cells.
Smooth muscle cells in the intima and in culture can express
VCAM-1.21 22 Expression of VCAM-1, but not of ICAM-1, was
frequently detected in medial smooth muscle cells adjacent to
lesions.
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Endothelial Cell VCAM-1 Expression Is Restricted to
Lesions, Whereas ICAM-1 Expression Extends Into the Uninvolved
Aorta
In hypercholesterolemic mice and rabbits,
VCAM-1 expression by endothelial cells was restricted
to intimal lesions (Figures 2 and 3). Expression by
endothelium was most intense at edges (borders) of
lesions and extended only several cells beyond lesions. In
endothelium over central portions of lesions,
expression of VCAM-1 was variable and different between mice and
rabbits. In mice, the majority of these endothelial
cells did not express VCAM-1, particularly in more advanced lesions,
whereas in rabbits, VCAM-1 was detected frequently in early and
advanced lesions (Figure 3). When
LDLR-/- cholesterol-fed mice were
injected with LPS, VCAM-1 (and ICAM-1) expression was found in all
endothelial cells over lesions (Figure 4), indicating that these cells have the
potential to express adhesion molecules when appropriately
activated.
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) in lesions. ICAM-1 is
expressed by endothelium over lesions and in the
uninvolved aorta. CD31 staining indicates an intact
endothelial cell layer. Bar=100 µm.
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Endothelial cell expression of ICAM-1 was upregulated
at edges and over lesions, but unlike VCAM-1, it extended much farther
into the uninvolved aorta (Figure 3). In regions such as the
aortic arch, ICAM-1 staining was detected in virtually all
endothelial cells, even when lesions involved only a
fraction of the lumen circumference.
In Normal Animals, Endothelial Cells at Sites
Predisposed to Lesion Formation Express VCAM-1 and ICAM-1
Initially, we evaluated adhesion molecule expression in
random cross sections of the descending thoracic aorta from normal
rabbits and C57BL/6 mice. VCAM-1 was expressed in occasional
endothelial and medial smooth muscle cells,
endothelial ICAM-1 expression was more abundant, and
E-selectin was not detected. (All endothelial cells
expressed these molecules after LPS treatment.) The descending thoracic
aorta provides abundant tissue for analysis, but only small
areas adjacent to intercostal artery ostia are predisposed to
atherosclerotic lesion formation. Cross sections of the aortic arch
were studied to determine whether a different adhesion molecule
expression pattern occurs in the lesser curvature, a site predisposed
to lesion formation. In mice and rabbits fed standard chow, expression
of VCAM-1 was observed in endothelial cells of the
lesser curvature and over intimal cushions of the brachiocephalic
artery (Figure 5). ICAM-1 expression
colocalized with VCAM-1 but was more diffuse. In many mice, ICAM-1
staining was nearly circumferential. Significant differences were not
seen between C57BL/6 and standard chow-fed
LDLR-/- mice (<6 months of age). VCAM-1 and
ICAM-1 expression was also observed in the thoracic and abdominal
aortas of rabbits and mice near ostia of intercostal, mesenteric, and
renal arteries (not shown).
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Endothelial cell surface expression of VCAM-1 in
normocholesterolemic mice was quantified in regions
with different probabilities for developing lesions. HP and LP regions
were mapped in the ascending aorta and proximal arch of
LDLR-/- mice (Figure 6). VCAM-1 expression on the
endothelial cell surface was quantified by
immunostaining and en face confocal microscopy in
standard diet-fed LDLR+/+ and
LDLR-/- mice (C57BL/6 background, 
10
generations). In every mouse, VCAM-1 expression was detected in HP
regions, and staining appeared more intense than in LP regions (Figure 7). Quantitative analysis of the
percentage area covered by specific VCAM-1 signal, as well as the
average intensity of the signal, revealed significant differences
between HP and LP regions (Table 3).
VCAM-1 staining was over the entire endothelial cell
surface in HP regions, and in some cells, staining was concentrated at
intercellular junctions (Figure 7). Previously, VCAM-1 was
observed at endothelial cell junctions in the rabbit
carotid artery.23
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Discussion |
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Immunostaining of normal aortas clearly demonstrated endothelial cell VCAM-1 expression in regions predisposed to atherosclerotic lesion formation. These observations are consistent with our Northern blot data, which showed readily detectable levels of VCAM-1 steady-state mRNA in normal mouse and rabbit aortas. Recently, Nakashima et al24 observed that endothelial VCAM-1 expression was upregulated by hypercholesterolemia found in ApoE-/- mice before lesion formation but did not detect significant expression in lesion-prone sites of normal C57BL/6 mice. The immunostaining approaches used in our study may have been more sensitive and may account for our ability to detect VCAM-1 expression in aortas of normal animals. Our observations of ICAM-1 expression in HP regions of normal animals and lower levels in LP regions are consistent with those of Nakashima et al.24
In normocholesterolemic animals, expression of VCAM-1 and ICAM-1 in endothelial cells at sites predisposed to lesion formation may be related to complex hemodynamics in these regions. In vitro, introduction of shear stress can activate various endothelial cell signal transduction pathways25 26 and influence the expression of adhesion molecules,27 28 and different shear stress profiles can induce unique repertoires of endothelial cell gene expression.29 30 Similar phenomena may occur at HP sites in vivo. Also, hemodynamics may increase local permeability or transport of lipoproteins by endothelium at HP sites and promote lipoprotein retention in the intima. Local oxidation of lipoproteins trapped in the intima may generate soluble factors that induce endothelial adhesion molecule expression.
The expression of VCAM-1 and ICAM-1 by aortic endothelium in normal animals may result in occasional recruitment of monocytes into the intima. Intimal monocytes/macrophages have been reported at lesion-predisposed sites of normal rabbits.31 These intimal cells may contribute to enhanced recruitment to the arterial intima of circulating monocytes after the initiation of hypercholesterolemia. A potential mechanism for this is production of chemokines and inflammatory cytokines during engulfment of oxidized lipoproteins and transformation into foam cells. Thus, the localized expression of VCAM-1 and ICAM-1 in aortic endothelium of normal animals may provide a milieu for atherosclerotic lesion formation.
In rabbits and mice, hypercholesterolemia
upregulates VCAM-1 and ICAM-1 expression in arterial
endothelium even before atherosclerotic lesion
formation.24 32 We found that expression of VCAM-1 and
ICAM-1, but not of E-selectin, was increased in the aorta of
hypercholesterolemic rabbits and mice and appeared to
be proportional to the extent of atherosclerotic lesion formation.
These data are consistent with and extend previous
observations.21 24 32 33 34 In small lesions, VCAM-1 and
ICAM-1 were expressed predominantly by endothelial
cells, whereas in large foam cell–rich lesions many intimal cells
expressed these molecules. Immunostaining of 12-week
lesions revealed abundant VCAM-1 and ICAM-1 expression by intimal
cells, as was found in 20-week lesions shown in Figure 2. It is
likely that expression by intimal cells accounted for increased VCAM-1
and ICAM-1 steady-state mRNA levels in Northern blots. Medial smooth
muscle cells adjacent to lesions also expressed VCAM-1, and this may
represent a phenotypic change in activated or migrating
cells.
Endothelial cell VCAM-1 and ICAM-1 expression patterns were highly reproducible and similar in rabbits and LDLR-/- mice fed cholesterol-containing diets. Expression was most pronounced in endothelial cells at edges (shoulders) of both large and small lesions and extended several cells beyond the edge. VCAM-1 expression was essentially restricted to lesions, whereas ICAM-1 expression extended into the uninvolved aorta. These data suggest that similar mechanisms may influence adhesion molecule expression in atherosclerotic lesions of mice and rabbits, but the regulation of VCAM-1 expression is controlled more precisely than ICAM-1 by lesion-derived factors. Although not presented, virtually identical VCAM-1 and ICAM-1 immunohistochemical staining patterns were obtained from ApoE-/- mice and Watanabe heritable hyperlipidemic rabbits fed standard laboratory chow. This indicates that hypercholesterolemia, and not other dietary factors, was responsible for upregulated VCAM-1 and ICAM-1 expression in lesions.
We found a difference between mice and rabbits regarding the extent of
VCAM-1 expression by endothelium overlying central
regions of lesions. In both species, VCAM-1 was expressed at edges of
lesions, but in rabbits, expression was relatively abundant over the
central regions of early and large foam cell lesions, although there
was variability between lesions. In LDLR-/-
mice, endothelial cells over the central portions of
lesions generally did not express VCAM-1 but retained the potential, as
was demonstrated by treating mice with LPS (Figure 4). The
mechanism for this species difference in VCAM-1 expression is not
obvious. One could speculate that a minor difference in the promoters
of the mouse and rabbit VCAM1 genes could result in slightly
different regulation of expression. Alternatively, the environment in
the central regions of mouse and rabbit lesions may be different, eg,
the degree of oxidative stress or production of a different
growth factor(s)/cytokine(s). In advanced human atherosclerotic
lesions, lumen endothelial VCAM-1 expression was
associated with subendothelial monocyte accumulation
and was not abundant.22
We did not detect E-selectin expression in mouse or rabbit atherosclerotic lesions. These data are consistent with other observations in rabbits,34 but they differ from reports of E-selectin expression in human lesions.35 36 37 Possible explanations are that human lesions were more advanced, some patients had associated conditions that induced E-selectin expression, and the antibody to human E-selectin used in some of these studies (BBA 1) was subsequently found to cross-react with P-selectin.
A comparison of endothelial cell VCAM-1 and ICAM-1
expression in lesions and lesion-predisposed sites suggests different
regulatory mechanisms. In normal animals, expression was scattered
throughout lesion-predisposed sites and probably was influenced by
local hemodynamic forces. However, even in early
lesions, expression was localized to lesion borders, and in mice,
VCAM-1 expression was absent from the center. Therefore, intrinsic
properties of lesions, rather than hemodynamics, are
likely the dominant factors responsible for this expression pattern.
Potential mechanisms include oxidative stress, NF-
B activation, and
altered nitric oxide production.
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Acknowledgments |
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This work was supported by the Heart and Stroke Foundation of Ontario (Grant T-3588) and the Medical Research Council of Canada (Grant MT-14151). M.I.C. holds an Established Investigatorship from the American Heart Association.
Received March 3, 1999; accepted April 26, 1999.
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A. Furnkranz, A. Schober, V. N. Bochkov, P. Bashtrykov, G. Kronke, A. Kadl, B. R. Binder, C. Weber, and N. Leitinger Oxidized Phospholipids Trigger Atherogenic Inflammation in Murine Arteries Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 633 - 638. [Abstract] [Full Text] [PDF]
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K. A. Kelly, J. R. Allport, A. Tsourkas, V. R. Shinde-Patil, L. Josephson, and R. Weissleder Detection of Vascular Adhesion Molecule-1 Expression Using a Novel Multimodal Nanoparticle Circ. Res., February 18, 2005; 96(3): 327 - 336. [Abstract] [Full Text] [PDF]
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 P.-Y. Chang, T.-L. Wu, K.-C. Tsao, C.-C. Li, C.-F. Sun, and J. T. Wu Microplate ELISAs for Soluble VCAM-1 and ICAM-1 Ann. Clin. Lab. Sci., January 1, 2005; 35(3): 312 - 317. [Abstract] [Full Text] [PDF]
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 M. P. Burns and N. DePaola Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H194 - H204. [Abstract] [Full Text] [PDF]
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R. P. Choudhury, J. X. Rong, E. Trogan, V. I. Elmalem, H. M. Dansky, J. L. Breslow, J. L. Witztum, J. T. Fallon, and E. A. Fisher High-Density Lipoproteins Retard the Progression of Atherosclerosis and Favorably Remodel Lesions Without Suppressing Indices of Inflammation or Oxidation Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1904 - 1909. [Abstract] [Full Text] [PDF]
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 S. Bro, F. Moeller, C. B. Andersen, K. Olgaard, and L. B. Nielsen Increased Expression of Adhesion Molecules in Uremic Atherosclerosis in Apolipoprotein-E-Deficient Mice J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1495 - 1503. [Abstract] [Full Text] [PDF]
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R. Khurana, S. Shafi, J. Martin, and I. Zachary Vascular Endothelial Growth Factor Gene Transfer Inhibits Neointimal Macrophage Accumulation in Hypercholesterolemic Rabbits Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 1074 - 1080. [Abstract] [Full Text] [PDF]
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S. SenBanerjee, Z. Lin, G. B. Atkins, D. M. Greif, R. M. Rao, A. Kumar, M. W. Feinberg, Z. Chen, D. I. Simon, F. W. Luscinskas, et al. KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation J. Exp. Med., May 17, 2004; 199(10): 1305 - 1315. [Abstract] [Full Text] [PDF]
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 J.-S. Choi, Y.-J. Choi, S.-H. Park, J.-S. Kang, and Y.-H. Kang Flavones Mitigate Tumor Necrosis Factor-{alpha}-Induced Adhesion Molecule Upregulation in Cultured Human Endothelial Cells: Role of Nuclear Factor-{kappa}B J. Nutr., May 1, 2004; 134(5): 1013 - 1019. [Abstract] [Full Text] [PDF]
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 A. K. Death, K. C. Y. McGrath, M. A. Sader, S. Nakhla, W. Jessup, D. J. Handelsman, and D. S. Celermajer Dihydrotestosterone Promotes Vascular Cell Adhesion Molecule-1 Expression in Male Human Endothelial Cells via a Nuclear Factor-{kappa}B-Dependent Pathway Endocrinology, April 1, 2004; 145(4): 1889 - 1897. [Abstract] [Full Text] [PDF]
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C. Kunsch, J. Luchoomun, J. Y. Grey, L. K. Olliff, L. B. Saint, R. F. Arrendale, M. A. Wasserman, U. Saxena, and R. M. Medford Selective Inhibition of Endothelial and Monocyte Redox-Sensitive Genes by AGI-1067: A Novel Antioxidant and Anti-Inflammatory Agent J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 820 - 829. [Abstract] [Full Text] [PDF]
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A. G. Passerini, D. C. Polacek, C. Shi, N. M. Francesco, E. Manduchi, G. R. Grant, W. F. Pritchard, S. Powell, G. Y. Chang, C. J. Stoeckert Jr., et al. Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta PNAS, February 24, 2004; 101(8): 2482 - 2487. [Abstract] [Full Text] [PDF]
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R. M. Porat, M. Grunewald, A. Globerman, A. Itin, G. Barshtein, L. Alhonen, K. Alitalo, and E. Keshet Specific Induction of tie1 Promoter by Disturbed Flow in Atherosclerosis-Prone Vascular Niches and Flow-Obstructing Pathologies Circ. Res., February 20, 2004; 94(3): 394 - 401. [Abstract] [Full Text] [PDF]
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S. Kaul and J. R. Lindner Visualizing coronary atherosclerosis in vivo: thinking big, imaging small J. Am. Coll. Cardiol., February 4, 2004; 43(3): 461 - 463. [Full Text] [PDF]
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R. Kiefmann, K. Heckel, M. Dorger, S. Schenkat, M. Stoeckelhuber, J. Wesierska-Gadek, and A. E. Goetz Role of poly(ADP-ribose) synthetase in pulmonary leukocyte recruitment Am J Physiol Lung Cell Mol Physiol, November 1, 2003; 285(5): L996 - L1005. [Abstract] [Full Text] [PDF]
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 B. OSTERUD and E. BJORKLID Role of Monocytes in Atherogenesis Physiol Rev, October 1, 2003; 83(4): 1069 - 1112. [Abstract] [Full Text] [PDF]
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 A.-L. Yang, C. J. Jen, and H.-i. Chen Effects of high-cholesterol diet and parallel exercise training on the vascular function of rabbit aortas: a time course study J Appl Physiol, September 1, 2003; 95(3): 1194 - 1200. [Abstract] [Full Text] [PDF]
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S.-P. Yang, L.-J. Ho, Y.-L. Lin, S.-M. Cheng, T.-P. Tsao, D.-M. Chang, Y.-L. Hsu, C.-Y. Shih, T.-Y. Juan, and J.-H. Lai Carvedilol, a new antioxidative {beta}-blocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-{kappa}B activity Cardiovasc Res, September 1, 2003; 59(3): 776 - 787. [Abstract] [Full Text] [PDF]
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C. L. Sundell, P. K. Somers, C. Q. Meng, L. K. Hoong, K.-L. Suen, R. R. Hill, L. K. Landers, A. Chapman, D. Butteiger, M. Jones, et al. AGI-1067: A Multifunctional Phenolic Antioxidant, Lipid Modulator, Anti-Inflammatory and Antiatherosclerotic Agent J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1116 - 1123. [Abstract] [Full Text] [PDF]
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A. Tailor and D. N. Granger Hypercholesterolemia Promotes P-Selectin-Dependent Platelet-Endothelial Cell Adhesion in Postcapillary Venules Arterioscler Thromb Vasc Biol, April 1, 2003; 23(4): 675 - 680. [Abstract] [Full Text] [PDF]
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J. Guo, M. Van Eck, J. Twisk, N. Maeda, G. M. Benson, P. H.E. Groot, and T. J.C. Van Berkel Transplantation of Monocyte CC-Chemokine Receptor 2-Deficient Bone Marrow Into ApoE3-Leiden Mice Inhibits Atherogenesis Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 447 - 453. [Abstract] [Full Text] [PDF]
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F. de Nigris, L. O. Lerman, S. W. Ignarro, G. Sica, A. Lerman, W. Palinski, L. J. Ignarro, and C. Napoli From the Cover: Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress PNAS, February 4, 2003; 100(3): 1420 - 1425. [Abstract] [Full Text] [PDF]
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 G. D. Angelini, C. Lloyd, R. Bush, J. Johnson, and A. C. Newby An external, oversized, porous polyester stent reduces vein graft neointima formation, cholesterol concentration, and vascular cell adhesion molecule 1 expression in cholesterol-fed pigs J. Thorac. Cardiovasc. Surg., November 1, 2002; 124(5): 950 - 956. [Abstract] [Full Text] [PDF]
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A. D. Pradhan, N. Rifai, and P. M. Ridker Soluble Intercellular Adhesion Molecule-1, Soluble Vascular Adhesion Molecule-1, and the Development of Symptomatic Peripheral Arterial Disease in Men Circulation, August 13, 2002; 106(7): 820 - 825. [Abstract] [Full Text] [PDF]
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I. Baranova, T. Vishnyakova, A. Bocharov, Z. Chen, A. T. Remaley, J. Stonik, T. L. Eggerman, and A. P. Patterson Lipopolysaccharide Down Regulates Both Scavenger Receptor B1 and ATP Binding Cassette Transporter A1 in RAW Cells Infect. Immun., June 1, 2002; 70(6): 2995 - 3003. [Abstract] [Full Text] [PDF]
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S. M. Lessner, H. L. Prado, E. K. Waller, and Z. S. Galis Atherosclerotic Lesions Grow Through Recruitment and Proliferation of Circulating Monocytes in a Murine Model Am. J. Pathol., June 1, 2002; 160(6): 2145 - 2155. [Abstract] [Full Text] [PDF]
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 A. R. Brooks, P. I. Lelkes, and G. M. Rubanyi Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow Physiol Genomics, April 10, 2002; 9(1): 27 - 41. [Abstract] [Full Text] [PDF]
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M. E. Rosenfeld Leukocyte Recruitment Into Developing Atherosclerotic Lesions: The Complex Interaction Between Multiple Molecules Keeps Getting More Complex Arterioscler Thromb Vasc Biol, March 1, 2002; 22(3): 361 - 363. [Full Text] [PDF]
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H. M. Dansky, C. B. Barlow, C. Lominska, J. L. Sikes, C. Kao, J. Weinsaft, M. I. Cybulsky, and J. D. Smith Adhesion of Monocytes to Arterial Endothelium and Initiation of Atherosclerosis Are Critically Dependent on Vascular Cell Adhesion Molecule-1 Gene Dosage Arterioscler Thromb Vasc Biol, October 1, 2001; 21(10): 1662 - 1667. [Abstract] [Full Text] [PDF]
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E. E. ERIKSSON, X. XIE, J. WERR, P. THOREN, and L. LINDBOM Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins FASEB J, May 1, 2001; 15(7): 1149 - 1157. [Abstract] [Full Text] [PDF]
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K. Y. Stokes, E. C. Clanton, J. M. Russell, C. R. Ross, and D. N. Granger NAD(P)H Oxidase-Derived Superoxide Mediates Hypercholesterolemia-Induced Leukocyte-Endothelial Cell Adhesion Circ. Res., March 16, 2001; 88(5): 499 - 505. [Abstract] [Full Text] [PDF]
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J. H. von der Thusen, T. J.C. van Berkel, and E. A.L. Biessen Induction of Rapid Atherogenesis by Perivascular Carotid Collar Placement in Apolipoprotein E-Deficient and Low-Density Lipoprotein Receptor-Deficient Mice Circulation, February 27, 2001; 103(8): 1164 - 1170. [Abstract] [Full Text] [PDF]
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G. W. Cockerill, T. Y. Huehns, A. Weerasinghe, C. Stocker, P. G. Lerch, N. E. Miller, and D. O. Haskard Elevation of Plasma High-Density Lipoprotein Concentration Reduces Interleukin-1-Induced Expression of E-Selectin in an In Vivo Model of Acute Inflammation Circulation, January 2, 2001; 103(1): 108 - 112. [Abstract] [Full Text] [PDF]
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