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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:310-316.)
© 1997 American Heart Association, Inc.

Articles

P-Selectin and Vascular Cell Adhesion Molecule-1 Are Focally Expressed in Aortas of Hypercholesterolemic Rabbits Before Intimal Accumulation of Macrophages and T Lymphocytes

Atsushi Sakai; Noriaki Kume; Eiichiro Nishi; Kenjiro Tanoue; Masayuki Miyasaka; Toru Kita

the Department of Geriatric Medicine (A.S., N.K., E.N., T.K.), Graduate School of Medicine, Kyoto University, Kyoto, the Department of Cardiovascular Research (K.T.), Tokyo Metropolitan Institute of Medical Science, Tokyo, and the Department of Bioregulation (M.M.), Biomedical Research Center, Osaka University Medical School, Osaka, Japan.

Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan. E-mail nkume@kuhp.kyoto-u.ac.jp.

	Abstract

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The accumulation of monocyte/macrophages and T lymphocytes in arterial intima is a hallmark of early atherogenesis. To investigate the temporal relationships between endothelial expression of adhesion molecules (eg, P-selectin and vascular cell adhesion molecule-1 [VCAM-1]) and intimal accumulation of macrophages and T lymphocytes, immunostaining was performed by using serial frozen sections from intercostal branch points of thoracic aortas of New Zealand White rabbits that had been fed a 0.3% cholesterol diet. After 1 week of cholesterol feeding, neither macrophages nor T lymphocytes were detected, although endothelial expression of P-selectin and VCAM-1 was observed. After 3 weeks, macrophages were detectable in 75% and T lymphocytes were present in 25% of the rabbits. Expression of P-selectin and VCAM-1 was sustained until 10 weeks. Infiltration of T lymphocytes was restricted in areas in which macrophages were accumulated and did not appear to precede macrophage infiltration. E-selectin expression was not detectable before accumulation of mononuclear leukocytes; however, very few endothelial cells covering foam cell lesions expressed E-selectin after 6 weeks. Similar results were obtained in Watanabe heritable hyperlipidemic rabbits aged 1, 2, and 3 months. Taken together, localized expression of P-selectin and VCAM-1 may play a key role in the initial recruitment of macrophages and T lymphocytes in early atherogenesis.

Key Words: atherosclerosis • endothelium • monocyte • E-selectin • MHC class II

	Introduction

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Atherogenesis consists of multiple and complex disease processes, including infiltration of inflammatory leukocytes, accumulation of cholesterol-laden macrophages, proliferation of smooth muscle cells, deposition of extracellular matrix, and thrombosis. Among these, one of the characteristic features of early atherosclerotic lesions appears to be localized accumulation of monocyte/macrophages and T lymphocytes in arterial intima. Macrophages accumulate cholesteryl ester and are subsequently transformed into foam cells. T lymphocytes accumulated in intima may secrete inflammatory cytokines that can activate vascular cells in situ and further modulate the progression of atherosclerosis.^{1 2 3 4 5 6} The molecular mechanisms responsible for mononuclear leukocyte infiltration may depend on multiple factors, including chemoattractants derived from the vascular wall^{6 7} ; however, adhesion of circulating blood monocytes and T lymphocytes appears to be one of the earliest steps in this process.^{4 8 9} Several different adhesion molecules have been identified in vascular endothelium that support the adhesion of mononuclear leukocytes.¹⁰ Furthermore, upregulated expression of VCAM-1,^{11 12 13 14} intercellular adhesion molecule-1,^{15 16 17 18} and E-selectin^{13 17} has been demonstrated in established lesions of atherosclerosis in both humans and experimental animal models. P-selectin has also been detected in advanced lesions of human atherosclerotic plaques.¹⁸ Among these molecules, VCAM-1 has been implicated in the initial recruitment of mononuclear leukocytes, since its expression has been detected in arterial endothelium before macrophage accumulation in rabbits with dietary-induced hypercholesterolemia.¹² In the present study, we explored the expression of P-selectin and E-selectin as well as VCAM-1 and their temporal relation to the intimal accumulation of monocyte/macrophages and T lymphocytes in the early stages of atherosclerotic lesions of both dietary and genetic models of hypercholesterolemia in the rabbit.

	Methods

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Materials
Four-month-old male NZW rabbits weighing 3.0 kg were purchased from Japan SLC. Sixteen of the rabbits were randomly divided into four groups (four rabbits per group) and fed a standard chow diet supplemented with 0.3% cholesterol for 1, 3, 6, or 10 weeks. Homozygous WHHL rabbits that had been bred in Kyoto were fed a standard chow diet and killed at the ages of 1, 2, and 3 months. The amount of chow was restricted to 100 g/d for each rabbit throughout the study, but water was supplied ad libitum. All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of Kyoto University. Serum TC levels were measured by using enzymatic methods (Cholesterol E-test, Wako Pure Chemical Co).

Immunohistochemistry
After 1, 3, 6, and 10 weeks on the cholesterol diet, the rabbits received heparin (300 U/kg body wt IV) and were anesthetized with sodium pentobarbital (25 mg/kg body wt IV). Vascular endothelium was fixed by systemic perfusion with 1% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.2) under a positive pressure of 100 cm H₂O. Thoracic aortas were isolated, and full-circumference segments around intercostal orifices, where early foam cell lesions are often found in a localized fashion,¹⁹ were snap-frozen in isopentane cooled with dry ice. Aortic samples were similarly prepared from untreated NZW rabbits and those killed 4 hours after intravenous injections of bacterial endotoxin (lipopolysaccharide: 100 µg/kg body wt IV); aortic samples from WHHL rabbits aged 1, 2, and 3 months were also prepared. Serial 6-µm-thick frozen segments were placed on poly-L-lysine–coated glass slides and fixed with acetone at -20°C for 2 minutes. After the segments had incubated with 0.1% BSA-PBS containing 2% horse serum for 20 minutes, aortic sections were incubated with primary antibodies directed to the target molecules for 30 minutes. The primary antibodies used to detect adhesion molecules,^{11 13 20} macrophages,²¹ and T lymphocytes²² in rabbits are listed in Table 1. Anti-CD68 antibody was purchased from Dako. Rb1/9, a monoclonal antibody for rabbit VCAM-1, and 14G2, a monoclonal antibody directed to rabbit E-selectin, were generous gifts from Dr Myron Cybulsky and Dr Barry Wolitzky, respectively. Concentrations of the primary antibodies were optimized by preliminary experiments. After the sections were washed with BSA-PBS, they were incubated with biotinylated secondary antibody (horse anti-mouse IgG) for 30 minutes and washed with BSA-PBS. Endogenous peroxidase activity was blocked by incubation with methanol containing 0.3% hydrogen peroxide, after which avidin-biotin peroxidase complexes (ABC Elite kit, Vector Labs) were added. Antibody binding was visualized with 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemical), and sections were then counterstained with Gill's hematoxylin.

View this table: [in this window] [in a new window]   Table 1. Monoclonal Antibodies Used in this Study

	Results

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Upregulated Expression of P-Selectin and E-Selectin in Aortic Endothelium by Bacterial Endotoxin In Vivo
Immunostaining for P-selectin, VCAM-1, and E-selectin was performed in aortic specimens from three untreated NZW rabbits and two NZW rabbits infused with bacterial endotoxin. In aortic endothelium from untreated NZW rabbits, neither P-selectin (Fig 1A), E-selectin, nor VCAM-1 (data not shown) was detectable; however, intact endothelial lining, which was uniformly stained with anti–MHC class I antigen (Fig 1B), was demonstrated. Staining of P-selectin (Fig 1C), E-selectin (Fig 1D), and VCAM-1 (data not shown) was shown clearly in aortic endothelium from endotoxin-injected rabbits. Positive staining disappeared when the primary antibody was replaced with class-matched irrelevant antibody (Fig 1E).

View larger version (113K): [in this window] [in a new window]   Figure 1. Photomicrographs. Frozen sections from thoracic descending aorta were prepared from untreated (A) or endotoxin-injected (C and D) NZW rabbits, and immunostaining using antibodies for P-selectin (A and C) and E-selectin (D) was performed. Expression of MHC class I antigen shows the integrity of endothelium in untreated rabbits (B). Staining with a class-matched irrelevant antibody served as negative control in endotoxin-injected rabbits (E) (original magnification x250).

P-Selectin and VCAM-1 Expression Before Accumulation of Macrophages or T Lymphocytes in Cholesterol-Fed Rabbits
To assess the temporal relationships between endothelial expression of adhesion molecules and intimal infiltration of macrophages and T lymphocytes in hypercholesterolemic rabbits, we focused on the intercostal orifices in the descending thoracic aorta, since these areas appear to be lesion-prone and consistently develop foam cell lesions in a localized fashion.¹⁹ Fig 2 summarizes serum TC levels in both cholesterol-fed NZW and WHHL rabbit models. Serum TC levels were time dependently elevated in both models and were comparable between cholesterol-fed (1 to 10 weeks) and WHHL (1- to 3-month-old) rabbits.

View larger version (16K): [in this window] [in a new window]   Figure 2. Line graphs show serum (s-) TC levels of cholesterol-fed NZW and WHHL rabbits. Blood samples were drawn from rabbits before perfusion fixation, and TC levels were measured by using a standard enzymatic assay. Each point indicates mean±SD.

After 1 week of cholesterol feeding, both P-selectin (Fig 3A) and VCAM-1 (Fig 3B) were expressed, in a similarly localized fashion, in these lesion-prone areas. Neither macrophages (Fig 3C) nor T lymphocytes (Fig 3D) were accumulated at this time. After 3 weeks of cholesterol feeding, expression of P-selectin (Fig 4A) and VCAM-1 (Fig 4B) was observed, and accumulation of macrophages was demonstrated underneath aortic endothelium positive for P-selectin and VCAM-1 (Fig 4C). In these early foam cell lesions, macrophages expressed MHC class II antigen (Fig 4D), and accumulation of very few T lymphocytes was detected (Fig 4E). Intimal thickening, which consisted of macrophage foam cells, became more prominent after 6 and 10 weeks of cholesterol feeding. T lymphocytes were also detected in these lesions, although their population was much less than macrophages. P-selectin and VCAM-1 remained expressed in aortic endothelium covering these foam cell lesions until at least 10 weeks after the initiation of cholesterol feeding. After 6 weeks, expression of E-selectin, in some cases, was detectable in a few ECs overlying foam cell lesions; however, there were fewer E-selectin–positive cells than those positive for P-selectin or VCAM-1, and they were never detected at 1 or 3 weeks of cholesterol-feeding in serial sections, when both P-selectin and VCAM-1 expression was demonstrated (Table 2).

View larger version (113K): [in this window] [in a new window]   Figure 3. Photomicrographs show immunostaining of P-selectin (A), VCAM-1 (B), macrophages (C), and T lymphocytes (D) in serial sections of the lesion-prone area of thoracic aorta from a NZW rabbit fed cholesterol for 1 week (original magnification x250).

View larger version (139K): [in this window] [in a new window]   Figure 4. Photomicrographs show immunostaining of P-selectin (A), VCAM-1 (B), macrophages (C), MHC class II antigen (D), and T lymphocytes (E) in serial sections of thoracic aorta from a NZW rabbit fed cholesterol for 3 weeks (original magnification x250).

View this table: [in this window] [in a new window]   Table 2. Summary of Immunohistochemical Study

P-Selectin and VCAM-1 Expression in Early Lesions of WHHL Rabbits
In 1-month-old WHHL rabbits, both P-selectin (Fig 5A) and VCAM-1 (Fig 5B) were demonstrated in areas around the intercostal orifices. E-selectin expression, in contrast, was not detectable in the serial sections where both P-selectin and VCAM-1 were expressed (Fig 5C). At this time, infiltration of a very few macrophages (Fig 5D) expressing MHC class II antigen (Fig 5E) but not T lymphocytes (Fig 5F) was detected in these lesion-prone areas. In 3-month-old WHHL rabbits, the expression of P-selectin (Fig 6A) and VCAM-1 (Fig 6B) was more prominent and uniformly detected in endothelium covering foam cell lesions, and accumulation of T lymphocytes was shown (Fig 6C). Expression of E-selectin was detected in some ECs covering foam cell lesions (Fig 6D); however, the pattern of E-selectin expression was much different from that of P-selectin or VCAM-1.

View larger version (165K): [in this window] [in a new window]   Figure 5. Photomicrographs show immunostaining of P-selectin (A), VCAM-1 (B), E-selectin (C), MHC class II macrophages (D), MHC class II antigen (E), and T lymphocytes (F) in serial sections of the lesion-prone area of thoracic aorta from a 1-month-old WHHL rabbit (original magnification x250).

View larger version (114K): [in this window] [in a new window]   Figure 6. Photomicrographs show immunostaining of P-selectin (A), VCAM-1 (B), T lymphocytes (C), and E-selectin (D) in serial sections of the thoracic aorta from a 3-month-old WHHL rabbit. Arrowheads indicate expression of E-selectin (original magnification x250).

Table 2 summarizes the number of rabbits positive for each antigen at different stages of early atherogenesis. Expression of both P-selectin and VCAM-1 was detectable as early as 1 week on the cholesterol diet and remained until 10 weeks. Infiltration of macrophages was not detectable at 1 week; however, most of the rabbits were positive for macrophages after 3 weeks. T lymphocytes were detected in one of four rabbits at 3 weeks on the diet. After 6 weeks, T-lymphocyte infiltration was shown in most of the rabbits. E-selectin expression, in contrast, was detectable in very few individual ECs in some rabbits (two and one of four rabbits after 6 and 10 weeks on the diet, respectively). In WHHL rabbits, expression of P-selectin and VCAM-1 and intimal accumulation of macrophages were demonstrated in all the rabbits from 1 month of age. T lymphocytes, in contrast, were not detectable in 1-month-old rabbits; however, all the rabbits were positive for T lymphocytes after 2 months of age. E-selectin was occasionally detected in one and three of four rabbits at 2 and 3 months of age, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Localized accumulation of macrophages and T lymphocytes in arterial intima is a hallmark of early atherogenesis. Endothelial expression of adhesion molecules directed to mononuclear leukocytes appears to play a role in the recruitment of these blood cells into arterial intima. In this study, we examined the expression of P-selectin, E-selectin, and VCAM-1 in both dietary and genetic models of hypercholesterolemia in the rabbit. To the best of our knowledge, this is the first evidence that P-selectin is focally expressed in arterial endothelium before infiltration of macrophages and T lymphocytes in animal models of atherosclerosis in vivo. In both models, serum TC levels were comparable to those in a study¹² that showed endothelial VCAM-1 expression before macrophage accumulation in arterial intima. Although P-selectin and VCAM-1 were similarly expressed in these hypercholesterolemic models, E-selectin expression was not observed before entry of macrophages or T lymphocytes. These results indicate that P-selectin and VCAM-1 may play more significant roles than E-selectin in the initial recruitment of mononuclear leukocytes in the very early stages of atherogenesis in hypercholesterolemic rabbit models.

P-selectin is one of the adhesion molecules expressed in vascular ECs that can support adhesion of leukocytes, including monocytes and lymphocytes. P-selectin is stored in Weibel-Palade bodies of ECs and is rapidly transferred to the cell surface in response to various stimuli.^{23 24} P-selectin expression, in the long term, can also be induced by inflammatory cytokines and bacterial endotoxin.^{25 26} Experiments with P-selectin knock-out mice have provided direct evidence that P-selectin can support leukocyte rolling, which appears to be an initial endothelial-leukocyte interaction before firm adhesion, in mesenteric postcapillary venules in vivo.²⁷ The role of P-selectin in atherogenesis has not been fully clarified; however, it might play a role in the initial interaction between leukocytes and endothelium. Furthermore, given that P-selectin expression apparently precedes intimal accumulation of macrophages or T lymphocytes, stimuli other than inflammatory cytokines derived from mononuclear leukocytes may be responsible for the initial expression of P-selectin as well as VCAM-1. Our studies^{28 29} have demonstrated that lysophosphatidylcholine, a polar phospholipid generated during oxidative modification of lipoproteins, can induce expression of adhesion molecules and smooth muscle growth factors in cultured ECs. VCAM-1 expression can also be enhanced by oxidized fatty acids in oxidized lipoproteins.³⁰ P-selectin expression is upregulated by oxidized LDLs and lysophosphatidylcholine in cultured vascular ECs and arterial endothelium ex vivo.^{31 32 33} Furthermore, by using intravital microscopy of a skinfold chamber model, Lehr et al³⁴ have revealed that injection of oxidized LDLs causes P-selectin–dependent leukocyte rolling in arterioles and venules in vivo. Therefore, lipids related to atherogenic lipoproteins might be responsible for VCAM-1 and P-selectin expression in the initial stage of atherogenesis.

In later stages, very little expression of E-selectin was detected where significant numbers of macrophages and T lymphocytes had already been accumulated. These results are comparable with those of Richardson et al,¹³ who used an en face Hautchen preparation in which E-selectin–positive cells were distributed randomly in endothelium covering foam cells as either single or groups of two or three cells in hyperlipidemic rabbits. These also appear to be compatible with studies by O'Brien et al,³⁵ who have shown that E-selectin expression is much less frequent than VCAM-1 expression in the luminal endothelium of human coronary atherosclerotic lesions. In these lesions, inflammatory cytokines derived from infiltrated blood cells might stimulate endothelium in situ to induce expression of E-selectin and other adhesion molecules, including VCAM-1 and P-selectin, and may further enhance leukocyte adhesion mechanisms.

In our hypercholesterolemic rabbit models, intimal infiltration of T lymphocytes did not appear to precede macrophage accumulation. Furthermore, the presence of T lymphocytes was restricted where macrophages had accumulated. Some macrophages in arterial intima were positive for MHC class II antigen; therefore, immunologically activated macrophages might play a role in the recruitment of T lymphocytes. Other mechanisms selective only for monocyte/macrophages might also be involved in the initial recruitment of macrophages. These may include another inducible adhesion molecule that remains to be identified.³⁶

In summary, the present immunohistochemical study suggests that P-selectin as well as VCAM-1 may, at least in part, be responsible for the initial mononuclear leukocyte recruitment into atherosclerotic lesions. Furthermore, factors other than cytokines derived from macrophages and T lymphocytes might act as stimuli for the initial expression of these adhesion molecules. Multiple factors might be involved in mononuclear leukocyte recruitment into atherosclerotic lesions; further studies to modulate expression of adhesion molecules, such as VCAM-1 and P-selectin, might provide new insights into the molecular mechanisms of mononuclear leukocyte recruitment in early atherogenesis.

	Selected Abbreviations and Acronyms

 

BSA-PBS = bovine serum albumin–supplemented Dulbecco's phosphate-buffered saline EC = endothelial cell NZW = New Zealand White TC = total cholesterol WHHL = Watanabe heritable hyperlipidemic VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
We would like to thank Dr Myron Cybulsky, Brigham and Women's Hospital, Boston, Mass, and Dr Barry Wolitzky, Hoffmann-LaRoche, Nutley, NJ, for their generous gifts of monoclonal antibodies for rabbit VCAM-1 (Rb1/9) and E-selectin (14G2), respectively. We also would like to acknowledge Drs Hiroshi Hamaguchi and Kazuo Kanai, Nihon Nohyaku, Osaka, Japan, for their helpful advice in immunohistochemistry.

Received April 23, 1996; revision received June 26, 1996;

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