© 1999 American Heart Association, Inc.
Original Contributions |
Focal Increases in Vascular Cell Adhesion Molecule-1 and Intimal Macrophages at Atherosclerosis-Susceptible Sites in the Rabbit Aorta After Short-Term Cholesterol Feeding
From the Department of Biomedical Engineering, Duke University, Durham, NC.
Correspondence to George A. Truskey, Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Box 90281, Durham, NC 27708-0281. E-mail gtruskey{at}acpub.duke.edu
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Abstract |
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Abstract—We tested the hypotheses that vascular cell adhesion molecule-1 (VCAM-1) expression on endothelium at lesion-prone sites in the rabbit aorta correlates with exposure to plasma cholesterol and that macrophage accumulation is associated with endothelial cells expressing VCAM-1. After rabbits were fed 0.25% cholesterol for 2 weeks, VCAM-1 expression was selectively increased at the distal and lateral portions of the major abdominal branches. In the arch and the celiac, superior mesenteric, and renal artery branches, VCAM-1 expression was positively correlated with the plasma cholesterol integrated over the duration of the experiments. After 2 weeks of cholesterol feeding, more macrophages were present around distal and lateral portions of the intercostal arteries and major abdominal branches relative to nonbranch regions. In the arch and around the intercostals and major abdominal branches, macrophage densities were positively correlated with the integrated plasma cholesterol. VCAM-1 and macrophage levels were correlated in lesion-prone regions. In normocholesterolemic rabbits, 23±4% (mean±SEM) of the macrophages were directly associated with VCAM-1–positive endothelium. After 2 weeks of 0.25% cholesterol feeding, the association increased to 37±4% (P<0.015). Associations were highest around the lateral and distal regions of the major abdominal branches. These results suggest that (1) VCAM-1 expression and intimal macrophage densities are influenced by plasma cholesterol and regional factors such as arterial fluid dynamics and (2) VCAM-1 plays a significant role in the localization of macrophages.
Key Words: atherosclerosis • endothelium • monocyte • hypercholesterolemia
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Introduction |
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Macrophage and LDL accumulation in the arterial intima is the earliest event during atherogenesis. Before the first visible lesions appear around the lateral and distal portions of arterial branches in hypercholesterolemic animals,1 lesion-prone sites differ from other arterial regions. In normocholesterolemic animals, lesion-prone regions exhibit elevated permeability to macromolecules. Within 2 weeks after onset of a hypercholesterolemic diet, lipoprotein and cholesterol accumulation begins and monocytes attach to endothelial cells.1 2 3
Monocyte adhesion to endothelium is stimulated by the expression of adhesion molecules on the endothelium, such as VCAM-1,4 5 6 7 8 E-selectin,6 P-selectin,7 8 9 intercellular adhesion molecule-1 (ICAM-1),8 and, a recently discovered glycoprotein, vascular monocyte adhesion-associated protein (VMAP-1).10 Considerable interest exists in the role of VCAM-1 that is specific for monocytes and leukocytes.4 Shortly after onset of a hypercholesterolemic diet, rabbits express VCAM-1 in the ascending aortic arch,5 around the intercostal arteries,7 and throughout the abdominal and thoracic aortas.5 VCAM-1 expression appeared to precede macrophage accumulation.5 7 Adhesion receptor expression and monocyte adhesion are affected by the local fluid dynamics11 as well as normal12 or oxidized13 LDL.
In the normal rabbit, the density of intimal macrophages is higher in the arch and around the lesion-prone regions of major orifices in the abdominal aorta than in nonbranch regions of the aorta.14 In rabbits fed a hypercholesterolemic diet, monocyte densities were greater near intercostal orifices than in nonbranch regions.15 The localized nature of monocyte adhesion suggests that hemodynamic factors and alterations in the vessel wall induced by lipoproteins influence where monocytes attach to the arterial endothelium.
Although a hypercholesterolemic diet is known to induce VCAM-1 expression, little information is available about the relation between plasma cholesterol and VCAM-1 expression and the distribution and association of VCAM-1 and macrophages around vessel branches where lesions first occur. We hypothesize that focal expression of VCAM-1 on endothelium around vessel branches correlates with the exposure to plasma cholesterol and that macrophage accumulation is associated with endothelial cells expressing VCAM-1. To test these hypotheses, rabbits were fed 0.25% cholesterol for 1 and 2 weeks. We examined the distribution of VCAM-1 expression and intimal monocytes in the arch and at lesion-prone branch sites and lesion-free sites in the thoracic aorta and abdominal aorta.
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Methods |
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Animal Procedures
A total of 23 healthy male New Zealand White rabbits were fed rabbit chow with or without 0.25% (wt/wt) cholesterol (Harlan Teklad) for 1 or 2 weeks. Total plasma cholesterol was determined with an enzymatic assay (Sigma Chemical Co). On the day of the experiment, rabbits received 2 mL of sodium heparin (710 U/mL) in 0.15 mol/L sterile saline through the marginal ear vein. Two minutes later, this was followed by an overdose of sodium pentobarbital (340 mg/kg, Delmarva Laboratories, Inc). The thoracic cavity was opened, and a 1/8-in catheter with a 3/16-in tubing tip was inserted through the left ventricle into the arch. The inferior vena cava was quickly opened. The aorta was perfused with 60 mL of sterile saline, followed by 800 to 1000 mL 10% formalin at 100 mm Hg for 15 minutes. Blood was removed by aspiration. After fixation, the aorta, from the brachiocephalic artery branch to the branches of the iliac arteries, was removed, cleaned, and opened.
Tissue Processing
For en face preparations, connective tissue and adherent blood
were gently removed from the adventitial surface under a dissecting
microscope. The vessel was cut open along the ventral aspect opposite
the intercostal arteries and the celiac, superior mesenteric, and right
and left renal arteries. The aorta was divided into 12 to 15 regions,
which were each pinned onto wax stages with the lumen side exposed.
For frozen sections, 1-cm pieces of aortic tissue from the arch, thoracic aorta, and abdominal aorta in the region of the celiac and mesenteric branches were snap-frozen with OCT compound (Miles Inc) in isopentane chilled in liquid nitrogen. Blocks, 3 mm on each side, of the lung and thymus were frozen in a similar manner. Sectioning was performed at the Immunopathology Laboratory at Duke University Medical Center. Frozen sections 4 µm thick were placed on glass slides and stored at -70°C until processed further. Five slides, each containing 4 frozen sections, were cut from each of the 3 aortic regions for a total of 60 sections from each animal. Within the arch and abdominal regions, the proximity to large branches was indicated by the presence of the flow divider and the branch itself within the section. To verify that the frozen sections retained endothelium, frozen sections were stained with FITC–labeled anti–von Willebrand factor antibody and visualized using fluorescence microscopy.
Immunohistochemistry
The following monoclonal antibodies were used: Rb1/9, a mouse
IgG1 that recognizes rabbit VCAM-14
(M.I. Cybulsky, MD, Brigham and Women's Hospital, Boston, Mass);
RAM-11 (Dako, Carpinteria, Calif), a mouse IgG1 that recognizes rabbit
macrophages; L11/135, a mouse antibody that binds to CD43 and
recognizes all thymocytes and T lymphocytes16 17 ; and 2C4,
a mouse IgG2a that recognizes major histocompatibility complex
(MHC)-II. L11/135 and 2C4 were harvested from high-density hybridoma
cultures using cells from the American Type Culture Collection
(Manassas, Va).18 19 Primary antibodies were diluted in
PBS or Tris-buffered saline (TBS) (Sigma) as follows: Rb1/9 (1:50 or
1:100), RAM-11 (1:50), L11/135 (1:100), and 2C4 (1:100).
For en face preparations, aortic sections were washed 2 times with PBS for 10 minutes each wash. Endogenous peroxidases were blocked with an incubation of 15 minutes in 0.3% H2O2 in methanol. Sections were washed 2 times with PBS, 10 minutes each. Nonspecific binding was blocked by incubation of tissue with diluted horse serum for 20 minutes. Sections were incubated at room temperature for 1 hour with primary antibody, followed by 3 washes for 3 minutes each with PBS containing 2% to 4% fetal bovine serum. Secondary antibody from Vectastain Elite ABC Kit (anti-murine IgG, PK-6102, Vector Laboratories) was applied to sections for 30 minutes. Sections were washed 2 times with PBS for 3 minutes each. The avidin-biotin complex was applied for 30 minutes, and sections were washed 2 times with PBS for 3 minutes each. Sections were incubated for 8 minutes 45 seconds in DAB substrate (DAB kit SK-4100, Vector Laboratories). After staining, tissue pieces were rinsed with PBS for 5 minutes and counterstained with Gill's hematoxylin. Sections were stored in scintillation vials in a solution of one-third water, one-third glycerol, and one-third 70% EtOH.
For frozen sections, TBS, pH 7.2, was used for all dilutions and washings. The slides were fixed for 10 minutes in acetone at -20°C and then air-dried. Then the same immunohistochemical procedure as en face was used, but after the final rinse, slides were dehydrated with ethanol, cleared with xylene, and coverslipped with Permount (Sigma).
Positive controls for Rb1/9, RAM-11, and 2C4 were rabbits that received 40 µg/kg lipopolysaccharide endotoxin (Sigma) for 4 hours. Rabbit lung tissue was used as a positive control for both macrophages and MHC-II complex. Rabbit thymus served as a positive control for T cells and MHC-II complex. Negative controls consisted of isotype-matched mouse IgG1 and IgG2a as well as the absence of the primary antibody.
Determination of VCAM-1 Expression and Intimal White Blood
Cell Density
To determine the density of VCAM-1–positive
endothelium, aortic sections 10 to 35
mm2 in area were placed between glass slides and
viewed at x100 magnification. Starting in one corner of the tissue,
nonoverlapping horizontal rows were scanned. At roughly 1-mm intervals,
1-mm2 regions were examined for VCAM-1–positive
endothelium. A second scan began in the opposite corner
on nonoverlapping vertical columns. Both scans were averaged for the
VCAM-1 density per section. When the section included intercostal
arteries or abdominal branches, data were separated into branch regions
within 1 mm of the branch opening and nonbranch regions. For
branch regions, multiple samples were obtained from the proximal,
distal, and lateral regions around each branch.
Intimal white blood cells were identified on the basis of nuclear morphological examination according to the method of Malinauskas et al.14 White blood cell nuclei were horseshoe-shaped, lobular, or elongated. Compared with elliptical nuclei for endothelium or smooth muscle cells, the nuclei of these cells generally stained a darker blue with the hematoxylin. By light microscopy (Axioplan, Carl Zeiss, Inc) at x400 oil immersion, aortic sections were scanned as described above. Grids of 0.0625 mm2 were examined and separated into branch (within 1 mm of branch opening) and nonbranch regions. For branch regions, samples were taken at proximal, distal, and 2 lateral locations. Densities (cells/mm2) of VCAM-1–positive cells and intimal macrophages, as well as any associations, defined as a macrophage touching a VCAM-1–positive cell, were determined.
Statistical Methods
All values are reported as the mean±SEM. The change in
plasma cholesterol with feeding and the effect of the
duration of cholesterol feeding on VCAM-1 and
macrophage densities were examined by ANOVA with repeated
measures. Tukey's test was used to detect differences between control
and cholesterol-fed animals.20 For VCAM-1 and
white blood cell densities around branches, differences among proximal,
lateral, and distal samples were assessed by use of the
2 goodness-of-fit test.20 Because
variances were dissimilar, changes in VCAM-1 levels and
macrophage densities as a function of time were determined with
the nonparametric Kruskal-Wallis test, and multiple
comparisons were performed with Dunn's test.20
Comparisons between branch and nonbranch locations were performed with
a repeated measures ANOVA and Bonferroni's test.
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Results |
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Plasma Cholesterol Concentrations
Plasma cholesterol levels in rabbits fed normal rabbit chow were 1.77±0.76 mmol/L (mean±SD, n=12). After 1 and 2 weeks on a 0.25% cholesterol diet, plasma cholesterol increased to 5.07±0.82 mmol/L (P<0.05) and 8.57±1.84 mmol/L (P<0.01), respectively. To account for the effect of plasma cholesterol and exposure time, the integrated plasma cholesterol for each animal was calculated as follows:
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to
obtain the integrated plasma cholesterol.
Identification of VCAM-1–Positive Endothelium and
Intimal Macrophages
In normocholesterolemic rabbits, low levels of
VCAM-1 were observed in the descending arch, thoracic aorta, and
abdominal aorta. VCAM-1 was often expressed in individual
endothelial cells. After 0.25% cholesterol
was fed for 2 weeks, visible lesions did not form. VCAM-1 was often
observed as clusters around vessel branches (Figure 1A
). Transmural sections indicated that
VCAM-1 expression was limited to the endothelium
(Figure 1B). No staining was observed when the primary antibody
was absent. Faint staining, not associated with individual cells, was
observed with isotype-matched control IgG1 (Figure 1C). No
staining was observed with isotype-matched control IgG2a.
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MHC-II– (Figure 1D and 1E) and RAM-11– (Figure 1F)
positive white blood cells showed numerous cytoplasmic processes and
irregularly shaped nuclei (Figure 1A, arrows, and Figure 1F), as noted previously.14 After 2 weeks of
feeding with 0.25% cholesterol, many of these white blood
cells had numerous lipid droplets. In transmural sections, RAM-11– or
MHC-II–positive cells were present in the intima and adjacent
media (Figure 1E). Adjustment of the focal planes of en face
sections also indicated that these cells were located beneath the
endothelium. Few smooth muscle cells or
endothelial cells stained positive for MHC-II. The
L11/135 antibody, which binds to CD43, was used to identify T
cells. Although CD43 is present on a number of different cell
types, L11/135 appears to be specific for thymocytes and all classes of
T cells, possibly because of differences in CD43 among cell
types.17 L11/135-positive T cells were round, without any
cytoplasmic processes or lipid droplets. Cholesterol
feeding did not stimulate T-cell accumulation, which accounted for
19.1±7.8% of the population of intimal white blood cells. Smooth
muscle cells were not observed in the intima, as judged by the absence
of HHF35 staining (not shown).
Intimal white blood cells were detected around the major abdominal
branches and intercostal arteries by MHC-II staining and morphological
examination. Even after 2 weeks of cholesterol feeding,
however, RAM-11–positive macrophages were not observed around
the intercostal branches. In 3 rabbits fed 0.25%
cholesterol for 1 week, RAM-11–positive
macrophages accounted for 13±10% of the intimal
macrophages. This is similar to the value reported for
normocholesterolemic rabbits (10±3%).14
In contrast, the density of MHC-II–positive macrophages was
similar to the density of white blood cells identified by the nuclear
shape (Figure 2A). On the basis of these
observations and reports that T cells represent a small
percentage of lymphocytes in the vessel wall during early
atherosclerosis7 21 22 and that
MHC-II–positive cells in lesions are mostly
macrophages,5 7 23 MHC-II–positive cells and
white blood cells identified by nuclear shape or morphological
examination are assumed to represent macrophages.
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Effect of Cholesterol Feeding Duration on VCAM-1
Expression and Intimal Macrophage Densities
In the aortic arch, the density of VCAM-1–positive
endothelium increased significantly after 2 weeks of
cholesterol feeding (P<0.05) (Figure 3A). For the thoracic and abdominal
aortas, data were subdivided into lateral, distal, proximal, and
nonbranch regions. The nonbranch regions correspond to regions >1
mm from the branch. Only at the lateral portions of the intercostal
branches did the density of VCAM-1–positive
endothelial cells increase significantly after feeding
of 0.25% cholesterol for 2 weeks (P<0.05)
(Figure 3B). The distribution of VCAM-1–positive
endothelium around the intercostal arteries was
significantly different from the uniform distribution for control
animals (P<0.001) and animals fed 0.25%
cholesterol for 1 (P<0.025) or 2
(P<0.001) weeks (Figure 3B). For all conditions, no
differences were found in VCAM-1 densities between branch and nonbranch
regions.
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In the abdominal aorta, the density of VCAM-1–positive
endothelium was nonuniformly distributed around the
branches (P<0.001). The density of VCAM-1–positive
endothelium lateral and distal to the branches
increased significantly from 1 to 2 weeks on a 0.25%
cholesterol diet (P<0.005) (Figure 3C).
The density of VCAM-1–positive endothelium lateral to
the branch was significantly higher than in nonbranch regions for
control (P<0.05) and after 2 (P<0.001) weeks of
0.25% cholesterol feeding. In addition, after 2 weeks of
0.25% cholesterol feeding, VCAM-1 densities distal to the
branch and around abdominal branches were significantly higher
than VCAM-1 densities around intercostal arteries.
Macrophage densities in the arch did not increase
(P=0.068) after a 0.25% cholesterol diet for 1
or 2 weeks (Figure 4A). In the thoracic
and abdominal aortas, macrophages distal and lateral to the
branch increased significantly from 1 to 2 weeks after initiation of a
0.25% cholesterol diet (Figure 4B and 4C). Around
the intercostal arteries, macrophage densities were nonuniform
(P<0.001) but were higher in the distal and lateral regions
than in the proximal region (Figure 4B). Macrophage
distributions around the celiac, superior mesenteric, and renal
arteries were nonuniform in control rabbits and in rabbits receiving
0.25% cholesterol for 1 or 2 weeks (P<0.001).
Macrophage densities at nonbranch regions did not change as a
result of cholesterol feeding. In the thoracic aorta,
macrophage densities distal and lateral to the branch were
significantly greater than macrophage densities at nonbranch
regions after 2 weeks of 0.25% cholesterol feeding
(P<0.001). No significant differences were detected in
control animals or after 1 week of cholesterol feeding. In
the abdominal aorta of normocholesterolemic rabbits,
macrophage densities lateral to the branch were greater than
densities in nonbranch regions (P<0.05). After 2 weeks of
cholesterol feeding, macrophage densities distal
(P<0.01) and lateral (P<0.001) to the abdominal
branches were significantly greater than macrophage densities
at nonbranch regions.
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Relationship Between Cumulative Cholesterol Exposure
and VCAM-1 Expression and Macrophage Densities
Because plasma cholesterol concentrations showed
significant variability among animals, the densities of
VCAM-1–positive endothelium and macrophages at
various locations were correlated with the integrated plasma
cholesterol (Equation 1). In the arch, the density
of VCAM-1–positive endothelium (Figure 5A) and macrophages (Figure 6A) were both significantly correlated
with the integrated plasma cholesterol (Table 1). In the thoracic aorta, however,
neither VCAM-1–positive endothelium (Figure 5B)
nor macrophages (Figure 6B) were correlated with the
integrated plasma cholesterol. In the abdominal aorta,
VCAM-1 and macrophage densities around the lateral superior
mesenteric or distal celiac and renal arteries were significantly
correlated with the integrated plasma cholesterol (Figures 5C and 6C and Table 1). Except for the lateral region
around the celiac branch, every other location that exhibited a
significant correlation between the density of VCAM-1–positive
endothelium and integrated plasma
cholesterol also exhibited a significant correlation
between macrophages and integrated plasma
cholesterol.
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Correlation Between VCAM-1 Expression and Macrophage
Densities
Every region in which VCAM-1 and macrophages were
correlated with integrated plasma cholesterol also showed a
significant correlation between VCAM-1 and macrophage densities
(Table 1 and Figure 7). In
addition, statistically significant correlations were also observed in
the proximal region of the intercostal arteries and the nonbranch
region in the vicinity of the celiac artery (Table 1).
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These correlations were based on the densities of VCAM-1–positive
endothelium and macrophages within the same
0.0625-mm2 region and did not consider whether
macrophages were directly associated with VCAM-1–positive
endothelium. We also examined the percentage of
macrophages that were either completely or partially beneath
VCAM-1–positive endothelium. In control animals,
22.3±3.8% (mean±SEM) of the macrophages were directly
associated with VCAM-1–positive endothelium. This
direct association increased to 36.6±3.7% in rabbits receiving 0.25%
cholesterol for 2 weeks. Around the abdominal branches, the
spatial association of intimal macrophages with
VCAM-1–positive endothelium was nonuniform, with a
higher association lateral to the branch than in the nonbranch region
(Table 2).
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Discussion |
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In this study, we obtained new information on the effect of plasma cholesterol levels and the duration of feeding on the spatial distribution and association between VCAM-1 and macrophage densities in the rabbit aorta. Two weeks after onset of a 0.25% cholesterol diet, VCAM-1 expression is selectively increased around the major branches in the abdominal aorta, whereas macrophage accumulation increases around the intercostal arteries and the major branches in the abdominal aorta. Around the abdominal branches and in the arch, VCAM-1 expression and macrophage accumulation are correlated with the integral of the plasma cholesterol over the duration of feeding. These correlations are strongest where visible lesions first develop.1 24 On the basis of the direct association between VCAM-1–positive endothelium and macrophages, VCAM-1 accounts for 37% of the accumulation of macrophages after onset of a hypercholesterolemic diet.
Others report that VCAM-1 expression increases in the ascending aorta after 4 days of a 0.3% cholesterol diet5 and at the intercostal orifices7 and at fatty streaks in abdominal aorta5 1 week after onset of the 0.3% cholesterol diet. The plasma cholesterol and integrated plasma cholesterol levels in these studies were 1.5 to 2 times higher than values we obtained after 1 week. These studies did not report VCAM-1 staining in normocholesterolemic animals, whereas others found VCAM-1 protein6 11 and gene expression25 present at low levels in arteries of normocholesterolemic rabbits. Longer-term diets may lead to significant increases in VCAM-1 in nonbranch regions of the aorta.6
Two studies report that VCAM-1 expression preceded RAM-11–positive
macrophage accumulation.5 7 In contrast, 2 weeks
after the start of the high-cholesterol diet, we found
increases in VCAM-1 and macrophages coincident around the major
branches. This difference in the temporal relationship between VCAM-1
and macrophages observed between the present study and past
studies may result from methodological differences. The en face
approach we used permitted examination of larger areas of the vessels
than could be analyzed with transmural sections. In addition,
we found that in normocholesterolemic animals and
during the first 2 weeks of feeding 0.25% cholesterol,
RAM-11 stained 
10% of the macrophages identified by MHC-II
staining and visual examination. Because approximately one third of the
macrophages are associated with VCAM-1, far fewer VCAM-1
positive endothelium would be directly associated with RAM-11–positive
macrophages. This low association might not be easily detectable with
techniques that do not sample large areas of tissue.
Our results agree well with other reports that show that
macrophage accumulation begins 2 weeks after the onset of a
hypercholesterolemic diet.1 26 27 The
present results confirm our previous observation that
macrophage densities in normocholesterolemic
rabbits are elevated around the celiac orifice.14 Back et
al15 found that in the thoracic aorta of
normocholesterolemic rabbits and rabbits fed 2%
cholesterol for 2 weeks, the density of
monocytes/macrophages in the nonbranch region was significantly
less than the macrophage density in a
4-mm2 region surrounding the intercostal
orifices. There was no difference between the macrophage
density in normocholesterolemic rabbits and after 2
weeks of cholesterol feeding, although only 2 animals were
studied. In contrast, we detected significant increases in
macrophage density around the intercostals after 2 weeks of
cholesterol feeding (Figure 4).
Localization of VCAM-1 expression and macrophage accumulation to the lesion-prone regions around the orifices suggests that fluid mechanics influences VCAM-1 expression and monocyte adhesion. Lateral to the branch, flow reversal begins earliest, and these regions exhibit low and oscillating shear stresses.28 Proximal to the branch and at the flow divider lip, shear stresses are higher. VCAM-1 expression appears to be increased at high- and low-shear-stress regions, but regions in which the shear stresses are lowest exhibited the highest association between VCAM-1–positive endothelium and intimal macrophages. This is consistent with a recent study11 in which surgically altering the rabbit carotid artery to either increase or decrease the wall shear stress increased VCAM-1 expression within 5 days. Only in the low-shear-stress vessels did monocytes adhere, and 65% were associated with VCAM-1–positive endothelium.
The association of VCAM-1 with 37% of the macrophages could
indicate that macrophages remained in the intima after the
overlying endothelium no longer expressed VCAM-1 or
that other receptors may be involved in binding to monocytes. Some or
all of the VCAM-1 expression might, however, result from
activated macrophages that entered the vessel wall by
another adhesion molecule. In culture, VCAM-1 expression is transient
and persists for as long as 48 hours after application of a
cytokine.4 It is not known how long VCAM-1
expression persists in vivo. The high values of
r2 for VCAM-1 and macrophage
densities in 1-mm2 regions (Table 1)
suggest that 10% to 30% of the macrophages may have persisted
in the intima after VCAM-1 expression returned to normal.
Alternatively, this association could arise because other receptors are
sensitive to the same stimuli. Possible candidates include
P-selectin,7 ICAM-1,8 and
VMAP-1.10
The correlation between endothelial cell expression of VCAM-1 or macrophage accumulation at lesion-prone sites around vessel branches with plasma cholesterol is consistent with activation of VCAM-1 expression by normal12 or oxidized13 LDL. Support for a direct role for VCAM-1 in adhesion is the in vitro observation that monocytes can arrest on activated endothelium through VCAM-1. Because the correlation is site specific, the correlation suggests that cholesterol or macrophages in the vessel wall serve as the agent that activates endothelium. Sites at which early lesions develop exhibit an increased frequency of elevated LDL permeability.29 Although endothelial permeability to LDL is not increased after 2 weeks of cholesterol feeding,30 LDL residence times30 31 and accumulation32 and aortic cholesterol24 all increase at lesion-prone sites. Possibly, lipoproteins may be modified by localized oxidation within the vessel wall or because of interactions with proteoglycans. These modified forms of LDL may activate the endothelium to express VCAM-1.
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Acknowledgments |
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This study was supported by NIH grant HL-41372. The authors gratefully acknowledge Dr Myron Cybulsky for providing the Rb1/9 antibody and Dr Richard Malinauskas for helpful suggestions.
Received February 11, 1998; accepted July 20, 1998.
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