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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:393-401.)
© 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

George A. Truskey; Robert A. Herrmann; Jason Kait; Kevin M. Barber

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

	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

	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.

	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 lymphocytes^{16 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.

	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:

(1)

where [Chol] is the plasma cholesterol level at time t and [Chol]₀ is the plasma cholesterol before onset of the diet. For individual animals, [Chol]-[Chol]₀ was fit to a polynomial and then integrated according to Equation 1 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.

View larger version (128K): [in this window] [in a new window]   Figure 1. En face (A, C, D, F) and transmural (B, E) views showing VCAM-1–positive endothelium (A, B), tissue stained with the isotype-matched control antibody (IgG1) (C), MHC-II–positive macrophages (D, E), and RAM-11–positive macrophages (F). En face sections were from rabbits fed 0.25% cholesterol for 2 weeks, and transmural sections were from rabbits fed 0.25% cholesterol for 1 week. Arrows in panel A identify macrophage-identified nuclei. Bars=40 µm.

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 atherosclerosis^{7 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.

View larger version (142K): [in this window] [in a new window]   Figure 2. En face views showing VCAM-1–positive endothelium at branch (A, C, E) and nonbranch (B, D, F) regions in normocholesterolemic rabbits (A, B) and rabbits after 1 (C, D) and 2 (E, F) weeks of 0.25% cholesterol feeding.

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.

View larger version (24K): [in this window] [in a new window]   Figure 3. VCAM-1 densities in the aortic arch (A), descending thoracic aorta (B), and abdominal aorta (C) for normocholesterolemic rabbits (n=4) and rabbits fed 0.25% cholesterol for 1 (n=5) or 2 weeks (n=7). For the thoracic and abdominal aorta, data were divided into branch and nonbranch regions. The branch regions are further subdivided into proximal, lateral, and distal portions as described in Methods. *P<0.005 for the effect of cholesterol feeding duration by the Kruskal-Wallis nonparametric test and Dunn's multiple comparison test, and +P<0.05, ++P<0.01 for lesion-susceptible branch versus lesion-resistant nonbranch sites by a repeated measures ANOVA and Bonferroni's test.

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.

View larger version (20K): [in this window] [in a new window]   Figure 4. Macrophage densities in the aortic arch (A), descending thoracic aorta (B), and abdominal aorta (C) for normocholesterolemic rabbits (n=4) and rabbits fed 0.25% cholesterol for 1 (n=5) or 2 weeks (n=8). For the thoracic and abdominal aorta, data were divided into branch and nonbranch regions. The branch regions are further subdivided into proximal, lateral, and distal portions as described in Methods. *P<0.01 and **P<0.005 for the effect of cholesterol feeding duration by the Kruskal-Wallis nonparametric test and Dunn's multiple comparison test, and +P<0.05, ++P<0.01, and +++P<0.001 for lesion-susceptible branch vs lesion-resistant nonbranch sites by a repeated measures ANOVA and Bonferroni's test.

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.

View larger version (23K): [in this window] [in a new window]   Figure 5. Dependence of VCAM-1 expression in the arch (A), thoracic aorta (B), and around the celiac region (C) on the integrated plasma cholesterol.

View larger version (22K): [in this window] [in a new window]   Figure 6. Dependence of macrophage density in the arch (A), thoracic aorta (B), and around the celiac branch (C) on the integrated plasma cholesterol.

View this table: [in this window] [in a new window]   Table 1. Correlation Between Integrated Plasma Cholesterol and VCAM-1–Positive Endothelium or Macrophage Densities

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).

View larger version (23K): [in this window] [in a new window]   Figure 7. Correlation between VCAM-1 and macrophage densities in the arch (top) and around the superior mesenteric flow divider (bottom).

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).

View this table: [in this window] [in a new window]   Table 2. Association Between VCAM-1 and Intimal Macrophages Around Vessel Branches of Rabbits Fed 0.25% Cholesterol for 2 Weeks (Mean±SEM)

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 protein^{6 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 times^{30 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.

	Acknowledgments

 
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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