Animal models of atherosclerosis and magnetic resonance imaging for monitoring plaque progression

Abstract

Atherosclerosis, the main cause of heart attack and stroke, is the leading cause of death in most modern countries. Preventing clinical events depends on a better understanding of the mechanism of atherosclerotic plaque destabilization. Our knowledge on the characteristics of vulnerable plaques in humans has grown past decades. Histological studies have provided a precise definition of high-risk lesions and novel imaging methods for human atherosclerotic plaque characterization have made significant progress. However the pathological mechanisms leading from stable lesions to the formation of vulnerable plaques remain uncertain and the related clinical events are unpredictable. An animal model mimicking human plaque destablization is required as well as an in vivo imaging method to assess and monitor atherosclerosis progression. Magnetic resonance imaging (MRI) is increasingly used for in vivo assessment of atherosclerotic plaques in the human carotids. MRI provides well-characterized morphological and functional features of human atherosclerotic plaque which can be also assessed in animal models. This review summarizes the most common species used as animal models for experimental atherosclerosis, the techniques to induce atherosclerosis and to obtain vulnerable plaques, together with the role of MRI for monitoring atherosclerotic plaques in animals.

Keywords

atherosclerosis animal models MRI

Introduction

Cardiovascular disease is widely recognized as the leading cause of morbidity and mortality in developed countries and is predicted to become the predominant disease worldwide in the near future.¹ Atherosclerosis is the most important single factor contributing to this disease.² Evolution of atherosclerotic plaque can lead to advanced lesions causing severe stenosis or to atherothrombosis upon plaque rupture. The precise mechanisms at play in atherosclerotic plaque progression are yet to be fully understood. One solution to this major public health problem is improving the prevention, screening and treatment. Better understanding of the underlying causes and mechanisms that lead to the development of vulnerable plaque and clinical events are essential while technological innovations for diagnosis and treatment are still needed.

Histological features of vulnerable plaque are now well described in human coronary and carotid arteries.^3–5 Recent development of morphological and functional imaging improved our knowledge of vulnerable plaque. Imaging techniques allowing precise characterization of human atherosclerotic plaque in vivo such as magnetic resonance imaging (MRI) are available.⁶

These human observations provide insights into the state of plaque evolution but remain limited for ethical reasons and are insufficient to understand the mechanisms leading up to plaque rupture, emboli or thrombosis. Cell culture is essential to try to elucidate specific signalling pathways, but is inadequate to reproduce the conditions of the disease in vivo. Animal models in which the pathophysiology of atherosclerosis can be reproduced are an established alternative to cell culture and are routinely used in preclinical research settings.

The ideal animal model of vulnerable plaque should develop advanced atherosclerosis lesions similar to those seen in humans, eventually leading to clinical events (myocardial infarction or stroke) in a reasonable time-frame. Atherosclerotic plaque development in preclinical animal models should be monitored with the same diagnostic tools that are used clinically.

In this current review we will present the different techniques to induce atherosclerosis in an animal model, the different animal models of vulnerable plaque and survey the MRI methods used to monitor the time course of the developing pathology.

Search methods

An electronic literature search of the US National Library of Medicine public domain database (MEDLINE) was performed for articles published from 1950 to present using the following keywords: animal models, mice, rabbit, pig, atherosclerosis, vulnerable plaque, MRI. The references of the retrieved articles were also manually searched for any additional relevant articles. These articles were supplemented with textbooks of animal models, atherosclerosis and imaging methods.

Part I. Animal models of atherosclerosis

Animal models of atherosclerosis started emerging in the literature at the beginning of the 20th century with rabbits being the first animal species to be described. Subsequently, a wide variety of birds, primates and non-primates (rodent, pig, dog, etc.) were characterized and since the 1990s transgenic strains of mice, rabbits and rats have been developed. Initially, studies focused on morphology, physiology and underlying causes of atherosclerosis. For a long period of time, animal models were developed to try to resolve technical intervention issues such as re-stenosis. Currently, most studies focus on the vulnerable plaque. Since many characteristics of the animal models are prescribed by the aim of the research, a large number of animal models have been reported. Our review will focus on animal models of atherosclerosis commonly used to study the evolution of atherosclerosis and the vulnerable plaque.

The choice of animal species

Experimental models of atherosclerosis are required to understand the natural history of the disease and the factors leading to plaque progression/rupture. Susceptibility to atherosclerosis varies not only between animal species but also between genetic strains within the same species. The ideal animal model does not exist, but it is critical that models mimic the human disease as closely as possible.

Animal models must meet a few general criteria such as size, docility, ease of handling, ease of breeding and housing, known genetic profile, analogies with humans (anatomic, physiological, metabolic and pathophysiological), reliable homologies with human diseases and cost.^7–9 In addition to those criteria, animal models of atherosclerosis should present a progressive development of arterial lesions throughout life, from initial fatty streak to advanced complicated lesions (ulceration, thrombus, etc.) with shared histological features, and clinical events (myocardial infarction, stroke) similar to humans.^10–12

A wide range of animals has been used to study atherosclerosis (rodents, rabbits, dogs, monkeys, birds and pigs) but three animal models currently dominate these applications: mice, rabbits and pigs. Advantages and drawbacks are summarized in Table 1.

Table 1

Advantages and drawbacks of the most widely used non-primate mammal species for atherosclerosis model

Animal	Advantages	Drawback
Mice	Small size, docile, easy to handle	Naturally resistant to atherosclerosis
	Large groups (statistical significance)	Technical challenge (small)
	Genome completely sequenced	Few biological samples (blood, tissues)
	Many strains available through genetic manipulation	Different sensitivity to diet-induced atherosclerosis
	Wide variety of atherosclerotic lesions induced	Different lipid profile
	Small amount of pharmacological agents requiredand low cost
Rabbit	Small size, easy to handle	Herbivorous
	Large enough to allow monitoring physiological changes	Different gastrointestinal anatomy and pathology
	Easy to obtain biological samples	Low hepatic lipase activity and no analog of human Apo AII–
	Phylogenetically closer to humans than rodents
	Lipid profile close to human (VLDL, Apo B, CETP+)
	Transgenic strains available
Pig	Size close to human	Housing and handling difficulties
	Omnivorous	Large amount of pharmacological agents necessary
	High sequence homology with human chromosome	Small group (statistically insignificant)
	Cardiovascular system very close to human	High cost
	Lipid profile similar to human
	Spontaneous atherosclerosis
	Lesion topography and morphology close to human
	Iterative sampling of biological samples
	Imaging of vessels with clinical imagers

CETP, cholesteryl ester transfer protein

Mice

Although mice do not develop spontaneous atherosclerosis, it is the most commonly used animal species to study the disease. Among all strains, the C57BL/6 mouse is currently the most used background to develop genetically modified atherosclerosis models, as it is also the most susceptible strain to diet-induced atherosclerosis.¹³ The majority of mouse models are based on disturbance of lipid metabolism through genetic manipulation from the C57BL/6 strain. Among those, the ApoE − / − mouse is the most distributed worldwide. Arterial lesions observed in ApoE − / − are similar to lesions found in humans: from foam cell rich fatty streaks to atheroma with large necrotic core, and fibro-fatty nodules. The most advanced lesions have been described to be located in the innominate artery (brachiocephalic trunk).¹⁴ Luminal thromboses are uncommon in Apo E − / − mice because of a spontaneous high fibrinolytic activity. Mice have a low level of plasma plasminogen activator inhibitor-1 and thrombin-activated fibrinolysis inhibitor.^15–17

However, mouse models present major disadvantages. Firstly, mice are naturally resistant to atherosclerosis progression and therefore advanced genetic manipulation is required.¹⁸ In addition, the lipid profile and metabolism are not similar to humans. Mice have high plasma levels of high-density lipoprotein (HDL) cholesterol and the cholesteryl ester transfer protein (CETP) is absent.¹⁹ One other major limitation of transgenic mouse models is the non-physiological high expression level of hypercholesterolemia, which may not be similar to the more frequent human pathological form.²⁰ Also, the small size of the animal makes blood sampling as well as dissection of the vessels difficult. Moreover the size of the arteries makes it extremely difficult to image with sufficient resolution in clinical scanners used for humans, precluding the efficient translation of techniques from animal model to human scanning.

Rabbit

Rabbit models of atherosclerosis have also been widely described. Being herbivorous, the rabbit is not inherently prone to the development of atherosclerosis. Among all rabbit strains, the New Zealand White (NZW) is commonly used.²¹ Rabbits have low total cholesterol concentration in the plasma, and HDL is the predominant lipoprotein.²² Similarly to humans, Apo A-II is absent and hepatic lipase activity is low.^23,24 These animals show Apo B levels and high CETP activity comparable to humans.^23,25,26 The NZW rabbit rarely shows spontaneous lesions and induction of vascular lesions necessitates high-fat high-cholesterol diet.^11,27

The NZW also serves as a background for two genetically altered rabbit models: the Watanabe Heritable Hyper Lipidemic (WHHL) rabbit and the St Thomas’ Hospital strains, which present genetic abnormalities in their lipid metabolism.^28,29 The WHHL rabbits develop spontaneous hypercholesterolemia and atherosclerosis at five months of age. These rabbits have an abnormal low-density lipoprotein (LDL) receptor responsible for pathological changes in cholesterol metabolism, leading to atherosclerosis development.^11,30

The St Thomas’ Hospital rabbits have a familial dysbeta-lipoproteinemia responsible for an overproduction of VLDL and LDL. They have xanthomatous arterial lesions similar to those of species subject to atherogenic diets.³¹

Transgenic rabbits are widely distributed. The different models of hyperlipidemia have been created to study atherosclerosis and its correlation with lipid metabolism.^32–34

Microscopically, the lesions observed are thick fatty streaks with most of the lipid localized within the foam cells. Advanced lesions such as fibrosis, hemorrhage, ulceration and thrombosis are rarely observed. Lesions are preferentially localized in the aorta, iliac and coronary arteries.

One major advantage of this model is the animal's size: small (easy to feed, care for, handle and inexpensive) but large enough to monitor physiological changes. Imaging techniques such as ultrasound (US) or MRI can be effectively applied to determine the plaque composition and its vulnerability.^35,36

One limitation of this animal model remains the absence of hepatic lipase and the lack of Human Apo A-II. Arterial lesions observed are more fatty and macrophage-rich compared with human plaques and also differ in location.³⁷

Pig and mini-pig

With the requirement to obtain close-to-clinical models, large animal models, such as the mini-pigs have become increasingly popular.³⁸ However, many options have been described and the best model is still a matter of debate.

Available evidence indicates that the pig is an optimal model for atherosclerosis because of the many important similarities with humans. The pig genome is highly similar to humans. Its chromosome structure is comparable to that found in humans.³⁹ The similarity with humans in cardiovascular anatomy and physiology,^40–42 in lipid profile and lipoprotein metabolism have also been demonstrated.⁴³ In addition, arterial structure and its response to hypercholesterolemia and platelet aggregation resembles humans.⁴⁴ Moreover, unlike mice, rabbits or rats, pigs develop spontaneous atherosclerosis.¹⁹ Lesions observed are characterized by necrotic cores, calcification, neovascularization and intraplaque hemorrhage (IPH) that closely mimics advanced human atherosclerosis.^45,46 Atherosclerotic lesions are preferentially localized in the aorta, coronary and iliac arteries.

The major drawback of the domestic pig model of atherosclerosis is its large size resulting in management difficulties. To overcome this limitation, development of downsized pig and mini-pigs resulting from selection and crossing between different strains has been described. Different strains of mini-pigs such as Yucatan, Hanford, Göttingen, Sinclair Hormel and Ossabaw are already in use for preclinical research in atherosclerosis. These animals present different susceptibility to diet-induced hypercholesterolemia, as well as to accelerated atherosclerosis development; differences are observed between strains and often in the same strain.^47,48 Among published studies, very few accurately described the origin of mini-pig strains and their corresponding lipoprotein profile.¹²

Recently, a down-sized pig model was obtained by a crossing between a Rapacz farm pig (R84C LDL receptor mutation) a smaller pig (Chinese Meishan) and a French minipig.⁴⁹ Even more recently a novel micro-minipig model of atherosclerosis was described in Japan, illustrating the growing importance of the minipig model.⁵⁰

Others animal models have been developed using avian, hamster, guinea pigs, rats and non-human primates.

Strategies to induce experimental atherosclerosis in animal models

Only few animals spontaneously develop atherosclerosis and often these lesions are rare, not advanced and not similar to those seen in humans.¹⁹ Moreover, they require a long time, typically many years, to develop. An experimental atherogenesis strategy is necessary. To approach the ‘ideal’ model, the intervention should lead to the development of atherosclerosis lesions similar to those seen in humans, and in a reasonable time frame of a few months.

Atherogenesis involves many systemic factors, such as hypertension, hyperlipidemia, diabetes mellitus and smoking.⁵¹ Local hemodynamic perturbations or ‘traumatic’ factors are also implicated. We will summarize the two main categories of inducing accelerated atherosclerosis: systemic or local intervention.

Systemic intervention

The systemic intervention approach is dominated by atherogenic diets, which are described among the major risk factors of atherogenesis. This results in hyperlipidemia (hypercholesterolemia and hypertriglyceridemia), diabetes mellitus (hyperglycemia) or metabolic syndrome depending on the diet composition and the individual susceptibility.

Hyperlipidemia

The role of hypercholesterolemia (LDL-Cholesterol) in atherogenesis is well established.² A strong association between certain types of dyslipidemia including hypercholesterolemia, hypertrygliceridemia, and combined hyperlipidemia and the development of atherosclerotic lesions has been documented by numerous clinical trials, as well as epidemiological and experimental studies.

High cholesterol and high-fat diets lead to atherosclerosis lesions development in mice, rabbit and pig. However results of numerous studies report that stable high LDL cholesterol plasma level is not easily reproducible even with the same animal species and with the same diet.

There are qualitative differences of the atherogenecity of various dietary fats.⁵² Coconut oil is more atherogenic than other saturated, non-saturated and polyunsaturated fats.⁵³ The modified diet called the Paigen diet, the most used for mouse models, contains 50% sucrose, 15% fat, 1% cholesterol and 0.5% cholic acid.⁵⁴

Diabetes mellitus

Diabetes is a well-recognized independent risk factor of atherosclerosis.^55,56

Diabetes can be induced by sugar-enriched diet to maintain constant and regular hyperglycemia or by injection of β-cell cytotoxic chemicals resulting in a greater than 80% reduction in β-cells and an increase in plasma glucose, such as with streptozotocin or alloxan.^43,57,58 The dose of these agents required for inducing diabetes depends on the animal species, route of administration and nutritional status.

Diabetes mellitus combined with a diet-induced hypercholesterolemia leads to increased development of atherosclerosis lesions compared with hypercholesterolemia alone.^43,58

Metabolic syndrome

Metabolic syndrome (hypertension or HTA, associated to diabetes, dyslipidemia and/or obesity) is known to accelerate atherosclerosis lesions in human.⁵⁹ It has been reported in Ossabaw pigs with HF/HC diet.^12,60,61 Unfortunately, induction of atherosclerosis lesions was disappointing.

Hypertension

Hypertension is clearly associated with an increased risk of cardiovascular diseases and accelerated atherosclerosis. Sekhara Rao and co-workers⁶² demonstrated that Carneau pigeons selected for increased blood pressure are more susceptible to moderate hypercholesterolemia acting as a stimulus for atherosclerosis progression. Sugiyama et al.⁶³ reported an acceleration of atherosclerotic lesions in transgenic mice with hypertension by the activated renin–angiotensin system. Hypertension can be induced in mice by Angiotensin II injection,^64,65 Deoxycorticosterone acetate salt administration,⁶⁶ or clipping of the renal artery.⁶⁷ Such models have also been developed in rabbits.^68,69

Tobacco and stress exposure

Others risk factors like tobacco or stress were studied but none of these solely led to a reproducible animal model of atherosclerosis.⁷⁰

Infection

General infection seems to be involved in atherogenesis related to inflammatory and immunological reaction. Chlamydia pneumoniae, Helicobacter pylori, Cytomegalovirus, herpes simplex virus, Streptococcus sanguis and Porphyromonas gingivalis have been detected in human atheroma. Many models have been proposed with intravenous inoculation.^71,72

Local intervention

The creation of local lesions is proposed by many authors to accelerate atherosclerosis development. Even if theses kinds of models are not completely representative of spontaneous atherosclerosis, they reduce the time required for the development of advanced plaques as well as the associated costs. Another advantage is the possibility to increase the total number of available lesions and pick their location.

There are two main approaches for local intervention: direct arterial injury or indirect mechanism reproducing hemodynamic conditions favourable for atherosclerosis development.

Direct mechanism

Endothelial denudation by balloon injury: Different methods are described. The most used and described is the endothelial denudation with a balloon injury. Experimental studies in various animal models have consistently shown that arterial wall injury leads to smooth muscle cell proliferation and migration into the intima that eventually provokes luminal stenosis and thrombosis.^73–76 However, lesion progression is characterized by fibrosis and rather stable lesions on histology. Balloon injury leads to smooth muscle cell proliferation in the intima with formation of extracellular matrix in response to mechanical intimal-to-medial injury in virtually any site within the vascular tree in any species.¹¹ This approach was primarily designed for the re-stenosis experimental model.⁴⁴

A combined injury induced by balloon angioplasty in the setting of hypercholesterolemia causes both intimal smooth muscle cell proliferation and intense macrophage infiltration in the intima, media and adventitia, with severe impairment of endothelial vasodilator function resulting in atherosclerotic lesions.

Endothelial denudation can be performed with compliant or not compliant balloons and can be associated with medial injury. With concomitant dietary hypercholesterolemia, more advanced atherosclerotic lesions can be observed.^77,78 According to Thim,⁷⁸ degree of injury seemed easier to control with non-compliant balloon injury compared with compliant balloon.

Drawbacks of this method are the rate of acute arterial thrombotic occlusion and the inability to analysis disease progression. It has been proposed that endothelial dysfunction plays a role in initiating atherosclerosis through alterations in expression of adhesion factors and inflammatory mediators.⁷⁹ More recently Turk et al.⁸⁰ in a high-fat cholesterol diet-induced porcine model report that early stage disease development may precede the development of endothelial dysfunction. However, because of the disruptive nature of mechanical injury, the role of endothelial function has been difficult to assess.

Others types of injury: Other localized vascular injuries have been proposed to accelerate atherosclerosis formation: external radiation,⁸¹ electric injury,^82,83 thermal balloon injury,⁸⁴ photochemical injury and cold injury.^85,86

Intramural delivery of lipids has also been proposed to initiate the process of atherosclerotic disease.^87,88

Indirect mechanism by hemodynamic manipulation

Although the entire vasculature is exposed to the atherogenic effect of systemic risk factors, atherosclerotic lesions form at specific regions of the arterial tree: near bifurcations, bends and branch ostia. Local hemodynamic factors, in particular low endothelial shear stress, play a fundamental role in the localization of atherosclerotic plaques.^89–91 Cheng et al.⁹² report large variations in absolute wall shear stress levels along the arterial tree within one species and between species. Wall shear stress is inversely related to the vessel diameter.

Reproduction of these hemodynamic perturbations in animal models exposed to systemic factors (hypercholesteremia, diabetes, hypertension) leads to the acceleration of atherosclerosis. Different techniques have been proposed.

Vein graft: The purpose of this technique was to understand aortocoronary vein graft bypass failure.^93–97 Rapid atherosclerosis observed in vein bypass leading to thrombosis was not similar to atherosclerosis observed in the arteries. Vein graft atherosclerotic lesions are more diffuse, concentric and friable with a poorly developed or absent fibrous cap, whereas native vessel atheroma are proximal, focal, eccentric and non-friable with a well-developed fibrous cap.^98,99 Accelerated atherosclerotic lesions contain more foam cells with varying degrees of lipid accumulation and macrophage/mononuclear and inflammatory cell infiltration than native atherosclerotic lesions.¹⁰⁰ Therefore, vein graft is not a good model for human arterial atherosclerosis.

The blood flow cessation model with a total ligation of the artery: This model consists of total ligation of the common carotid artery near the bifurcation in the mouse model.^101,102 It is characterized by rapid and reproducible arterial remodelling with the development of significant intimal thickening. The lesions observed consist of intimal hyperplasia. Such models seem to be interesting in arterial remodelling analysis.

Partial ligation of the artery: This model consists of partial ligation of a straight arterial segment inducing hemodynamic perturbation. Ishii et al.^103,104 described this model in the common carotid artery of Yucatan mini-pigs also undergoing a high-fat diet. They obtained advanced atherosclerotic lesions comparable to those observed in humans.

Stenosis collar: Perivascular collar placement offers the advantage of maintaining the structural integrity of the endothelium while inducing rapid, site-controlled lesion formation.^105–108 The neointima observed within the collar is generally fibroproliferative, with limited foam cell formation and extracellular lipid deposition.^109–111

Cholesterol feeding or local (oxidized) LDL application in conjunction with the placement of a perivascular Silastic collar has been shown to promote the development of more atherosclerosis-like lesions in the rabbit carotid artery and more recently in LDL r − / − mice.^{90,106,107,112}

Atherosclerosis induced by ligation or stenosis collar can also be explained by local ischemia secondary to the adventitial vasa vasorum occlusion, direct media injury resulting in smooth muscle cell proliferation and migration to the intima, local inflammatory reaction and endothelial NO synthesis inhibition.

In our opinion, the ideal technique to induce atherosclerosis similar to humans is the systemic approach, which exposes the animals to well known risk factors. However, the level of exposure and development of hyperlipidemia and hyperglycemia should be as close as possible to the levels observed in humans. The major limitation of this approach is the time necessary to obtain atherosclerosis, particularly advanced lesions. The major drawback of the local intervention approach is the direct disruption of the arterial wall leading to difficulties interpreting the resulting lesions and the underlying causes of the normal lesion etiology. Reproduction of the hemodynamic conditions leading up to atherogenesis appears to be the best method, accelerating atherosclerosis development without direct mechanical endothelial injury.

Part II. Animal models of vulnerable plaque

Human vulnerable plaque

Human vulnerable plaque is an atherosclerotic plaque prone to rupture, thrombosis and emboli leading to the occurrence of cardiovascular events. Coronary and carotid vulnerable plaques have been extensively described through histopathological analysis and autopsic studies.^3–5

Morphological criteria of human vulnerable plaque are the following:

Large lipid rich-necrotic core associated with thin fibrous cap;

Intraplaque hemorrhage;

Fibrous cap rupture;

Plaque ulceration;

Inflammation with the presence of macrophages.

The underlying mechanisms of plaque vulnerability remain unknown. Preclinical animal models are essential to improve our understanding of the processes that influence the development of vulnerable plaque. There are two ways to develop such animal models of advanced atherosclerotic lesions:

Develop atherosclerotic plaque and look for spontaneous complications;

Develop atherosclerotic plaque and induce a complication.

Animal models of induced vulnerable plaque

This approach ignores the complexity of the mechanism of destabilization. They are rather models of rupture and athero-thrombosis than models of vulnerable plaque. After atherosclerotic plaque has been obtained, different methods are available to induce advanced lesions.

Pharmacological triggering with procoagulant Russel's viper venom and vasopressor histamine (Constantinides model)

This method was developed by Constantinides 40 years ago.¹¹³ Intraperitoneal injection of Russel's viper venom (RVV) (procoagulant with endothelial toxin) followed by intravenous injection of histamine (vasopressor) in high-fat diet rabbits led to plaque rupture and thrombosis. This model was reproduced by Abela and Chen.^114,115 Nakamura used serotonin or angiotensin II combined with RVV.¹¹⁶ Plaque rupture and luminal thrombosis are frequently observed and resemble human lesions. However the triggering process is not physiological and does not reproduce human pathophysiology.

Direct mechanical injury to plaques

Plaque rupture can be obtained by inflating an angioplasty balloon into the plaques or by directly squeezing the plaques with forceps.^117–119 Cold induced endothelial injury,⁸⁶ perivascular electrical injury¹²⁰ or radiation are also described to obtain vulnerable plaque in NZW Rabbits.^121,122 Erythrocyte injection into existing plaque has been reported to lead to vulnerable plaque.^123,124 Increased lipid content and augmentation of oxidative stress and inflammation in the plaque are the probable pathological mechanisms.

Such models are complex to perform, far from human physiology and expensive.

Histological analysis of arterial lesions that have been induced by pharmacological or mechanical intervention is difficult to interpret.

Transgenic destabilization of atherosclerotic plaque

Some studies propose a transgenic approach to promote plaque destabilization using inflammatory or apoptosis effects. Gene transfection has been reported directly in atherosclerotic plaque with the p53 gene or the MMP-9 gene or in systematic transfection with the IL-18 gene.^106,125,126

These models are complex, expensive and difficult to reproduce.

Exposure to stress

Rekther et al.¹²⁷ induced a central stress by intracerebral injection of corticotropin releasing factor in ApoE − / − mice. They observed plaque rupture and thrombosis. The signal pathway involved remains unknown.

Exposure to hypertension or Angiotensin II

Mazzolai et al.¹²⁸ showed that Angiotensin II, beyond its hemodynamic effect, modulates the atherosclerotic phenotype from stable to vulnerable in ApoE − / − mice.

Exposure to a modified wall shear stress

Ishii et al.¹⁰³ reported an animal model of vulnerable carotid atherosclerotic plaque. Carotid atherosclerotic models were performed in Yucatan mini-pigs by using a combination of partial ligation and high cholesterol diet. Typical features of vulnerable plaque (necrotic core with thin fibrous cap, IPH) were observed three months after surgery. Distal embolism in rete mirabile were identified and correlated with advanced plaques.

Cheng et al.^90,129 obtained vulnerable plaque with a constrictive collar around carotid arteries in ApoE − / − mice.

Animal models of spontaneous vulnerable plaque

There is no ideal animal model of spontaneous vulnerable plaque. Spontaneous ruptures are rare and fleeting. Such models are time consuming. It implies long-term experiments and the percentage of advanced lesions is expected to be low (Table 2).

Table 2

Animal models of spontaneous vulnerable plaque

Animal	Methods	Arterial site	Lesions observed	Delay	Limits
Apo E − / − miceRosenfeld (131)	Chow diet	Brachiocephalic arteries	Intraplaque hemorrhage IPH	42–54 weeks	No human like plaque rupture
			Plaque fissure or disruption		No thrombus
			Fibrotic conversion of necrotic zones		Long-term experiment
Apo E − / − miceJohnson–Williams (132,134,135)	High-fat diet	Brachiocephalic arteries	IPH	5–59 weeks	Low rate of thrombosis
			Fibrous cap rupture
			Thin fibrous cap with large lipid core
			Thrombosis
LDLR − / − and Apo E − / − miceCalara (138)	Normal or high-fat diet	Aorta (origin)	IPH	9–20 months	Frequent absence of a clearly defined single fibrous cap
		Coronary arteries	Deep plaque ruptures
			Thrombus

Rhesus MonkeysKusumi (139)	LDL receptor deficiency	Aorta	Thin fibrous cap	22 years	Only one case described
		Coronary arteries	Large lipid core
			Plaque rupture
Rapacz pigs Prescott (46)	Inherited hyper LDL	Coronary, iliac and femoral arteries	IPH	39–54 months	Media destruction is less pronounced than in human lesions
			Plaque rupture
			Neovascularization
			Thrombus
PigsGerrity (59)	Diabetes induced	Coronary	Lipid rich-necrotic core	9 months
	Normal or high-fat diet	Femoral	IPH
		Aorta	Severe calcification
MinipigsThim (65)	HFD	Coronary arteries	Thin fibrous cap	18 weeks
			Large lipid core
			IPH
			Neovascularization
			Calcification

IPH, intraplaque hemorrhage

Transgenic mice models of spontaneous vulnerable plaque

Rosenfeld et al.¹³⁰ described advanced lesions in the innominate artery of chow-fed Apo E − / − mice aged 42–54 weeks. Advanced arterial lesions were characterized by large necrotic core, fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap and calcifications. A high frequency of IPH was observed. The hemorrhage occurs at all stages of atherosclerosis and leads to fissures of lateral fatty streaks that form adjacent to or on top of the established plaques. There was no plaque rupture as observed in human lesions (rupture of a well formed fibrous cap) but rather plaque disruption (fissures of fatty streak). Moreover, no thrombus was observed in association with this disruption while occlusive thrombus is the predominant marker of plaque rupture in human lesions. This model has to be considered as a model for studying vascular fibrosis and calcification and not for studying vulnerable plaque. Johnson and Jackson have also reported advanced lesions in the innominate arteries of Apo E − / − mice fed a high-fat high-cholesterol diet which died after 46 ± 3 weeks (range 37–59).¹³¹ They report a high frequency of IPH associated with plaque rupture and luminal thrombus formation. The plaque rupture was characterized by fragmentation and loss of elastin in the fibrous cap of relatively small and lipid-rich plaques overlying large complex lesions.^132–134 According to Jackson and in contrary to the Rosenfeld hypothesis, this is a model for studying events leading to plaque rupture in humans.¹³⁵ Other mice models of advanced atherosclerotic plaque have been published with the low-density lipoprotein deficient mouse (LDL R(−/−)). LDL R(−/−) mice under a high-fat diet present similar lesions in the innominate arteries in size and composition to those described by Rosenfeld in chow fed Apo E −/− mice.¹³⁶

Calara et al.¹³⁷ reported, in 2001, IPH, deep plaque ruptures (or erosions reaching necrotic core areas) and large thrombus originating from the core of a disrupted atherosclerotic lesion in aorta and coronary arteries of Apo E (−/−) and LDL R (−/−) mice (9–20 months old). Lesions were rare and the frequent absence of a clearly defined single fibrous cap limits their usefulness as a model of fibrous cap rupture.

Large-animal models of spontaneous vulnerable plaque

Advanced atherosclerotic lesions were reported in a female rhesus monkey with spontaneous hypercholesterolemia related to a genetically determined LDL receptor deficiency which died at 22 years of age.¹³⁸ Lesions observed were multifocal in the aorta and coronary arteries and were characterized by heavy lipid deposition, fibrous cap, necrotic core, fibrinogen/fibrin in plaques and spontaneous rupture. This animal model published in 1993 has never been reproduced.

Prescott et al.^45,46 reported advanced atherosclerotic lesions similar to those found in humans in inherited hypercholesterolemia pigs at 39–54 months. Lesions were observed in coronary, iliac and femoral arteries and were characterized by fibrous cap, necrotic core, IPH, neovascularization, plaque rupture and rare luminal thrombosis. Necrotic core and eccentricity of lesions are very similar to those observed in humans, but media destruction is less pronounced. Gerrity et al.⁵⁸ developed a pig model (Yorkshire) of advanced atherosclerotic lesions in coronary, femoral arteries and aorta with hypercholesterolemia induced by high-fat diet and diabetes induced by intravenous injection of the β cell cytotoxin streptozotocin. Advanced lesions occurred in a relatively short time (9 months) and were characterized by fibroatheroma, IPH and severe calcifications. No rupture and no luminal thrombosis were described. Chatzizisis et al.⁸⁹ reproduced this model with success. The time necessary to obtain lesions and the rapid growth and size of the domestic pig limit the use of these models (90–120 kg at six months; adult body weight > 300 kg). To overcome size-related problems, models of spontaneous vulnerable atherosclerotic plaque in downsized, mini or micropigs are under development. Thim⁷⁸ recently generated a mini-pig with familial hypercholesterolemia by crossing the Rapacz pig (inherited hypercholesterolemia pigs) with a smaller pig and then with an even smaller mini-pig. After 18 weeks of atherogenic diet, body weights were between 30 and 50 kg. Spontaneous atherosclerotic lesions were found at 18 weeks in the proximal segments of the coronary arteries. These lesions were typically eccentric and characterized by necrotic core, thin and inflamed fibrous cap, IPH, expansive remodelling and calcifications. However plaque rupture or luminal thrombosis was not observed.

Part III. MRI and animal models of atherosclerosis

Post mortem histological analysis of atherosclerotic plaque in animal models is fundamental to validate the model and remains the gold standard. However, one of the major endpoints of animal models is to obtain a better knowledge of the mechanism of destabilization of the atherosclerotic plaque. Thus direct visualization of plaque in situ and in vivo over time could bring valuable insights into the processes involved.

Morphological and functional imaging of atherosclerotic plaques in animals must satisfy many constraints. These constraints are related to the animals themselves: the need for anesthesia, the difficulty of handling (animal size and weight) and the size and location of the arteries in question in relation to the geometry and sensitivity of the imaging coils available to achieve the necessary spatial resolution to characterize the lesion.

MRI: the imaging modality of choice in atherosclerosis assessment

Different non-invasive imaging modalities to evaluate atherosclerosis in animal models have been described. Ultrasound imaging techniques have been widely used for atherosclerotic plaque detection in the animal models of atherosclerosis. Recent advances with contrast enhanced techniques allow detection of neovascularization in atherosclerotic plaque.^139,140 Determination of plaque composition is based on tissue echogenecity. However precise plaque characterization (lipid core, intraplaque hemorrhage or fibrous cap rupture) is not possible. Image acquisition is complex and operator dependent.

The use of computed tomography (CT) has also been described in this field. The main advantages of CT include its high spatial resolution and short scan time but its major disadvantage for atherosclerotic plaque characterization is the limited discrimination of soft tissue. Micro and nano-CT allow higher spatial resolution but are limited for in vivo imaging.¹⁴¹ Recent experimental studies on animal models of atherosclerosis using CT report fused CT and positron emission tomography (with 18F Fluoro deoxyglucose [¹⁸FDG]) imaging which gives anatomic and metabolic assessement of atherosclerotic plaque.^142,143

MRI is well known to be a reliable tool for the analysis of atherosclerotic components in human carotid arteries.^144,145 It allows precise description of plaque: lipid-rich-necrotic core, fibrous cap, calcification and fibrosis. Vulnerability criteria such as ulceration, cap rupture and IPH are also easily identifiable. Furthermore, contrast agents (gadolinium, USPIO) give information on the inflammatory activity and the presence of macrophages in the plaque. In brief, the main advantages of MRI include its high spatial resolution, great soft-tissue contrast, lack of radiation exposure, good clinical translatability and the ability to combine functional, anatomic and molecular information. Moreover, anesthesia can be performed with continuous gas inhalation through a mask without invasive maneuver and without close monitoring allowing a long scan time. Thus MRI is well-suited to monitor atherosclerotic plaque formation and destabilization in preclinical animal models.

Morphological imaging: atherosclerotic plaque composition

Several authors have already used MRI to study atherosclerotic lesions in animal models, such as mice, rabbit or pig. Skinner et al.¹⁴⁶ have demonstrated the usefulness of high resolution MRI to show atherosclerosis progression and to determine composition of atherosclerotic lesions in the rabbit abdominal aorta. Fayad et al.¹⁴⁷ developed a non-invasive MR microscopy technique to study in vivo atherosclerotic lesions in ApoE −/− mice with an excellent correlation between MRI and histopathology. Another study demonstrates aneurysm development over atherosclerotic lesions in the abdominal aorta of ApoE −/− mice.¹⁴⁸ Helft et al.¹⁴⁹ used in vivo MRI to quantify atherosclerotic components in the thoracic and abdominal aorta of rabbits. There was a significant correlation for plaque composition (P < 0.05) between MRI and histopathology for the analysis of lipidic and fibrous components. Johnstone et al.¹⁵⁰ reported in vivo MRI of thrombus formation after pharmacological triggering of plaque disruption in the modified Constantinides rabbit model of plaque disruption. MRI data correlated with the histopathology regarding aortic wall thickness (R = 0.77, P < 0.0005), thrombus size (R = 0.82, P < 0.0001), thrombus length (R = 0.86, P < 0.005) and anatomic location (R = 0.98, P < 0.0001). More recently Phinikaridou et al.¹⁵¹ demonstrated that in vivo MRI at 3.0 T detects features of vulnerable plaques in NZW rabbits aorta. It has been also demonstrated that MRI is a useful tool in pig models to determine arterial wall thickness and area, wall hematoma and thrombus in carotid and coronary atherosclerotic lesions.^152–155

Intraplaque neovascularization and inflammation can also be assessed with gadolinium enhanced MRI in animal models.^152,156,157 Furthermore, the ability of dynamic contrast enhanced (DCE) MRI to detect neovessels has recently been reported in atherosclerotic lesions of rabbit abdominal aorta.^158,159 DCE-MRI consists in repeated MRI acquisitions of the same imaging slice/volume serially with high temporal resolution after the injection of a Gd-based contrast agent.

Molecular imaging

In addition to morphological characterization obtained with MRI, several novel molecular enhancers have been developed in animal models targeting different cells, molecules and biological process which allow a more detailed characterization of biological process at play in atherosclerosis. Molecular targets for macrophages, elastin, fibrin and HDL have been tested in animal models. Ultra-small iron oxide particle enhanced MRI has been developed for macrophage imaging in animal models of atherosclerosis with a good correlation with histology.^35,160,161 MRI of coronary wall using an elastin-binging contrast agent in a swine model of coronary atherosclerosis has been recently reported.¹⁶² The authors demonstrated the ability of this agent to detect and quantify the vascular remodeling. Botnar et al.¹⁶³ demonstrated the feasibility of in vivo molecular MRI for the detection of acute and subacute thrombosis using a fibrin-binding MRI contrast agent in an animal model of atherosclerosis. HDL-based MRI contrast agent in atherosclerotic mice have been also reported.¹⁶⁴ This contrast agent which enhances macrophage-rich areas of atherosclerotic plaque allows a more detailed plaque composition description.

Multimodality and functional imaging

Myeloperoxidase, a central inflammatory enzyme secreted by activated macrophages is involved in multiple stages of plaque destabilization in human atherosclerotic plaques.¹⁶⁵

A myelopreoxydase-activatable contrast agent has been reported in atherosclerotic rabbits with a good histological correlation with myeloperoxidase-rich areas infiltrated by macrophages.¹⁶⁶

Promising combined MR-PET (18F Fluoro deoxyglucose) is under development. By combining the excellent anatomical (MRI) with functional information (PET), these advancements may allow a more comprehensive and accurate quantification of inflammation in atherosclerotic plaques.

Imaging of plaque biomechanics

Analysis of biomechanical features of atherosclerotic plaques revealed the role played in atherosclerotic plaque development and destabilization. MRI with specific sequences can provide information on flow velocity and wall shear stress in the arterial tree. Thus, some authors report the use of MRI to assess the biomechanical process of atherosclerosis development in animal models of atherosclerosis induced by partial ligation or stenosis collar.^167–170

Technical aspects

Vessel wall MRI imaging in these animal models requires high spatial resolution and high signal-to-noise ratio and with minimal acquisition time. The luminal diameter and the wall thickness of the mouse aorta are around 0.5 mm and 40–200 μm (Figure 1). Visualization of such small features requires very high field strength magnets, high-spatial and temporal resolution three-dimensional imaging, respiratory and cardiac gating and continuous anesthesia administration to allow longer imaging. A number of groups have developed high-field MRI microscopy studies of atherosclerosis in mice at 9.4 and 17.6 T,^147,171,172 and at 4.7 and 7 T.¹⁷³

Figure 1

Magnetic resonance imaging and animal models of atherosclerosis. (a) Mouse aorta (Apo E−/−), internal diameter = 0.5 mm and arterial wall thickness = 40–200 μm. (b) Rabbit aorta (Watanabe), internal diameter = 5 mm and arterial wall thickness = 0.4–1.3 mm. (c) Minipigs carotid artery, internal diameter = 6 mm and arterial wall thickness = 1 mm

The use of rabbit or pig models, with their larger arteries, has permitted the use of clinical MRI imagers (1.5 or 3 Tesla), which are more readily available and allow for translation of imaging protocols to studies in humans (Figure 1).

Future directions

Vulnerable atherosclerotic plaques are responsible for triggering critical cardiovascular events. Clinical imaging of patients can provide much useful information, for example the identification of the lipid-rich necrotic core, and we have gained much knowledge from such studies in humans, but imaging of the development of disease from the early stages of stable plaque through the time the plaque becomes vulnerable and presents with clinical events is lacking and it is hard to imagine such a study. In vivo imaging studies of animal models of atherosclerosis provide valuable opportunities to investigate the pathophysiological etiology of plaque. The non-invasive nature of many imaging modalities, particularly MRI, allows frequent assessment enabling intensive monitoring of plaque formation and progression, and may provide critical information on the processes involved in vulnerable plaque development and rupture. Going forward, in both preclinical and clinical settings, the combination of multiple imaging modalities, including high resolution MRI, PET, duplex US and biological markers may be useful and complementary tools in the understanding of vulnerable atherosclerosis and cardiovascular disease.

Conflicts of interest: None.

References

Writing Group Members, Lloyd-Jones

Adams

Brown

American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics 2010 update: a report from the American Heart Association. Circulation 2010;121:46–215

Libby

Ridker

Maseri

. Inflammation and atherosclerosis. Circulation 2002;105:1135–43

Stary

Chandler

Dinsmore

A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995;92:1355–74

Virmani

Kolodgie

Burke

Farb

Schwartz

. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol 2000;20:1262–75

Redgrave

Lovett

Gallagher

Rothwell

. Histological assessment of 526 symptomatic carotid plaques in relation to the nature and timing of ischemic symptoms: the Oxford plaque study. Circulation 2006;113:2320–8

Underhill

Hatsukami

Fayad

Fuster

Yuan

. MRI of carotid atherosclerosis: clinical implications and future directions. Nat Rev Cardiol 2010;7:165–73

Elkhaili

Salmon

Interest of use of miniature pig in pharmacokinetic studies. J Pharm Clin 1997;16:219–23

Uginbuhl

Pauli

Ratcliffe

. Atherosclerosis in swine and swine as a model for the study of atherosclerosis. Adv Cardiol 1974;13:119–26

Rekhter

. How to evaluate plaque vulnerability in animal models of atherosclerosis? Cardiovasc Res 2002;54:36–41

10.

Cefalu

Wagner

. Aging and atherosclerosis in human and nonhuman primates. Age 1997;20:15–28

11.

Narayanaswamy

Wright

Kanadarpa

. Animal models for atherosclerosis, restenosis, and endovascular graft research. J Vasc Interv Radiol 2000;11:5–17

12.

Russel

Proctor

. Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis. Cardiovasc Pathol 2006;15:318–30

13.

Schreyer

Wilson

LeBoeuf

. C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis. Atherosclerosis 1998;136:17–24

14.

Rosenfel

Kauser

Martin-McNulty

Polinsky

Schwartz

Rubanyi

. Estrogen inhibits the initiation of fatty streaks throughout the vasculature but does not inhibit intra-plaque hemorrhage and the progression of established lesions in apoliporpotein E deficient mice. Atherosclerosis 2002;164:251–9

15.

Zhu

Carmeliet

Fay

. Plasminogen activator inhibitor-1 is a major determinant of arterial thrombolysis resistance. Circulation 1999;99:3050–5

16.

Bouma

Meijers

. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). J Thromb Haemost 2003;1:1566–74

17.

Te Velde

Wagenaar

Reijerkerk

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2003;1:2087–96

18.

Breslow

. Mouse models of atherosclerosis. Science 1996;272:685–8

19.

Moghadasian

Frohlich

McManus

. Advances in experimental dyslipidemia and atherosclerosis. Lab Invest 2001;81:1173–83

20.

Singh

Tiwari

Dikshit

Barthwal

. Models to study atherosclerosis: a mechanistic insight. Curr Vasc Pharmacol 2009;7:75–109

21.

Thiery

Nebendahl

Rapp

Kluge

Teupser

Seidel

. Low atherosclerotic response of a strain of rabbits to diet-induce hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995;15:1181–8

22.

Taylor

Fan

. Transgenic rabbit models for the study of atherosclerosis. Front Biosci 1997;2:298–308

23.

Chapman

. Animal lipoproteins: chemistry, structure, and comparative aspects. J Lipid Res 1980;21:789–853

24.

Warren

Elbert

Mitchell

Barter

. Rabbit hepatic lipase cDNA sequence: low activity is associated with low messenger RNA levels. J lipid Res 1991;32:1333–9

25.

Nagashima

McLean

Lawn

. Cloning and mRNA tissue distribution of rabbit cholesteryl ester transfer protein. J Lipid Res 1988;29:1643–9

26.

Tall

. Plasma cholesteryl transfer protein. J lipoid Res 1993;34:1255–74

27.

Kamimura

Suzuki

Sakamoto

Miura

Misumi

Miyahara

. Development of atherosclerotic lesions in cholesterol-loaded rabbits. Exp Anim 1999;48:1–7

28.

Watanabe

. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL rabbit). Atherosclerosis 1980;36:261–8

29.

Seddon

Woolf

La Ville

Hereditary hyperlipidemia and atherosclerosis in the rabbit due to overproduction of lipoproteins. II. Preliminary report of arterial pathology. Arteriosclerosis 1987;7:113–24

30.

Finking

Hanke

. Nikolaj Nikolajewitsch Anitschkow (1885–1964) established the cholesterol-fed rabbit as a model for atherosclerosis research. Atherosclerosis 1997;135:1–7

31.

Hadjiisky

Bourdillon

Grosgogeat

. Experimental models of atherosclerosis. Contribution, limits and trends. Arch Mal Coeur Vaiss 1991;84:1593–603

32.

Brousseau

Hoeg

. Transgenic rabbits as models for atherosclerosis research. J Lipid Res 1999;40:365–75

33.

Fan

Wang

Bensadoun

Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins. Proc Natl Acad Sci USA 1994;91:8724–8

34.

Huang

Schwendner

Rall

Jr Sanan

Mahley

. Apolipoprotein E2 transgenic rabbits. Modulation of teh type III hyperlipoproteinemic phenotype by estrogen and occurrence of spontaneous atherosclerosis. J Biol Chem 1997;272:22685–94

35.

Sigovan

Boussel

Sulaiman

Rapid-clearance iron nanoparticles for inflammation imaging of atherosclerotic plaque: initial experience in animal model. Radiology 2009;252:401–9

36.

Wetterholm

Caidahl

Volkmann

Brandt-Eliasson

Fritsche-Danielson

Gan

. Imaging of atherosclerosis in WHHL rabbits using high-resolution ultrasound. Ultrasound Med Biol 2007;33:720–6

37.

Badimon

. Atherosclerosis and thrombosis: lessons from animal models. Thromb Haemost 2001;86:356–65

38.

Dolgin

. Minipig, minipig, let me in. Nat Med 2010;16:1349

39.

Lunney

. Advances in swine biomedical model genomics. Int J Biol Sci 2007;3:179–84

40.

White

Bloor

. Coronary collateral circulation in the pig: correlation of collateral flow with coronary bed size. Basic Res Cardiol 1981;76:189–96

41.

Swindle

Horneffer

Gardner

Anatomic and anesthetic considerations in experimental cardiopulmonary surgery in swine. Lab Anim Sci 1986;36:357–61

42.

Smith

Spinale

Swindle

. Cardiac function and morphology of Hanford miniature swine and Yucatan miniature and micro swine. Lab Anim Sci 1990;40:47–50

43.

Dixon

Stoops

Parker

Laughlin

Weisman

Sturek

. Dyslipidemia and vascular dysfunction in diabetic pigs fed an atherogenic diet. Arterioscler Thromb Vasc Biol 1999;19:2981–92

44.

Lam

Lacoste

Bourassa

. Cilazapril and early atherosclerotic changes after balloon injury of porcine carotid arteries. Circulation 1992;85:1542–7

45.

Prescott

McBride

Hasler-Rapacz

Von Linden

Rapacz

. Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for Apolipoprotein B. Am J Pathol 1991;139:139–47

46.

Prescott

Hasler-Rapacz

von Linden-Reed

Rapacz

. Familial hypercholesterolemia associated with coronary atherosclerosis in swine bearing different alleles for apolipoprotein B. Ann N Y Acad Sci 1995;748:283–92

47.

Lewis

Page

. Hereditary obesity: relation to serum lipoproteins and protein concentrations in swine. Circulation 1956;14:55–9

48.

Detweiler

. Genetic aspects of cardiovascular diseases in animals. Circulation 1964;30:114–27

49.

Thim

Hagensen

Drouet

Familial hypercholesterolaemic downsized pig with human-like coronary atherosclerosis: a model for preclinical studies. EuroIntervention 2010;6:261–8

50.

Miyoshi

Horiuchi

Inokuchi

Novel microminipig model of atherosclerosis by high fat and high cholesterol diet, established in Japan. In Vivo 2010;24:671–80

51.

Ehtisham

Chimowitz

Furlan

Lafranchise

. Systemic risk factors associated with progression of atherosclerosis from the coronary to the carotid arteries. J Stroke Cerebrovasc Dis 2005;14:182–5

52.

Aguilera

Ramírez-Tortosa

Mesa

Ramírez-Tortosa

Gil

. Sunflower, virgin-olive and fish oils differentially affect the progression of aortic lesions in rabbits with experimental atherosclerosis. Atherosclerosis 2002;162:335–44

53.

Kritchevsky

Tepper

Wright

Czarnecki

Wilson

Nicolosi

. Cholesterol vehicle in experimental atherosclerosis 24: avocado oil. J Am Coll Nutr 2003;22:52–5

54.

Nishina

Verstuyft

Paigen

. Synthetic low and high fat diets for the study of atherosclerosis in the mouse. J Lipid Res 1990;3:859–69

55.

Garcia

McNamara

Gordon

Kannel

. Morbidity and mortality in diabetics in the Framingham population. Sixteen-year follow-up study. Diabetes 1974;23:105–11

56.

Rönnemaa

Laakso

Puukka

Kallio

Pyörälä

. Atherosclerotic vascular disease in middle-aged, insulin-treated, diabetic patients. Association with endogenous insulin secretion capacity. Arteriosclerosis 1988;8:237–44

57.

Yin

Wang

A minipig model of high-fat/high-sucrose diet-induced diabetes and atherosclerosis. Int J Exp Pathol 2004;85:223–31

58.

Gerrity

Natarajan

Nadler

Kimsey

. Diabetes-induced accelerated atherosclerosis in swine. Diabetes 2001;50:1654–65

59.

Klein

Lee

. Components of the metabolic syndrome and risk of cardiovascular disease and diabetes in Beaver Dam. Diabetes Care 2002;25:1790–4

60.

Neeb

Edwards

Alloosh

Long

Mokelke

Sturek

. Metabolic syndrome and coronary artery disease in Ossabaw compared with Yucatan swine. Comp Med 2010;60:300–15

61.

Dyson

Alloosh

Vuchetich

Mokelke

Sturek

. Components of excess atherogenic diet. Comp Med 2006;56:35–45

62.

Sekhara Rao

Wagner

Hutchins

Rowe

Edwards

. Spontaneous and diet-aggravated atherosclerosis in White Carneau pigeons genetically selected for discordant blood pressure. Atherosclerosis 1987;65:29–35

63.

Sugiyama

Haraoka

Watanabe

Acceleration of atherosclerotic lesions in transgenic mice with hypertension by the activated renin-angiotensin system. Lab Invest 1997;76:835–42

64.

Weiss

Kools

Taylor

. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in apoE-deficient mice. Circulation 2001;103:448–54

65.

Daugherty

Manning

Cassis

. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest 2000;105:1605–12

66.

Weiss

Taylor

. Deoxycorticosterone acetate salt hypertension in apolipoprotein E − / − mice results in accelerated atherosclerosis: the role of angiotensin II. Hypertension 2008;51:218–24

67.

Wiesel

Mazzolai

Nussberger

Pedrazzini

. Two-kidney, one clip and one-kidney, one clip hypertension in mice. Hypertension 1997;29:1025–30

68.

Campbell

Day

Skinner

Tume

. The effect of hypertension on the accumulation of lipids and the uptake of (3H)cholesterol by the aorta of normal-fed and cholesterol-fed rabbits. Atherosclerosis 1973;18:301–19

69.

Chobanian

Lichtenstein

Nilakhe

Haudenschild

Drago

Nickerson

. Influence of hypertension on aortic atherosclerosis in the Watanabe rabbit. Hypertension 1989;14:203–9

70.

Chen

Quan

Hwang

Atherosclerosis lesion progression during inhalation exposure to environmental tobacco smoke: a comparison to concentrated ambient air fine particles exposure. Inhal Toxicol 2010;22:449–59

71.

Messas

Batista

Jr Levine

Amar

. Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model. Circulation 2002;105:861–7

72.

Muhlestein

Anderson

Hammond

Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model. Circulation 1998;97:633–6

73.

Ross

Glomset

. The pathogenesis of atherosclerosis. N Engl J Med 1976;295:369–77

74.

Moore

. Thromboatherosclerosis in normolipemic rabbits: a result of continued endothelial damage. Lab Invest 1973;29:478–87

75.

Steele

Chesebro

Stanson

Balloon angioplasty: natural history of the pathophysiologic response to injury in a pig model. Circ Res 1985;57:105–12

76.

Block

Baughman

Pasternack

Fallon

. Transluminal angioplasty: correlation of morphologic and angiographic findings in an experimental model. Circulation 1980;61:778–85

77.

Recchia

Abendschein

Saffitz

Wickline

. The biologic behavior of balloon hyperinflation-induced arterial lesions in hypercholesterolemic pigs depends on the presence of foam cells. Arterioscler Thromb Vasc Biol 1995;15:924–9

78.

Thim

. Human-like atherosclerosis in minipigs: a new model for detection and treatment of vulnerable plaques. Dan Med Bull 2010;57:B4161

79.

Vita

Loscalzo

. Shouldering the risk factor burden: infection, atherosclerosis, and the vascular endothelium. Circulation 2002;106:164–6

80.

Turk

Henderson

Vanvickle

Watkins

Laughlin

. Arterial endothelial function in a porcine model of early stage atherosclerotic vascular disease. Int J Exp Pathol 2005;86:335–45

81.

Lee

Jarmolych

Kim

Production of advanced coronary atherosclerosis, myocardial infarction and ‘sudden death’ in swine. Exp Mol Pathol 1971;15:170–90

82.

Betz

Schlote

. Responses of vessel walls to chronically applied electrical stimuli. Basic Res Cardiol 1979;74:10–20

83.

Kling

Holzschuh

Strohschneider

Betz

. Enhanced endothelial permeability and invasion of leukocytes into the artery wall as initial events in experimental arteriosclerosis. Int Angiol 1987;6:21–8

84.

Suzuki

Lyons

Yeung

Ikeno

. The porcine restenosis model using thermal balloon injury: comparison with the model by coronary stenting. J Invasive Cardiol 2008;20:142–6

85.

Kondo

Umemura

Miyaji

Nakashima

. Milrinone. A phosphodiesterase inhibitor suppresses intimal thickening after photochemically induced endothelial injury in the mouse femoral artery. Atherosclerosis 1999;142:133–8

86.

Fang

Zhang

Jiang

. Developing a novel rabbit model of atherosclerotic plaque rupture and thrombosis by cold-induced endothelial injury. J Biomed Sci 2009;16:39–48

87.

Keelan

Bayes-Genis

Kantor

A novel porcine model for in vivo detection of vulnerable plaque: deposition and localization of lipid-rich necrotic lesions in the coronary arterial wall. Circulation 2001;104:11–67

88.

Granada

Moreno

Burke

Schulz

Raizner

Kaluza

. Endovascular needle injection of cholesteryl linoleate into the arterial wall produces complex vascular lesions identifiable by intravascular ultrasound: early development in a porcine model of vulnerable plaque. Coron Artery Dis 2005;16:217–24

89.

Chatzizisis

Jonas

Coskun

Prediction of the localization of high-risk coronary atherosclerotic plaques on the basis of low endothelial shear stress: an intravascular ultrasound and histopathology natural history study. Circulation 2008;117:993–1002

90.

Cheng

Tempel

van Haperen

Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation 2006;113:2744–53

91.

Chatzizisis

Coskun

Jonas

Edelman

Feldman

Stone

. Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling: molecular, cellular, and vascular behavior. J Am Coll Cardiol 2007;49:2379–93

92.

Cheng

Helderman

Tempel

Large variations in absolute wall shear stress levels within one species and between species. Atherosclerosis 2007;195:225–35

93.

Zwolak

Kirkman

Clowes

. Atherosclerosis in rabbit vein grafts. Arteriosclerosis 1989;9:374–9

94.

Lardenoye

de Vries

Löwik

Accelerated atherosclerosis and calcification in vein grafts: a study in APOE*3 Leiden transgenic mice. Circ Res 2002;91:577–84

95.

Dietrich

Zou

Rapid development of vein graft atheroma in ApoE-deficient mice. Am J Pathol 2000;157:659–69

96.

Hanyu

Kume

Ikeda

Minami

Kita

Komeda

. VCAM-1 expression precedes macrophage infiltration into subendothelium of vein grafts interposed into carotid arteries in hypercholesterolemic rabbits – a potential role in vein graft atherosclerosis. Atherosclerosis 2001;158:313–9

97.

Zou

Dietrich

Metzler

Wick

. Mouse model of venous bypass graft arteriosclerosis. Am J Pathol 1998;153:1301–10

98.

Kalan

Roberts

. Morphologic findings in saphenous veins used as coronary arterial bypass conduits for longer than 1 year: necropsy analysis of 53 patients, 123 saphenous veins, and 1865 five-millimeter segments of veins. Am Heart J 1990;119:1164–84

99.

Neitzel

Barboriak

Pintar

Qureshi

. Atherosclerosis in aortocoronary bypass grafts. Morphologic study and risk factor analysis 6 to 12 years after surgery. Arteriosclerosis 1986;6:594–600

100.

Kockx

De Meyer

Bortier

Luminal foam cell accumulation is associated with smooth muscle cell death in the intimal thickening of human saphenous vein grafts. Circulation 1996;94:1255–62

101.

Ivan

Khatri

Johnson

Expansive arterial remodeling is associated with increased neointimal macrophage foam cell content: the murine model of macrophage-rich carotid artery lesions. Circulation 2002;105:2686–91

102.

Sasaki

Kuzuya

Nakamura

A simple method of plaque rupture induction in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2006;26:1304–9

103.

Ishii

Viñuela

Murayama

Swine model of carotid artery atherosclerosis: experimental induction by surgical partial ligation and dietary hypercholesterolemia. AJNR Am J Neuroradiol 2006;27:1893–9

104.

Shi

Feng

Vulnerable plaque in a swine model of carotid atherosclerosis. AJNR Am J Neuroradiol 2009;30:469–72

105.

Booth

Martin

Honey

Hassall

Beesley

Moncada

. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis 1989;76:257–68

106.

von der Thüsen

van Berkel

Biessen

. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation 2001;103:1164–70

107.

Bentzon

Sondergaard

Kassem

Falk

. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation 2007;116:2053–61

108.

Folts

Crowell

Rowe

. Platelet aggregation in partially obstructed vessels and its elimination with aspirin. Circulation 1976;54:365–70

109.

Booth

RFG

Martin

Honey

Hassall

Beesley

Moncada

. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis 1989;76:257–68

110.

Kockx

De Meyer

GRY

Jacob

Bult

Herman

. Triphasic sequence of neointimal formation in the cuffed carotid artery of the rabbit. Arterioscler Thromb 1992;12:1447–57

111.

Akishita

Ouchi

Miyoshi

Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells. Atherosclerosis 1997;130:1–10

112.

Matthys

Van Hove

Kockx

Local application of LDL promotes intimal thickening in the collared carotid artery of the rabbit. Arterioscler Thromb Vasc Biol 1997;17:2423–29

113.

Constantinides

Charkracarti

. Rabbit arterial thrombosis production by systemic procedures. Arch Pathol 1961;72:197–208

114.

Abela

Picon

Friedl

Triggering of plaque disruption and arterial thrombosis in an atherosclerotic rabbit model. Circulation 1995;91:776–84

115.

Chen

Zhang

Liu

Prediction of atherosclerotic plaque ruptures with high-frequency ultrasound imaging and serum inflammatory markers. Am J Physiol Circ Physiol 2007;293:2836–44

116.

Nakamura

Abe

Kinukawa

. Aortic medial necrosis with or without thrombosis in rabbits treated with Russell's viper venom and angiotensin II. Atherosclerosis 1997;128:149–56

117.

Gertz

Fallon

Gallo

Hirudin reduces tissue factor expression in neointima after balloon injury in rabbit femoral and porcine coronary arteries. Circulation 1998;98:580–7

118.

Rekhter

Hicks

Brammer

Animal model that mimics atherosclerotic plaque rupture. Circ Res 1998;83:705–13

119.

Reddick

Zhang

Maeda

. Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice. Atherosclerosis 1998;140:297–305

120.

Chiesa

Di Mario

Colombo

Development of a lipid-rich, soft plaque in rabbits, monitored by histology and intravascular ultrasound. Atherosclerosis 2001;156:277–87

121.

Pakala

Leborgne

Cheneau

Radiation-induced atherosclerotic plaque progression in a hypercholesterolemic rabbit: a prospective vulnerable plaque model?

Cardiovasc Radiat Med 2003;4:146–51

122.

Leborgne

Pakala

Dilcher

Effect of antioxidants on atherosclerotic plaque formation in balloon-denuded and irradiated hypercholesterolemic rabbits. J Cardiovasc Pharmacol 2005;46:540–7

123.

Lin

Pathological mechanisms and dose dependency of erythrocyte-induced vulnerability of atherosclerotic plaques. J Mol Cell Cardiol 2007;43:272–80

124.

Kolodgie

Gold

Burke

Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med 2003;349:2316–25

125.

de Nooijer

Verkleij

von der Thüsen

Lesional overexpression of matrix metalloproteinase-9 promotes intraplaque hemorrhage in advanced lesions but not at earlier stages of atherogenesis. Arterioscler Thromb Vasc Biol 2006;26:340–6

126.

de Nooijer

von der Thüsen

Verkleij

Overexpression of IL-18 decreases intimal collagen content and promotes a vulnerable plaque phenotype in apolipoprotein-E-deficient mice. Arterioscler Thromb Vasc Biol 2004;24:2313–9

127.

Rekhter

Labatzky

Meltzer

. Plaque rupture, thrombosis and paraplegia are triggered by corticotropin releasing factor: a pilot study in mice. J Submicrosc Cytol Pathol 2000;32:415

128.

Mazzolai

Duchosal

Korber

Endogenous angiotensin II induces atherosclerotic plaque vulnerability and elicits a Th1 response in ApoE − / − mice. Hypertension 2004;44:277–82

129.

Cheng

Tempel

van Haperen

Activation of MMP8 and MMP13 by angiotensin II correlates to severe intra-plaque hemorrhages and collagen breakdown in atherosclerotic lesions with a vulnerable phenotype. Atherosclerosis 2009;204:26–33

130.

Rosenfeld

Polinsky

Virmani

Kauser

Rubanyi

Schwartz

. Advanced atherosclerotic lesions in innominate artery of apoE knockout mouse. Arterioscler Thromb Vasc Biol 2000;20:2587–92

131.

Johnson

Jackson

. Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse. Atherosclerosis 2001;154:399–406

132.

Jackson

Bennett

Biessen

Johnson

Krams

. Assessment of unstable atherosclerosis in mice. Arteioscler Thromb Vasc Biol 2007;27:714–20

133.

Williams

Johnson

Carson

Jackson

. Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 2002;22:788–92

134.

Johnson

Carson

Williams

Plaque rupture after short periods of fat feeding in the apolipoprotein E-knockout mouse: model characterization and effects of pravastatin treatment. Circulation 2005;111:1422–30

135.

Jackson

. Defining and defending murine models of plaque rupture. Arterio Thromb Vasc Biol 2007;27:973–7

136.

MacDougall

Kramer

Polinsky

Aggressive very low-density lipoprotein (VLDL) and LDL lowering by gene transfer of the VLDL receptor combined with a low-fat diet regimen induces regression and reduces macrophage content in advanced atherosclerotic lesions in LDL receptor deficient mice. Am J Pathol 2006;168:2064–73

137.

Calara

Silvestre

Casanada

Yuan

Napoli

Palinski

. Spontaneous plaque rupture and secondary thrombosis in apolipoprotein E-deficient and LDL receptor deficient mice. J Pathol 2001;195:257–63

138.

Kusumi

Scanu

McGill

Wissler

. Atherosclerosis in a rhesus monkey with genetic hypercholesterolemia and elevated plasma Lp(a). Atherosclerosis 1993;99:165–74

139.

Giannarelli

Ibanez

Cimmino

Contrast-enhanced ultrasound imaging detects intraplaque neovascularization in an experimental model of atherosclerosis. JACC Cardiovasc Imaging 2010;3:1256–64

140.

Nitta-Seko

Nitta

Shiomi

Utility of contrast-enhanced ultrasonography for qualitative imaging of atherosclerosis in Watanabe heritable hyperlipidemic rabbits: initial experimental study. Jpn J Radiol 2010;28:656–62

141.

Awan

Denis

Bailey

The LDLR deficient mouse as a model for aortic calcification and quantification by micro-computed tomography. Atherosclerosis 2011;219:455–62

142.

Vucic

Dickson

Calcagno

Pioglitazone modulates vascular inflammation in atherosclerotic rabbits noninvasive assessment with FDG-PET-CT and dynamic contrast-enhanced MR imaging. JACC Cardiovasc Imaging 2011;4:1100–9

143.

Mehta

Torigian

Gelfand

Saboury

Alavi

. Quantification of atherosclerotic plaque activity and vascular inflammation using [18-F] Fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT). J Vis Exp 2012:3777

doi: 10.3791/3777

144.

Moody

. Characterization of complicated carotid plaque with magnetic resonance direct thrombus imaging in patients with cerebral ischemia. Circulation 2003;107:3047–52

145.

Saam

Hatsukami

Takaya

The vulnerable, or high-risk, atherosclerotic plaque: noninvasive MR imaging for characterization and assessment. Radiology 2007;244:64–77

146.

Skinner

Yuan

Mitsumori

Serial magnetic resonance imaging of experimental atherosclerosis detects lesion fine structure, progression and complications in vivo. Nat Med 1995;1:69–73

147.

Fayad

Fallon

Shinnar

Noninvasive In vivo high-resolution magnetic resonance imaging of atherosclerotic lesions in genetically engineered mice. Circulation 1998;98:1541–7

148.

McFadden

Chaabane

Contard

In vivo magnetic resonance imaging of large spontaneous aortic aneurysms in old apolipoprotein E-deficient mice. Invest Radiol 2004;39:585–90

149.

Helft

Worthley

Fuster

Atherosclerotic aortic component quantification by non-invasive magnetic resonance imaging: an in vivo study in rabbits. J Am Coll Cardiol 2001;37:1149–54

150.

Johnstone

Botnar

Perez

In vivo magnetic resonance imaging of experimental thrombosis in a rabbit model. Arterioscler Thromb Vasc Biol 2001;21:1556–60

151.

Phinikaridou

Ruberg

Hallock

In vivo detection of vulnerable atherosclerotic plaque by MRI in a rabbit model. Circ Cardiovasc Imaging 2010;3:323–32

152.

Lin

Abendschein

Haacke

. Contrast-enhanced magnetic resonance angiography of carotid arterial wall in pigs. J Magn Reson Imaging 1997;7:183–90

153.

Worthley

Helft

Fuster

High resolution ex vivo magnetic resonance imaging of in situ coronary and aortic atherosclerotic plaque in a porcine model. Atherosclerosis 2000;150:321–9

154.

Worthley

Helft

Fuster

Noninvasive in vivo magnetic resonance imaging of experimental coronary artery lesions in a porcine model. Circulation 2000;101:2956–61

155.

Worthley

Helft

Fuster

Serial In vivo MRI documents arterial remodeling in experimental atherosclerosis. Circulation 2000;101:586–9

156.

Koktzoglou

Harris

Tang

Gadofluorine-enhanced magnetic resonance imaging of carotid atherosclerosis in Yucatan miniswine. Invest Radiol 2006;41:299–304

157.

Chaabane

Pellet

Bourdillon

Contrast enhancement in atherosclerosis development in a mouse model: in vivo results at 2 Tesla. MAGMA 2004;17:188–95

158.

Calcagno

Vucic

Mani

Goldschlager

Fayad

. Reproducibility of black blood dynamic contrast-enhanced magnetic resonance imaging in aortic plaques of atherosclerotic rabbits. J Magn Reson Imaging 2010;32:191–8

159.

Calcagno

Mani

Ramachandran

Fayad

. Dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) of atherosclerotic plaque angiogenesis. Angiogenesis 2010;13:87–99

160.

Sigovan

Bessaad

Alsaid

Assessment of age modulated vascular inflammation in ApoE − / − mice by USPIO-enhanced magnetic resonance imaging. Invest Radiol 2010;45:702–7

161.

Bessaad

Sigovan

Alsaid

M1-activated macrophages migration, a marker of aortic atheroma progression: a preclinical MRI study in mice. Invest Radiol 2010;45:262–9

162.

von Bary

Makowski

Preissel

MRI of coronary wall remodeling in a swine model of coronary injury using an elastin-binding contrast agent. Circ Cardiovasc Imaging 2011;4:147–55

163.

Botnar

Perez

Witte

In vivo molecular imaging of acute and subacute thrombosis using a fibrin-binding magnetic resonance imaging contrast agent. Circulation 2004;109:2023–9

164.

Cormode

Briley-Saebo

Mulder

An ApoA-I mimetic peptide high-density-lipoprotein-based MRI contrast agent for atherosclerotic plaque composition detection. Small 2008;4:1437–44

165.

Chen

Pham

Weissleder

Bogdanov

Jr . Human myeloperoxidase: a potential target for molecular MR imaging in atherosclerosis. Magn Reson Med 2004;52:1021–8

166.

Ronald

Chen

Enzyme-sensitive magnetic resonance imaging targeting myeloperoxidase identifies active inflammation in experimental rabbit atherosclerotic plaques. Circulation 2009;120:592–9

167.

Zheng

Abendschein

Okamoto

MRI-based biomechanical imaging: initial study on early plaque progression and vessel remodeling. Magn Reson Imaging 2009;27:1309–18

168.

van Bochove

Straathof

Krams

Nicolay

Strijkers

. MRI-determined carotid artery flow velocities and wall shear stress in a mouse model of vulnerable and stable atherosclerotic plaque. MAGMA 2010;23:77–84

169.

Kuhlmann

Cuhlmann

Hoppe

Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice. J Vis Exp 2012:13

170.

Thim

Hagensen

Hørlyck

Wall shear stress and local plaque development in stenosed carotid arteries of hypercholesterolemic minipigs. J Cardiovasc Dis Res 2012;3:76–83

171.

Wiesmann

Szimtenings

Frydrychowicz

High-resolution MRI with cardiac and respiratory gating allows for accurate in vivo atherosclerotic plaque visualization in the murine aortic arch. Magn Reson Med 2003;50:69–74

172.

Herold

Wellen

Ziener

In vivo comparison of atherosclerotic plaque progression with vessel wall strain and blood flow velocity in apoE(−/−) mice with MR microscopy at 17.6 T. MAGMA 2009;22:159–66

173.

Hockings

Roberts

Galloway

Repeated three-dimensional magnetic resonance imaging of atherosclerosis development in innominate arteries of low-density lipoprotein receptor-knockout mice. Circulation 2002;106:1716–21