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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2630.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

ICAM-1 Deficiency Reduces Atherosclerotic Lesions in Double-Knockout Mice (ApoE^-/-/ICAM-1^-/-) Fed a Fat or a Chow Diet

Marie-Claude Bourdillon; Robin N. Poston; Chantal Covacho; Elza Chignier; Giampiero Bricca; John Louis McGregor

From INSERM U331/Faculté de Médecine RTH Laënnec, Lyon, France (M.-C.B., C.C., E.C., G.B., J.L.M.); and the Department of Experimental Pathology, UMDS, Guy’s Hospital, London, UK (R.N.P.).

Correspondence to Marie-Claude Bourdillon, MD, INSERM, Unit 331/Site Cardiologique, 22 Avenue Doyen Lépine, Case Postale 18, F-69675 BRON Cedex France. E-mail bourdillon{at}lyon151.inserm.fr

	Abstract

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Abstract—Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE^-/- mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE^-/-) or double-knockout (DKO; ApoE^-/-, ICAM-1^-/-) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

Key Words: atherosclerosis • ICAM-1 • apolipoprotein E–deficient mice • aortic lesions

	Introduction

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Atherosclerosis may be the result of genetic susceptibility combined with environmental factors such as diet, lifestyle, or possibly microbial infections.^{1 2} Oxidized LDL, one of the factors thought to affect vessel wall integrity,³ can lead to an inflammatory response.⁴ Such a response will induce endothelial cell activation, extravasation of leukocytes, and migratory/reparative process by vascular smooth muscle.^{5 6} Activated endothelium will express in sequence a series of adhesion molecules and powerful cofactors such as chemokines, which together will tether and activate leukocyte integrin complexes.⁷ Monocytes or T-lymphocytes, in the above-described process, will be allowed to extravasate to sites of inflammation.⁸ These adhesion molecules include intercellular adhesion molecule-1 (ICAM-1 or CD54),⁹ platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD 31),¹⁰ vascular cell adhesion molecule-1 (VCAM-1 or CD106),^{11 12} and P-selectin (CD 62P).¹³ ICAM-1, an adhesion molecule constitutively expressed by endothelial cells, has been shown to be upregulated in human atherosclerotic lesions.⁹

Atherosclerotic mice models (apolipoprotein E–deficient, ApoE^-/-, and LDL receptor–deficient, LDLr^-/-) have been shown to mimic human lesions closely.^{14 15 16} Moreover, lesions in these animals can be accentuated by the use of a hypercholesterolemic diet.^{17 18} However, it was observed by Roselaar et al,¹⁹ in a comparative study between ApoE and LDLr deficient mice, that the total area of atherosclerotic lesions was greater at all times in ApoE^-/- than LDLr^-/-. It is conceivable that the expression and role of adhesion molecules and cofactors may slightly differ between ApoE^-/- and LDLr^-/- mice. Very little is known at this stage on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE^-/- mice fed a fat or chow diet. However, qualitative and quantitative increases in endothelial ICAM-1 expression in ApoE-deficient mice are observed.^{20 21} Moreover, an anti–ICAM-1 monoclonal antibody in ApoE^-/- mice greatly reduced the homing of monocytes to sites of atherosclerotic lesions.²² Finally, ICAM-1 knockout in a C57BL6 fat-fed model greatly reduced the size of vascular lesions.²³ This study has investigated the size of aortic arch lesions in mice deficient in ApoE^-/-, ICAM-1^-/- (double-knockout, DKO) and compared them with ApoE^-/--deficient (single-knockout, SKO) mice.

	Methods

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Animal Handling
Surgical procedures and animal care strictly followed the Guidelines of the National Institutes of Health and Medical Research (decree No. 87-848 of October 19, 1987). All animals used in this study were ether-anesthetized before organ sampling.

Mice
The generation of the double target mutation mice model was carried out with 2 original knockout lines: the Icam1^{tm1Bay24 25 26} and the ApoE^tm1Unc.^{1 15 27} Dr N. Maeda (University of North Carolina, Chapel Hill) and Dr A.L. Beaudet (Baylor College of Medicine, Houston, Tex) kindly made the ApoE and ICAM-1–deficient mice available to the European Network on Atherosclerosis. Briefly, ApoE^tm1Unc chimeras were mated to C57BL/6J to obtain B6129F1 animals homozygous for the disrupted gene. These founders were back-crossed 6 times to C57BL/6J mice. A redirection by embryo transfer and 3 additional back-cross generations on C57BL/6JIco were conducted before intercrossing to obtain C57BL/6JIco-ApoE^tm1Unc breeding. The C57BL/6JIco-Icam1^tm1Bay breeding colony was also established after embryo rederivation and back-cross with the C57BL/6J mice colony. Both lines were maintained in specific and opportunistic pathogen-free (SOPF) status by transfer of the SOPF mice and housing in a filter-top cage system coupled with a laminar flow working station in a full barrier unit. The double-mutant colony was generated under the same housing conditions. Homozygous C57BL/6JIco-ApoE^tm1Unc and C57BL/6JIco-Icam1^tm1Bay were mated to obtain heterozygous mutants. These double heterozygotes were intercrossed to produce homozygous animals. These animals were identified by a double protocol: total cholesterol level assay for ApoE mutants and a polymerase chain reaction genotyping specific for ICAM-1. This is, to our knowledge, the first reported production of a double ApoE and ICAM-1 gene–deleted mouse.²⁸ Male mice were used throughout the entire study. The weight of ApoE^-/- animals at 3, 5, and 15 weeks was 9, 20, and 28 g, respectively; weight of ApoE^-/- and ICAM-1^-/- animals at 5, 15, and 20 weeks was 21, 30, and 31 g, respectively.

Identification of Knockout Animals
Total cholesterol measurement for ApoE-deficient animals was performed on a blood sample collected from the retro-orbital sinus of anesthetized animals. Commercially available assay kits (Boehringer Mannheim) were used (cholesterol is expressed in mmol/L). For identification of ICAM-1 mutants, polymerase chain reaction was done on tail fragments of animals. Genotyping and phenotyping of animals were performed as previously indicated.²⁶ The sequences of the oligonucleotides (Genosys), with 3 sets of primers, were as follows: -ICAM-1 #2: 5'-ggA CAg gTC ggT CTT gAC AA-3'; -ICAM-1 #2': 3'-CCT gTC CAg CCA gAA CTg TT-5'; -ICAM-1 #4: 5'-CAg CAC gTg CAg TTC CAg g-3'; -ICAM-1#4': 3'-gTC gTg CAC gTC AAg gTC C-5'; -ICAM-1 #5: 5'-gTT CTT CTg AgC ggC gTC-3'; -ICAM-1 #5': 3'-CAA gAA gAC TCg CCg CAg-5'.

Experimental Design
Both ApoE-deficient mice (n=45) and double ApoE and ICAM-1 homozygous mutants (n=45) were weaned at 3 weeks of age and maintained on chow ("Souriffarat" breeding diet, standard formulation, pellets, irradiated at 25 kGy) for 1 week (control group at time 0). Single ApoE^-/- and double ApoE^-/-, ICAM-1^-/- mutant animals were analyzed after 3, 6, 15, and 20 weeks of either chow or fat diet feeding (n=5 mice per group). The fat diet consisted in a Western type (21% fat, 0.15% cholesterol, special high fat formulation, and powder, irradiated at 25 kGy). The cholesterol levels of 5 animals were analyzed per time point and per diet.

Histological and Morphometric Analysis
Animals were killed after having been anesthetized by ether inhalation. The heart, after incision of the thorax, was briefly perfused with 4% paraformaldehyde in PBS solution. The heart and the contiguous thoracic aorta were then cut off and rapidly embedded in tissue-Tek OCT compound (Miles). OCT-treated samples were then frozen in liquid nitrogen. Sections (8 µm thick) were mounted on gelatin-coated slides and stored at -80°C. Before staining, sections were air-dried and fixed in cold acetone. Lipid staining was performed by oil red O (ORO). In addition, standard hematoxylin-phloxine-safranin staining was also performed.

Immunohistochemistry
Macrophages were stained with MOMA-2 (rat antibody directed against murine monocytes/macrophages, Serotec) monoclonal antibody. A nonimmune monoclonal antibody of the same species and class as the MOMA-2 antibody, a rat IgG2a (Pharmingen), was used as a negative control. A biotinylated rabbit anti-rat/mouse adsorbed IgG was used as second antibody (Vector Laboratory). Sections were developed with avidin-biotin-horseradish peroxidase complex before AEC (3-amino-9-ethylcarbazole) staining procedures as described.²⁹

Quantimetry
The ratio of lesion area to media area was performed through the use of a Quantimet 600 Leica analyzer. Morphometric analyses of intima lesion area over media area were carried out on the whole circumference of the proximal aortic arch, with the use of a previously described quantification method.^{14 23 30} The number of sections used per animal was at least 5 per animal and per time point. Measurements were performed on ORO-stained slides for the different weeks of feeding (5 mice per group). Results are expressed as mean±SEM of intima/media area ratio. The differences between SKO and DKO mice groups were computed by unpaired Student’s t test.

	Results

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Cholesterol Levels
Animals from the two groups, fed on a chow diet for various lengths of time (0, 3, 6, 15, and 20 weeks), showed no differences in cholesterol levels (Figure 1). In addition, no differences in cholesterol levels for animals fed a fat diet were observed at 3, 6, and 15 weeks. It should be noted that cholesterol levels in chow-fed animals were in general lower than those observed for fat-fed animals. We observed a drop in cholesterol levels in 15-week-old animals fed a fat diet. It is conceivable that in these animals, at such a time point and cholesterol level, there is more active cholesterol transfer/sequestration in tissues and hence a lower circulating level. In one instance, cholesterol levels differed between animal types in that SKO animals at 20 weeks, on a fat diet only, showed a significantly higher cholesterol level than in the DKO (Figure 1). At this time point, the SKO somehow differ from the DKO in their capacity to transfer cholesterol from circulation to tissues. However, in that case, no difference in lesion area was observed. All other cholesterol results showed no differences between animal types.

View larger version (37K): [in this window] [in a new window]   Figure 1. Plasma cholesterol levels in ApoE-/- (SKO) and ApoE-/-, ICAM-1-/- (DKO) mice. Levels of cholesterol are for SKO or DKO animals fed a chow or fat diet over 0 to 20 weeks. Values are mean±SEM.

Vascular Lesions
The aortic arch region in SKO and DKO was investigated in weaned animals and after feeding a chow or a fat diet (3, 6, 15, and 20 weeks). Lesions increased in severity through all time points of the experiment in both the SKO and DKO animals. They were qualitatively similar in both groups and resembled those previously described.¹⁴ However, those in the DKO mice were of less severity than in the SKO (Figure 2, A-B, E-F, G-H). Macrophages were immunostained in the core and at the surface of lesions (Figure 2, C-F). Moreover, macrophages were also observed beneath the endothelial cell layer. When classic advanced atherosclerotic lesions were produced in the aortic arch of animals fed a chow diet for 20 weeks, ORO-stained foam cells were seen in the shoulders and core of plaques (Figure 2, G-H). Smooth muscle proliferation, calcifications, and cholesterol clefts were also observed in extended lesions of mice fed a fat diet for 20 weeks (Figure 2, I-J).

View larger version (83K): [in this window] [in a new window]   Figure 2. ORO staining and immunohistochemistry of ApoE-/- (SKO) and ApoE-/-, ICAM-1-/- (DKO) mice. Size of ORO-stained aortic lesions are observed to be larger in SKO (A) compared with DKO (B) mice fed 20-week chow diet. Pulmonary artery, present next to aorta in (A), also shows presence of lesions (shown by white arrowheads). Macrophages, stained by MOMA-2, are shown penetrating endothelial layer in SKO (C) and DKO (D) animals after 15-week chow diet. Macrophages are also localized in core and edges (white arrows) of lesions after 15-week fat diet in SKO (E) and DKO (F) mice. Lipid ORO-stained droplets are present in aortic lesions from SKO (G) and DKO (H) fed 20-week chow diet. Respective ORO and hematoxylin-phloxine-safranin staining of complex lesions with cholesterol clefts are shown in SKO (I) and DKO (J) mice fed fat diet for 20 weeks. Calcified deposits (shown by *) are present in center of lesions (I, J). Black arrows, unless otherwise indicated, show atherosclerotic lesions (A, B), macrophages (C through F), lipid droplets (G, H) and cholesterol clefts (I, J). L indicates aorta lumen. Original magnification, x40 (A, B); x400 (C, D); and x250 (E through J).

Lesion Area/Media Area
The protective effect resulting from ICAM-1 deficiency is seen in all DKO animals compared with SKO throughout the whole feeding period. Indeed, at 3, 6, 15, and 20 weeks, chow-fed DKO mice showed a lesion/media area ratio that was significantly lower compared with the SKO (Figure 3A and Table). This protective effect, in the absence of ICAM-1 expression, was present at all times in chow-fed animals. Percentage reduction in lesion area/media area for 3, 6, 15, and 20 weeks was 77%, 55%, 62%, and 54%, respectively. Likewise, in fat-fed animals, the lack of ICAM-1 reduced lesion size to some extent at all time points (Figure 3B and Table). Percentage reduction in lesion/media area ratio for 3, 6, 15, and 20 weeks was 94%, 55%, 76%, and 17%, respectively. However, at 6 and 20 weeks only, the decreases were not significant (Table).

View larger version (9K): [in this window] [in a new window]   Figure 3. Lesions area in ApoE-/- (SKO) and ApoE-/-, ICAM-1-/- (DKO) mice. A, Ratios of lesion area over media area for SKO or DKO animals fed a chow diet over 0 to 20 weeks. Individual values and means of SKO and DKO area ratios are shown for each time. B, Ratios of lesion area over media area for SKO or DKO animals fed fat diet over 0 to 20 weeks. Individual values and means of SKO and DKO area ratios are shown for each time.

View this table: [in this window] [in a new window]   Table 1. Ratio of Lesion Area to Media Area

	Discussion

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This study shows for the first time that ICAM-1 plays a crucial role in modulating the size of lesions in aortic arch isolated from ApoE^-/- deficient mice fed either a chow or a fat diet. Indeed, results show a decreased ratio in intima to media area in all mice deficient in ApoE^-/-, ICAM-1^-/- compared with ApoE^-/- animals, although in 2 groups of the 8, the difference did not achieve statistical significance. The lack of significance in these groups may be attributable to the inherent variability of the atherosclerotic process and the small groups of 5 animals. In contrast, plasma cholesterol did not appear to be implicated in affecting lesion size. As previously reported,¹³ focal atherosclerotic lesions seen in this study resemble human lesions. Moreover, in line with previously published data,^{14 17 31} the appearances of the atherosclerotic lesions are characterized by typical lipid deposition, cellular infiltration, and smooth muscle cell proliferation.

Atherosclerotic lesions may be the result of some form of inflammation induced by the presence of oxidized LDL, Chlamydia pneumoniae,³² or viral or other factors occurring at the level of the vessel wall.³³ Vascular endothelial cells, activated at sites of inflammation, interact with different leukocyte subtypes through adhesion molecules and various cofactors and are thought to play a key role in the initiation and perpetuation of atherosclerotic lesions.³⁴ Several studies have observed an initial increase in intimal leukocyte accumulation³⁵ that is followed by a decreased density of leukocyte recruitment within atherosclerotic walls.¹⁹ Among a number of adhesion molecules implicated in the homing of leukocytes to sites of inflammation, endothelial ICAM-1 and its leukocyte ligand CD11a/CD18 (also known as _Lß₂ or LFA-1) play a major role in this process.^{36 37} Monocytes attracted by endothelial released MCP-1 can bind directly through CD11a/CD18 to vascular ICAM-1 and/or by fibrinogen through CD11b/CD18 (also known as _Mß₂ or MAC-1) and ICAM-1.³⁸

Constitutive levels of ICAM-1 in mice differ markedly from one organ to another. Endothelial ICAM-1 expression is highest in the heart vessels compared with the brain, mesentery, or small intestine.³⁹ Recent qualitative and quantitative data report a significant increase in ICAM-1 expression in the ApoE-deficient mouse.^{20 21} Moreover, and very significantly, observations made on human coronaries and carotids show ICAM-1 expression correlated to vascular lesions.⁹ ICAM-1 as well as VCAM-1 are expressed in a flow-dependent manner.⁴⁰ Upregulation of ICAM-1 in cultured human and rabbit arterial endothelial cells has been shown to occur after lysophosphatidylcholine treatment.⁴¹ Moreover, lysophosphatidylcholine induced the expression of ICAM-1 on endothelium derived from human iliac arteries but not from umbilical veins.⁴¹ It is of interest to note that high levels of lysophosphatidylcholine are present in hyperlipidemia. Deficiency of ICAM-1 expression in C57BL6 fat-fed animals showed a 75% protection against atherosclerosis when compared with wild-type animals.²³ However, one should note that ApoE-deficient animals fed a fat diet probably show lesions that are closer, compared with C57BL6, to those seen in human. Our data support a role for ICAM-1 at all stages of atherosclerosis in chow-fed animals. However, this does not appear to be the case for fat-fed animals. Indeed, ICAM-1 appears to have the greatest impact at the early stages of atherosclerosis in animals fed a fat diet. Interestingly, monoclonal antibodies directed to ICAM-1 in ApoE-deficient animals on a chow diet reduced the homing of macrophages to atherosclerotic plaques by 65%.²² In addition, the use of anti–ICAM-1 monoclonal antibodies in hypercholesterolemic rats significantly reduced the homing of macrophages to the intima by 42%.⁴² It is most probable that an absence of ICAM-1 greatly reduces the inflow of monocytes to sites of lesions and hence affects the ratio in intima to media area of aortic arch.

The work presented in this study and that from other teams clearly indicate that ICAM-1 is a key target implicated in cell-cell interactions leading to the initiation and perpetuation of atherosclerotic lesions. However, great care must be taken in blocking vascular ICAM-1 expression because it impairs inflammatory and immune responses in C57BL6 mice.²⁴ It should be noted that ICAM-1 expression is only part of the mechanism implicated in the homing of leukocytes to sites of vascular lesions; other adhesion molecules, for instance, P-selectin, have been implicated in gene-deleted mice.⁴³ Furthermore, knocking out MCP-1 or its receptor in LDL^-/- or ApoE^-/- mice, respectively, will also significantly decrease lesion formation.^{44 45} In conclusion, adhesion and transmigration mediated by several interacting molecular mechanisms appear to be essential for monocyte traffic in atherosclerosis, and this study provides further evidence of the importance of these phenomena in the pathogenesis of the disease.

While this manuscript was being revised, the work by Collins et al⁴⁶ has been published. The authors report atherosclerosis in ApoE knockout animals also having a deficiency in ICAM-1 or P-selectin or E-selectin. A part of this study focused on the effect of ICAM-1 deficiency on animals at 20 weeks of diet on a chow diet. A significant reduction in lesion area was also observed in both male and female mice.

	Acknowledgments

 
This work was supported by the French Ministry of Education Scientific Research (grant MESR ACC-SV9) and by the European Network on Atherosclerosis (BIOMED 2, grant PL 1195). We would like to thank George Canard, Dr Patrick Hardy (from Transgenic Alliance-Iffa Credo, L’Arbresle), Dr Catherine Souchier, Dr Jean-Claude Bernengo (Centre Commun de Quantimétrie, Université Claude-Bernard, Lyon), and Odile Gayet for their excellent help and advice during the course of this work.

Received March 21, 2000; accepted July 10, 2000.

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