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Leukocyte adhesion molecules in atherogenesis
Clinica Chimica Acta 286 (1999) 207–218
Leukocyte adhesion molecules in atherogenesis a, b a Myron I. Cybulsky *, Andrew H. Lichtman , Leena Hajra , Kaeko Iiyama a a Department of Laboratory Medicine and Pathobiology, University of Toronto, The Toronto General Hospital Research Institute, 200 Elizabeth Street, CCRW 1 -855, Toronto, ON, Canada, M5 G 2 C4 b Vascular Research Division, Department of Pathology, Brigham and Women’ s Hospital and Harvard Medical School, 75 Francis Street, Boston, MA, 02115 USA
Accepted 6 February 1999
Abstract Functions of mononuclear leukocytes and endothelial cell leukocyte adhesion molecules in the formation of early atherosclerotic lesions is discussed. The main transgenic mouse models developed to study cholesterol metabolism and atherosclerotic lesion formation, including apolipoprotein E knockout and low density lipoprotein receptor knockout (LDLR2 / 2) mice, are reviewed. Differences in their dependence on dietary cholesterol supplementation is emphasized and a new semi-purified, cholate-free mouse diet for LDLR2 / 2 mice is described. This diet is highly reproducible, versatile (pellet, powder or liquid formulations), inexpensive and promotes hypercholesterolemia and atherosclerotic lesion development despite absence of sodium cholate. We describe the expression patterns of leukocyte adhesion molecules in rabbit and mouse models of atherosclerosis and compare them to humans. Finally, ongoing studies are summarized which utilize transgenic mice to assess the roles of individual adhesion molecules in atherosclerotic lesion formation. 1999 Elsevier Science B.V. All rights reserved. Keywords: Leukocytes; Atherosclerosis; VCAM-1; ICAM-1; Mouse models
Abbreviations: Apo E 2 / 2 , apolipoprotein E knockout; ICAM-1, intercellular adhesion molecule-1; LDLR2 / 2 , low density lipoprotein receptor knockout; NF-kB, nuclear factor-kB; VCAM-1, vascular cell adhesion molecule-1. *Corresponding author. Tel. / fax: 1 1-416-340-3578. E-mail address: [email protected] (M.I. Cybulsky) 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00102-3
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1. Introduction Atherosclerosis is a disease of elastic and muscular arteries and is the principal antecedent to myocardial and cerebral infarctions and ischemia of the extremities [1]. These conditions generally occur in the setting of advanced atherosclerosis. Infarctions usually result from sudden occlusion of the vascular lumen by thrombus associated with a plaque that only partially impinges into the lumen, and ischemia is due to gradual severe luminal narrowing. In humans, atherosclerosis develops indolently for decades prior to onset of complications and clinical manifestations. However, understanding the pathogenesis of early atherosclerotic lesions and appropriate interventions in populations at risk may alleviate the significant morbidity and mortality associated with complications of advanced lesions. The development of atherosclerotic lesions can be subdivided into initiation (formation of small fatty streaks), expansion (vertical and lateral growth, as well as coalescence of small fatty streaks) and progression to fibrous plaques (intimal smooth muscle cell recruitment, collagen deposition and formation of a fibrous cap). The focus of our research efforts has been on lesion initiation and expansion. As others, we have used animal models of hypercholesterolemia, initially rabbit and more recently murine. Because hypercholesterolemia is only one of several known major risk factors that have been identified in humans, our animal models may not reflect the entire pathogenic spectrum of human atherosclerosis. Nevertheless, irrespective of the stimulus, the arterial wall can respond to injury with a limited number of biological programs, one of which is recruitment and accumulation of leukocytes in the intima.
2. Role of leukocytes in atherogenesis Leukocytes are involved in each step of atherosclerotic lesion development. The adherence of blood monocytes and lymphocytes to the intact endothelial lining of large arteries is one of the earliest detectable events in atherosclerosis in humans and various animal models (reviewed in Refs. [2,3]). Adherent monocytes transmigrate into the intima, transform into macrophages, engulf lipids and become foam cells. Since early fatty streaks are composed almost entirely of macrophage foam cells, recruitment of monocytes to the intima is thought to be critical to initiating lesion formation. Monocyte recruitment, particularly at the periphery of the lesion [4], may also contribute to lesion expansion. Within lesions, leukocytes may influence the organization of cells and produce cytokines / growth factors, that promote migration of medial smooth muscle cells into the intima and stimulate intimal cell replication [2]. Smooth muscle cells deposit matrix proteins and collagen during the formation of a
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fibrous cap. Leukocytes can also secrete matrix metalloproteases, which may contribute to remodeling and destabilization of advanced lesions. In species ranging from pigeon to man, leukocyte recruitment and atherosclerotic lesion formation occur reproducibly at specific sites in the arterial tree, such as arterial bifurcations or curvatures [5–7]. The composition of leukocytes in lesions appears to be tightly regulated and consists of monocytes and lymphocytes. Polymorphonuclear leukocytes are excluded.
3. Leukocyte adhesion molecules The distribution pattern of atherosclerotic lesions and mononuclear leukocyteselective recruitment suggest that early lesion formation may be governed by local events. Potential mechanisms include hemodynamic factors, induction of a unique pattern of adhesion molecule expression and production of chemokines specific for mononuclear leukocytes. The focus of our laboratory is on leukocyte adhesion molecules. We have evaluated the expression patterns of VCAM-1, ICAM-1 and E-selectin, adhesion molecules whose expression on vascular endothelial cells is regulated primarily by induction of transcription (reviewed in Ref. [8]). Cytokines such as interleukin-1 and tumor necrosis factor a, produced in many acute and chronic conditions, are important mediators of induced adhesion molecule expression. Other endothelial cell adhesion molecules may also be relevant to atherogenesis. Some, such as ICAM-2, are expressed constitutively by most endothelial cells [8], and therefore, not likely to contribute to the topographic pattern of lesion formation. The cell-surface expression of P-selectin can be upregulated regionally by a protein synthesis-independent mechanism. Under normal conditions P-selectin is stored in cytoplasmic Weibel-Palade granules and is able to rapidly translocate to the cell surface in response to a variety of stimuli [8]. The significance of detecting P-selectin protein or mRNA in endothelial cells is difficult to interpret, since only cell surface-expressed P-selectin is relevant to leukocyte recruitment. VCAM-1 and ICAM-1 have structural and functional similarities (reviewed in Ref. [8]). Both are transmembrane glycoproteins and members of the immunoglobulin (Ig) gene superfamily. VCAM-1 has seven extracellular Ig-like domains, whereas ICAM-1 has five. Both bind integrin ligands and have two distinct binding domains — VCAM-1 in Ig domains 1 and 4 and ICAM-1 in 1 and 3. VCAM-1 binds a4 integrins (a4b1 and to some extent a4b7). ICAM-1 Ig domain 1 interacts with aLb2 integrins (LFA-1) and Ig domain 3 with aMb2 integrins (Mac-1). In addition, ICAM-1 can interact with a variety of molecules including C3b, fibrinogen and bacterial lipopolysaccharide. Both VCAM-1 and ICAM-1 can stabilize leukocyte adhesion to endothelium and participate in
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diapedesis. VCAM-1 expression may contribute to the selective recruitment of mononuclear leukocytes, because these leukocytes, but not neutrophils, express a4 integrins. In addition to expression on vascular endothelium, both molecules can be expressed by other cell types, including vascular smooth muscle cells and macrophages. E-selectin interacts via its lectin domain with molecules containing sialylated CD15. It functions, as do other selectins, in mediating leukocyte tethering and rolling on endothelium. E-selectin expression is largely restricted to vascular endothelium and it is only expressed in activated endothelial cells.
4. Murine models of atherosclerosis The development of murine models defective in genes controlling lipid metabolism and lipoprotein expression offers valuable tools to dissect complex interactions between diet and genetics in atherosclerosis. In the last several years, embryonic stem cell and transgenic technologies have been used to alter the expression levels of various genes affecting lipoprotein metabolism and have led to the development of murine knockout and transgenic models of atherogenesis. The apolipoprotein E knockout (ApoE 2 / 2) [9,10], low density lipoprotein receptor knockout (LDLR2 / 2) [11] and human apolipoprotein B transgenic mice [12,13] develop lesions throughout the arterial tree. Their distribution pattern and morphologic features share many similarities with human atherosclerosis, suggesting that similar pathogenic mechanisms may be involved [14–16]. A significant difference in the ApoE 2 / 2 and LDLR2 / 2 models is the degree of hypercholesterolemia and extent of atherosclerosis found when mice are fed regular chow-based diets without lipid additives. ApoE 2 / 2 mice fed a normal chow mouse diet develop hypercholesterolemia (10.4–13.0 mmol / l, predominantly VLDL) and atherosclerotic lesions, however, if fed a ‘Western-type’ diet (0.15% cholesterol, 21% fat), the hypercholesterolemia is . 39.0 mmol / l and lesions develop more rapidly [9,14]. In contrast, LDLR2 / 2 mice fed a normal chow diet have only a twofold elevation in plasma cholesterol (primarily IDL / LDL fraction) and do not develop lesions in the short term [11]. When fed a diet consisting of 1.25% cholesterol, 7.5% cocoa butter, 7.5% casein and 0.5% cholic acid, these mice develop marked hypercholesterolemia ( . 39.0 mmol / l, with increased VLDL, IDL / LDL and decreased HDL) and lesions throughout the aorta [16]. Because hypercholesterolemia and aortic lesion formation in LDLR2 / 2 mice is highly dependent on initiating a hypercholesterolemic diet, the onset of lesion development can be precisely controlled and these mice provide a unique opportunity for studying early events in atherogenesis.
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5. Semi-purified, cholate-free mouse diets Prior to the development of atherosclerosis-prone gene-targeted mutant mice, many studies were performed with normal mice fed chow-based diets supplemented with varying amounts of saturated fats, cholesterol and cholate to induce atheromatous lesions. In particular, C57BL / 6 mice are susceptible to dietary intervention and develop foam-cell rich lesions in the aortic root, but not advanced atheromas [17–21]. Dietary cholate was required in order to achieve significant hypercholesterolemia, presumably by interfering with hepato-biliary excretion of cholesterol. Most published studies of atherosclerosis in LDLR2 / 2 mice have relied on the same chow-based diets supplemented with cholate, cholesterol and lipid that were used in the earlier C57BL / 6 mouse studies. This has led to criticisms that the LDLR2 / 2 mouse model may not accurately represent the human atherosclerotic disease process due to potential toxic metabolic effects of cholate. For example, cholate may cause hepatic steatosis that can progress to cirrhosis accompanied by a number of host metabolic, physiological, and hormonal changes that can cause misleading interpretation of studies focusing upon the histopathological and molecular events during atherogenesis. From a nutritional perspective, the dilution of a chow diet with purified lipids, such as hydrogenated coconut oil, increases the caloric density of the diet and reduces the ratio of essential nutrients to dietary energy, thereby potentially contributing to marginal nutrient intake in mice consuming the atherogenic diet. ‘Chow’ diets do not take advantage of the accumulated knowledge concerning nutritional requirements of mice and the experience of many investigators employing precisely controlled semi-purified or purified diets for studies of chronic disease processes in rodents [22–25]. Chow diets are carefully formulated from natural ingredients to provide adequate nutrition for growth and reproduction, but they vary seasonally, in different geographic locations, and between companies in the sources of ingredients included in the final product. These diets satisfy the minimum requirements for many nutrients in mice and rats, but the concentrations of individual nutrients may vary substantially in different products over time [26]. Furthermore, many man-made and natural toxins are detected in chow diets, such as: aflatoxins, nitrosamines, pesticides, herbicides and heavy metals [26–28]. Chow diets contain a variety of natural substances from grains, fruits, and vegetables that may modify lipid metabolism and atherogenesis, including a diverse array of soluble and insoluble fiber sources and a multitude of biologically active phytochemicals such as carotenoids and flavonoids. In order to avoid issues of diet reproducibility, essential nutrients and cholate supplementation, we designed a semi-purified AIN76A based diet that can be fed in powdered, pelleted, or liquid form and can be manipulated for the precise
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evaluation of diet-genetic interactions in murine atherosclerosis [29]. These cost-effective diets can help improve the quality of data obtained and the comparison of results among laboratories over time. Furthermore, the use of semi-purified diets in murine studies provides a method for precise control of dietary and nutritional factors, allowing for a meaningful comparison of the effects of specific nutritional interventions that may be relevant to human disease processes. We evaluated circulating lipid profiles and atherosclerotic lesion development in LDLR2 / 2 mice that were randomly assigned among four diets: control (10% kcal lipid), high fat / moderate cholesterol (40% kcal lipid, 0.5% cholesterol by weight), high fat / high cholesterol (1.25% cholesterol by weight), and high fat / high cholesterol / Na-cholate (0.5% w / w). In all cholesterol-supplemented groups, fasting serum cholesterol was increased compared to controls after 6 or 12 weeks of feeding (P , 0.01) and atherosclerotic lesions developed throughout the aorta. The percent of aortic surface area covered by oil red O-stained lesions was determined from digitally scanned photographs [30]. Few lesions were detected in mice fed the control diet. In contrast, lesions in the arch and descending thoracic aorta were found in all mice fed cholesterolsupplemented diets (7.01 to 12.79% of the aortic surface area versus 0.16% in controls, P , 0.002). Histological features of atherosclerotic lesions in mice fed cholate-free or cholate-containing diets were similar. This study showed that precisely defined cholesterol-supplemented diets can significantly enhance atherogenesis in LDLR2 / 2 mice, even in the absence of cholate. A recent study by other investigators is in agreement with these data [31].
6. Expression of leukocyte adhesion molecules in atherosclerotic lesions Our laboratory compared the expression of VCAM-1, ICAM-1 and E-selectin in aortas in hypercholesterolemic rabbits, LDLR2 / 2 and ApoE 2 / 2 mice. Northern blot analysis demonstrated increased VCAM-1 and ICAM-1, but not E-selectin, steady state mRNA levels in hypercholesterolemic mouse and rabbit aortas which correlated with the extent of atherosclerotic lesion formation (determined by oil red O staining). In small lesions, VCAM-1 and ICAM-1 were expressed predominantly by endothelial cells, whereas in large foam cell-rich lesions many intimal cells expressed these molecules. It is likely that expression by intimal cells accounted for increased VCAM-1 and ICAM-1 steady state mRNA levels in Northern blots. VCAM-1 was also expressed by medial smooth muscle cells adjacent to lesions. This phenotypic change may occur in activated smooth muscle cells or cells in the process of migration to the intima. Expression patterns of endothelial cell VCAM-1 and ICAM-1 were highly reproducible and similar in distribution among rabbits and LDLR2 / 2 mice fed
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cholesterol-containing diets. Endothelial cell expression was most pronounced at the periphery of both large and small lesions and extended several cells beyond the edge. VCAM-1 expression was essentially restricted to lesions, whereas ICAM-1 expression extended into the uninvolved aorta. These data suggest that adhesion molecule expression in atherosclerotic lesions of mice and rabbits may be under similar mechanistic control, however, the regulation of VCAM-1 expression is more tightly controlled than ICAM-1 by lesion-derived factors. We observed virtually identical VCAM-1 and ICAM-1 immunohistochemical staining patterns in Apo E 2 / 2 mice and Watanabe heritable hyperlipidemic rabbits fed standard laboratory chow, which indicates that hypercholesterolemia and not other dietary factors were responsible for upregulated VCAM-1 and ICAM-1 expression in lesions. The endothelium over central portions of rabbit, but not mouse, lesions frequently expressed VCAM-1. These observations on adhesion molecule expression patterns in atherosclerotic lesions are consistent with and extend previous observations in animal models [32–36]. In humans, VCAM-1, ICAM-1 and E-selectin expression was observed by several groups in advanced atherosclerotic plaques obtained at autopsy or from hearts of transplant recipients. In all cases, ICAM-1 expression, detected by immunohistochemical staining, was found in endothelial cells over plaques and in intimal smooth muscle cells and macrophages [37–41]. E-selectin expression was variable and restricted to vascular endothelium [39–41]. Caution should be exercised in interpreting the E-selectin data, since the anti-E-selectin antibody used in all of these studies (BBA1 from British Biotechnology) has been subsequently shown to cross-react with P-selectin (manufacturer’s information). Advanced human coronary artery plaques displayed focal VCAM-1 expression in luminal endothelial cells, usually in association with inflammatory infiltrates [41,42]. Focal endothelial VCAM-1 expression was also found in uninvolved vessels with diffuse intimal thickening. Within plaques, VCAM-1 was expressed by subsets of smooth muscle cells and macrophages and by endothelial cells of neovasculature at the base of plaques. Further experiments in our laboratory determined that aortic endothelial cells in regions predisposed to atherosclerotic lesion formation expressed VCAM-1 in both normal mice and rabbits. ICAM-1 was also expressed in these regions, however its expression (at slightly lower levels) extended into regions with a low probability of developing lesions. For example, in the aortic arch ICAM-1 expression was circumferential, whereas lesion-predisposed areas are confined to the lesser curvature and intimal cushions near ostia of brachiocephalic and carotid arteries. These data generally are consistent with those recently reported by Nakashima et al. [36]. They found endothelial cell ICAM-1 expression in lesion-prone sites of ApoE 2 / 2 and C57BL / 6 mouse aortic arches and lower levels of expression in lesion-protected sites. They also observed VCAM-1 expression in lesion-prone sites of ApoE 2 / 2 mice, however C57BL / 6 mice had
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only very weak expression. We found convincing expression of VCAM-1 in lesion predisposed sites of mice and rabbits and this may reflect a higher sensitivity of immunostaining aortic cross-sections, where cell-surface and cytoplasmic antigens are detected. Recent preliminary experiments in our laboratory using C57BL / 6 mice and confocal microscopy confirmed VCAM-1 cell surface expression on endothelial cells in lesion-predisposed regions of the aortic arch. In normocholesterolemic animals, complex hemodynamic profiles may induce expression of VCAM-1 and ICAM-1 in endothelial cells at sites predisposed to lesion formation. Shear stress can directly influence the expression of adhesion molecules in endothelium [43,44] and different shear stress profiles can induce unique repertoires of gene expression [45,46]. Hemodynamics may also increase local lipoprotein permeability or transport through the endothelial monolayer and retention in the intima. Lipoprotein oxidation may generate soluble factors that can induce endothelial adhesion molecule expression. After hypercholesterolemia was initiated by diet, early foam cell lesions began forming and coalescing at lesion-predisposed sites. At this point, endothelial cell expression patterns of VCAM-1 and ICAM-1 correlated with the distribution of lesions, and therefore, the intrinsic properties of lesions, rather than hemodynamics, are likely the dominant factors responsible for this expression pattern. For example, in control mice VCAM-1 expression was scattered throughout lesion-predisposed sites, but became localized to the borders of lesions in hypercholesterolemic animals. This expression pattern must be related to local factors in this region, and oxidative stress, NF-kB activation and / or decreased nitric oxide production are potential mechanisms. Irrespective of the mechanism, expression of VCAM-1 and ICAM-1 by aortic endothelium in normal animals may mediate occasional emigration of leukocytes. Others have reported that in normal rabbits the density of intimal macrophages is higher at lesion-predisposed sites [47]. Upon initiation of hypercholesterolemia and accumulation of lipoprotein particles in the arterial intima, these macrophages may become activated and secrete proinflammatory factors. Thus, the localized expression of VCAM-1 and ICAM-1 in aortic endothelium of normal animals may provide a milieu for atherosclerotic lesion formation.
7. Function of leukocyte adhesion molecules in atherogenesis The functions of leukocyte adhesion molecules in atherosclerotic lesion formation are being investigated by a number of laboratories including ours. The approach is to breed mice with deficient adhesion molecule expression into the
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ApoE 2 / 2 or LDLR2 / 2 background and evaluate the extent of lesion formation. Traditionally lesion formation in the mouse aorta has been evaluated in two ways. One involves cutting serial cross-sections through the aortic root, staining sections for lipid (e.g. oil red O or Sudan stains) and using morphometric techniques to estimate the total volume of the intimal lesion. The other is to determine the extent of the aortic surface area covered by lesions. To date, few reports have been published and they suggest that some adhesion molecules, such as P-selectin, can influence lesion formation, but not eliminate it [48]. Our preliminary data appear to indicate that absence of ICAM-1 has little if any effect on lesion formation. We are also evaluating the role of VCAM-1 in atherogenesis using VCAM-1 domain 4-deficient mice bred into the LDLR2 / 2 background and preliminary observations suggest a reduction in lesion formation. The development of early atherosclerotic lesions is dependent on several processes that include intimal lipid accumulation, leukocyte and smooth muscle cell recruitment, cell proliferation and apoptosis. Future studies are likely to evaluate each of these directly. Leukocyte recruitment to lesions will be studied using semi-quantitative morphologic or molecular biologic approaches. For example, Steinberg’s group has developed a PCR-based method that can detect monocytes localized in the aorta within days of transfusion [49]. Recent reports indicate that deficiencies of MCP-1, a mononuclear leukocytespecific chemoattractant or CCR2, its receptor, result in reduced atherosclerotic lesion formation [50,51]. In a way, this result is surprising considering the redundancy of leukocyte chemoattractants and receptors. It remains to be determined if reduced leukocyte recruitment accounted for decreased lesions in these mice. There is evidence suggesting that chemoattractants participate in leukocyte recruitment by a mechanism that is independent of upregulated transcription of leukocyte adhesion molecules in endothelial cells. Therefore, in future studies mice will be bred with combined deficiencies in chemoattractant receptors / chemoattractants and various adhesion molecules.
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Leukocyte adhesion molecules in atherogenesis a, b a Myron I. Cybulsky *, Andrew H. Lichtman , Leena Hajra , Kaeko Iiyama a a Department of Laboratory Medicine and Pathobiology, University of Toronto, The Toronto General Hospital Research Institute, 200 Elizabeth Street, CCRW 1 -855, Toronto, ON, Canada, M5 G 2 C4 b Vascular Research Division, Department of Pathology, Brigham and Women’ s Hospital and Harvard Medical School, 75 Francis Street, Boston, MA, 02115 USA
Accepted 6 February 1999
Abstract Functions of mononuclear leukocytes and endothelial cell leukocyte adhesion molecules in the formation of early atherosclerotic lesions is discussed. The main transgenic mouse models developed to study cholesterol metabolism and atherosclerotic lesion formation, including apolipoprotein E knockout and low density lipoprotein receptor knockout (LDLR2 / 2) mice, are reviewed. Differences in their dependence on dietary cholesterol supplementation is emphasized and a new semi-purified, cholate-free mouse diet for LDLR2 / 2 mice is described. This diet is highly reproducible, versatile (pellet, powder or liquid formulations), inexpensive and promotes hypercholesterolemia and atherosclerotic lesion development despite absence of sodium cholate. We describe the expression patterns of leukocyte adhesion molecules in rabbit and mouse models of atherosclerosis and compare them to humans. Finally, ongoing studies are summarized which utilize transgenic mice to assess the roles of individual adhesion molecules in atherosclerotic lesion formation. 1999 Elsevier Science B.V. All rights reserved. Keywords: Leukocytes; Atherosclerosis; VCAM-1; ICAM-1; Mouse models
Abbreviations: Apo E 2 / 2 , apolipoprotein E knockout; ICAM-1, intercellular adhesion molecule-1; LDLR2 / 2 , low density lipoprotein receptor knockout; NF-kB, nuclear factor-kB; VCAM-1, vascular cell adhesion molecule-1. *Corresponding author. Tel. / fax: 1 1-416-340-3578. E-mail address: [email protected] (M.I. Cybulsky) 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00102-3
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1. Introduction Atherosclerosis is a disease of elastic and muscular arteries and is the principal antecedent to myocardial and cerebral infarctions and ischemia of the extremities [1]. These conditions generally occur in the setting of advanced atherosclerosis. Infarctions usually result from sudden occlusion of the vascular lumen by thrombus associated with a plaque that only partially impinges into the lumen, and ischemia is due to gradual severe luminal narrowing. In humans, atherosclerosis develops indolently for decades prior to onset of complications and clinical manifestations. However, understanding the pathogenesis of early atherosclerotic lesions and appropriate interventions in populations at risk may alleviate the significant morbidity and mortality associated with complications of advanced lesions. The development of atherosclerotic lesions can be subdivided into initiation (formation of small fatty streaks), expansion (vertical and lateral growth, as well as coalescence of small fatty streaks) and progression to fibrous plaques (intimal smooth muscle cell recruitment, collagen deposition and formation of a fibrous cap). The focus of our research efforts has been on lesion initiation and expansion. As others, we have used animal models of hypercholesterolemia, initially rabbit and more recently murine. Because hypercholesterolemia is only one of several known major risk factors that have been identified in humans, our animal models may not reflect the entire pathogenic spectrum of human atherosclerosis. Nevertheless, irrespective of the stimulus, the arterial wall can respond to injury with a limited number of biological programs, one of which is recruitment and accumulation of leukocytes in the intima.
2. Role of leukocytes in atherogenesis Leukocytes are involved in each step of atherosclerotic lesion development. The adherence of blood monocytes and lymphocytes to the intact endothelial lining of large arteries is one of the earliest detectable events in atherosclerosis in humans and various animal models (reviewed in Refs. [2,3]). Adherent monocytes transmigrate into the intima, transform into macrophages, engulf lipids and become foam cells. Since early fatty streaks are composed almost entirely of macrophage foam cells, recruitment of monocytes to the intima is thought to be critical to initiating lesion formation. Monocyte recruitment, particularly at the periphery of the lesion [4], may also contribute to lesion expansion. Within lesions, leukocytes may influence the organization of cells and produce cytokines / growth factors, that promote migration of medial smooth muscle cells into the intima and stimulate intimal cell replication [2]. Smooth muscle cells deposit matrix proteins and collagen during the formation of a
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fibrous cap. Leukocytes can also secrete matrix metalloproteases, which may contribute to remodeling and destabilization of advanced lesions. In species ranging from pigeon to man, leukocyte recruitment and atherosclerotic lesion formation occur reproducibly at specific sites in the arterial tree, such as arterial bifurcations or curvatures [5–7]. The composition of leukocytes in lesions appears to be tightly regulated and consists of monocytes and lymphocytes. Polymorphonuclear leukocytes are excluded.
3. Leukocyte adhesion molecules The distribution pattern of atherosclerotic lesions and mononuclear leukocyteselective recruitment suggest that early lesion formation may be governed by local events. Potential mechanisms include hemodynamic factors, induction of a unique pattern of adhesion molecule expression and production of chemokines specific for mononuclear leukocytes. The focus of our laboratory is on leukocyte adhesion molecules. We have evaluated the expression patterns of VCAM-1, ICAM-1 and E-selectin, adhesion molecules whose expression on vascular endothelial cells is regulated primarily by induction of transcription (reviewed in Ref. [8]). Cytokines such as interleukin-1 and tumor necrosis factor a, produced in many acute and chronic conditions, are important mediators of induced adhesion molecule expression. Other endothelial cell adhesion molecules may also be relevant to atherogenesis. Some, such as ICAM-2, are expressed constitutively by most endothelial cells [8], and therefore, not likely to contribute to the topographic pattern of lesion formation. The cell-surface expression of P-selectin can be upregulated regionally by a protein synthesis-independent mechanism. Under normal conditions P-selectin is stored in cytoplasmic Weibel-Palade granules and is able to rapidly translocate to the cell surface in response to a variety of stimuli [8]. The significance of detecting P-selectin protein or mRNA in endothelial cells is difficult to interpret, since only cell surface-expressed P-selectin is relevant to leukocyte recruitment. VCAM-1 and ICAM-1 have structural and functional similarities (reviewed in Ref. [8]). Both are transmembrane glycoproteins and members of the immunoglobulin (Ig) gene superfamily. VCAM-1 has seven extracellular Ig-like domains, whereas ICAM-1 has five. Both bind integrin ligands and have two distinct binding domains — VCAM-1 in Ig domains 1 and 4 and ICAM-1 in 1 and 3. VCAM-1 binds a4 integrins (a4b1 and to some extent a4b7). ICAM-1 Ig domain 1 interacts with aLb2 integrins (LFA-1) and Ig domain 3 with aMb2 integrins (Mac-1). In addition, ICAM-1 can interact with a variety of molecules including C3b, fibrinogen and bacterial lipopolysaccharide. Both VCAM-1 and ICAM-1 can stabilize leukocyte adhesion to endothelium and participate in
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diapedesis. VCAM-1 expression may contribute to the selective recruitment of mononuclear leukocytes, because these leukocytes, but not neutrophils, express a4 integrins. In addition to expression on vascular endothelium, both molecules can be expressed by other cell types, including vascular smooth muscle cells and macrophages. E-selectin interacts via its lectin domain with molecules containing sialylated CD15. It functions, as do other selectins, in mediating leukocyte tethering and rolling on endothelium. E-selectin expression is largely restricted to vascular endothelium and it is only expressed in activated endothelial cells.
4. Murine models of atherosclerosis The development of murine models defective in genes controlling lipid metabolism and lipoprotein expression offers valuable tools to dissect complex interactions between diet and genetics in atherosclerosis. In the last several years, embryonic stem cell and transgenic technologies have been used to alter the expression levels of various genes affecting lipoprotein metabolism and have led to the development of murine knockout and transgenic models of atherogenesis. The apolipoprotein E knockout (ApoE 2 / 2) [9,10], low density lipoprotein receptor knockout (LDLR2 / 2) [11] and human apolipoprotein B transgenic mice [12,13] develop lesions throughout the arterial tree. Their distribution pattern and morphologic features share many similarities with human atherosclerosis, suggesting that similar pathogenic mechanisms may be involved [14–16]. A significant difference in the ApoE 2 / 2 and LDLR2 / 2 models is the degree of hypercholesterolemia and extent of atherosclerosis found when mice are fed regular chow-based diets without lipid additives. ApoE 2 / 2 mice fed a normal chow mouse diet develop hypercholesterolemia (10.4–13.0 mmol / l, predominantly VLDL) and atherosclerotic lesions, however, if fed a ‘Western-type’ diet (0.15% cholesterol, 21% fat), the hypercholesterolemia is . 39.0 mmol / l and lesions develop more rapidly [9,14]. In contrast, LDLR2 / 2 mice fed a normal chow diet have only a twofold elevation in plasma cholesterol (primarily IDL / LDL fraction) and do not develop lesions in the short term [11]. When fed a diet consisting of 1.25% cholesterol, 7.5% cocoa butter, 7.5% casein and 0.5% cholic acid, these mice develop marked hypercholesterolemia ( . 39.0 mmol / l, with increased VLDL, IDL / LDL and decreased HDL) and lesions throughout the aorta [16]. Because hypercholesterolemia and aortic lesion formation in LDLR2 / 2 mice is highly dependent on initiating a hypercholesterolemic diet, the onset of lesion development can be precisely controlled and these mice provide a unique opportunity for studying early events in atherogenesis.
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5. Semi-purified, cholate-free mouse diets Prior to the development of atherosclerosis-prone gene-targeted mutant mice, many studies were performed with normal mice fed chow-based diets supplemented with varying amounts of saturated fats, cholesterol and cholate to induce atheromatous lesions. In particular, C57BL / 6 mice are susceptible to dietary intervention and develop foam-cell rich lesions in the aortic root, but not advanced atheromas [17–21]. Dietary cholate was required in order to achieve significant hypercholesterolemia, presumably by interfering with hepato-biliary excretion of cholesterol. Most published studies of atherosclerosis in LDLR2 / 2 mice have relied on the same chow-based diets supplemented with cholate, cholesterol and lipid that were used in the earlier C57BL / 6 mouse studies. This has led to criticisms that the LDLR2 / 2 mouse model may not accurately represent the human atherosclerotic disease process due to potential toxic metabolic effects of cholate. For example, cholate may cause hepatic steatosis that can progress to cirrhosis accompanied by a number of host metabolic, physiological, and hormonal changes that can cause misleading interpretation of studies focusing upon the histopathological and molecular events during atherogenesis. From a nutritional perspective, the dilution of a chow diet with purified lipids, such as hydrogenated coconut oil, increases the caloric density of the diet and reduces the ratio of essential nutrients to dietary energy, thereby potentially contributing to marginal nutrient intake in mice consuming the atherogenic diet. ‘Chow’ diets do not take advantage of the accumulated knowledge concerning nutritional requirements of mice and the experience of many investigators employing precisely controlled semi-purified or purified diets for studies of chronic disease processes in rodents [22–25]. Chow diets are carefully formulated from natural ingredients to provide adequate nutrition for growth and reproduction, but they vary seasonally, in different geographic locations, and between companies in the sources of ingredients included in the final product. These diets satisfy the minimum requirements for many nutrients in mice and rats, but the concentrations of individual nutrients may vary substantially in different products over time [26]. Furthermore, many man-made and natural toxins are detected in chow diets, such as: aflatoxins, nitrosamines, pesticides, herbicides and heavy metals [26–28]. Chow diets contain a variety of natural substances from grains, fruits, and vegetables that may modify lipid metabolism and atherogenesis, including a diverse array of soluble and insoluble fiber sources and a multitude of biologically active phytochemicals such as carotenoids and flavonoids. In order to avoid issues of diet reproducibility, essential nutrients and cholate supplementation, we designed a semi-purified AIN76A based diet that can be fed in powdered, pelleted, or liquid form and can be manipulated for the precise
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evaluation of diet-genetic interactions in murine atherosclerosis [29]. These cost-effective diets can help improve the quality of data obtained and the comparison of results among laboratories over time. Furthermore, the use of semi-purified diets in murine studies provides a method for precise control of dietary and nutritional factors, allowing for a meaningful comparison of the effects of specific nutritional interventions that may be relevant to human disease processes. We evaluated circulating lipid profiles and atherosclerotic lesion development in LDLR2 / 2 mice that were randomly assigned among four diets: control (10% kcal lipid), high fat / moderate cholesterol (40% kcal lipid, 0.5% cholesterol by weight), high fat / high cholesterol (1.25% cholesterol by weight), and high fat / high cholesterol / Na-cholate (0.5% w / w). In all cholesterol-supplemented groups, fasting serum cholesterol was increased compared to controls after 6 or 12 weeks of feeding (P , 0.01) and atherosclerotic lesions developed throughout the aorta. The percent of aortic surface area covered by oil red O-stained lesions was determined from digitally scanned photographs [30]. Few lesions were detected in mice fed the control diet. In contrast, lesions in the arch and descending thoracic aorta were found in all mice fed cholesterolsupplemented diets (7.01 to 12.79% of the aortic surface area versus 0.16% in controls, P , 0.002). Histological features of atherosclerotic lesions in mice fed cholate-free or cholate-containing diets were similar. This study showed that precisely defined cholesterol-supplemented diets can significantly enhance atherogenesis in LDLR2 / 2 mice, even in the absence of cholate. A recent study by other investigators is in agreement with these data [31].
6. Expression of leukocyte adhesion molecules in atherosclerotic lesions Our laboratory compared the expression of VCAM-1, ICAM-1 and E-selectin in aortas in hypercholesterolemic rabbits, LDLR2 / 2 and ApoE 2 / 2 mice. Northern blot analysis demonstrated increased VCAM-1 and ICAM-1, but not E-selectin, steady state mRNA levels in hypercholesterolemic mouse and rabbit aortas which correlated with the extent of atherosclerotic lesion formation (determined by oil red O staining). In small lesions, VCAM-1 and ICAM-1 were expressed predominantly by endothelial cells, whereas in large foam cell-rich lesions many intimal cells expressed these molecules. It is likely that expression by intimal cells accounted for increased VCAM-1 and ICAM-1 steady state mRNA levels in Northern blots. VCAM-1 was also expressed by medial smooth muscle cells adjacent to lesions. This phenotypic change may occur in activated smooth muscle cells or cells in the process of migration to the intima. Expression patterns of endothelial cell VCAM-1 and ICAM-1 were highly reproducible and similar in distribution among rabbits and LDLR2 / 2 mice fed
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cholesterol-containing diets. Endothelial cell expression was most pronounced at the periphery of both large and small lesions and extended several cells beyond the edge. VCAM-1 expression was essentially restricted to lesions, whereas ICAM-1 expression extended into the uninvolved aorta. These data suggest that adhesion molecule expression in atherosclerotic lesions of mice and rabbits may be under similar mechanistic control, however, the regulation of VCAM-1 expression is more tightly controlled than ICAM-1 by lesion-derived factors. We observed virtually identical VCAM-1 and ICAM-1 immunohistochemical staining patterns in Apo E 2 / 2 mice and Watanabe heritable hyperlipidemic rabbits fed standard laboratory chow, which indicates that hypercholesterolemia and not other dietary factors were responsible for upregulated VCAM-1 and ICAM-1 expression in lesions. The endothelium over central portions of rabbit, but not mouse, lesions frequently expressed VCAM-1. These observations on adhesion molecule expression patterns in atherosclerotic lesions are consistent with and extend previous observations in animal models [32–36]. In humans, VCAM-1, ICAM-1 and E-selectin expression was observed by several groups in advanced atherosclerotic plaques obtained at autopsy or from hearts of transplant recipients. In all cases, ICAM-1 expression, detected by immunohistochemical staining, was found in endothelial cells over plaques and in intimal smooth muscle cells and macrophages [37–41]. E-selectin expression was variable and restricted to vascular endothelium [39–41]. Caution should be exercised in interpreting the E-selectin data, since the anti-E-selectin antibody used in all of these studies (BBA1 from British Biotechnology) has been subsequently shown to cross-react with P-selectin (manufacturer’s information). Advanced human coronary artery plaques displayed focal VCAM-1 expression in luminal endothelial cells, usually in association with inflammatory infiltrates [41,42]. Focal endothelial VCAM-1 expression was also found in uninvolved vessels with diffuse intimal thickening. Within plaques, VCAM-1 was expressed by subsets of smooth muscle cells and macrophages and by endothelial cells of neovasculature at the base of plaques. Further experiments in our laboratory determined that aortic endothelial cells in regions predisposed to atherosclerotic lesion formation expressed VCAM-1 in both normal mice and rabbits. ICAM-1 was also expressed in these regions, however its expression (at slightly lower levels) extended into regions with a low probability of developing lesions. For example, in the aortic arch ICAM-1 expression was circumferential, whereas lesion-predisposed areas are confined to the lesser curvature and intimal cushions near ostia of brachiocephalic and carotid arteries. These data generally are consistent with those recently reported by Nakashima et al. [36]. They found endothelial cell ICAM-1 expression in lesion-prone sites of ApoE 2 / 2 and C57BL / 6 mouse aortic arches and lower levels of expression in lesion-protected sites. They also observed VCAM-1 expression in lesion-prone sites of ApoE 2 / 2 mice, however C57BL / 6 mice had
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only very weak expression. We found convincing expression of VCAM-1 in lesion predisposed sites of mice and rabbits and this may reflect a higher sensitivity of immunostaining aortic cross-sections, where cell-surface and cytoplasmic antigens are detected. Recent preliminary experiments in our laboratory using C57BL / 6 mice and confocal microscopy confirmed VCAM-1 cell surface expression on endothelial cells in lesion-predisposed regions of the aortic arch. In normocholesterolemic animals, complex hemodynamic profiles may induce expression of VCAM-1 and ICAM-1 in endothelial cells at sites predisposed to lesion formation. Shear stress can directly influence the expression of adhesion molecules in endothelium [43,44] and different shear stress profiles can induce unique repertoires of gene expression [45,46]. Hemodynamics may also increase local lipoprotein permeability or transport through the endothelial monolayer and retention in the intima. Lipoprotein oxidation may generate soluble factors that can induce endothelial adhesion molecule expression. After hypercholesterolemia was initiated by diet, early foam cell lesions began forming and coalescing at lesion-predisposed sites. At this point, endothelial cell expression patterns of VCAM-1 and ICAM-1 correlated with the distribution of lesions, and therefore, the intrinsic properties of lesions, rather than hemodynamics, are likely the dominant factors responsible for this expression pattern. For example, in control mice VCAM-1 expression was scattered throughout lesion-predisposed sites, but became localized to the borders of lesions in hypercholesterolemic animals. This expression pattern must be related to local factors in this region, and oxidative stress, NF-kB activation and / or decreased nitric oxide production are potential mechanisms. Irrespective of the mechanism, expression of VCAM-1 and ICAM-1 by aortic endothelium in normal animals may mediate occasional emigration of leukocytes. Others have reported that in normal rabbits the density of intimal macrophages is higher at lesion-predisposed sites [47]. Upon initiation of hypercholesterolemia and accumulation of lipoprotein particles in the arterial intima, these macrophages may become activated and secrete proinflammatory factors. Thus, the localized expression of VCAM-1 and ICAM-1 in aortic endothelium of normal animals may provide a milieu for atherosclerotic lesion formation.
7. Function of leukocyte adhesion molecules in atherogenesis The functions of leukocyte adhesion molecules in atherosclerotic lesion formation are being investigated by a number of laboratories including ours. The approach is to breed mice with deficient adhesion molecule expression into the
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ApoE 2 / 2 or LDLR2 / 2 background and evaluate the extent of lesion formation. Traditionally lesion formation in the mouse aorta has been evaluated in two ways. One involves cutting serial cross-sections through the aortic root, staining sections for lipid (e.g. oil red O or Sudan stains) and using morphometric techniques to estimate the total volume of the intimal lesion. The other is to determine the extent of the aortic surface area covered by lesions. To date, few reports have been published and they suggest that some adhesion molecules, such as P-selectin, can influence lesion formation, but not eliminate it [48]. Our preliminary data appear to indicate that absence of ICAM-1 has little if any effect on lesion formation. We are also evaluating the role of VCAM-1 in atherogenesis using VCAM-1 domain 4-deficient mice bred into the LDLR2 / 2 background and preliminary observations suggest a reduction in lesion formation. The development of early atherosclerotic lesions is dependent on several processes that include intimal lipid accumulation, leukocyte and smooth muscle cell recruitment, cell proliferation and apoptosis. Future studies are likely to evaluate each of these directly. Leukocyte recruitment to lesions will be studied using semi-quantitative morphologic or molecular biologic approaches. For example, Steinberg’s group has developed a PCR-based method that can detect monocytes localized in the aorta within days of transfusion [49]. Recent reports indicate that deficiencies of MCP-1, a mononuclear leukocytespecific chemoattractant or CCR2, its receptor, result in reduced atherosclerotic lesion formation [50,51]. In a way, this result is surprising considering the redundancy of leukocyte chemoattractants and receptors. It remains to be determined if reduced leukocyte recruitment accounted for decreased lesions in these mice. There is evidence suggesting that chemoattractants participate in leukocyte recruitment by a mechanism that is independent of upregulated transcription of leukocyte adhesion molecules in endothelial cells. Therefore, in future studies mice will be bred with combined deficiencies in chemoattractant receptors / chemoattractants and various adhesion molecules.
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