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Animal models and potential mechanisms of plaque destabilisation and disruption
  1. M Ni,
  2. W Q Chen,
  3. Y Zhang
  1. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong, China
  1. Correspondence to Professor Yun Zhang, Shandong University Qilu Hospital, No107, Wen Hua Xi Road, Jinan, Shandong, 250012, People’s Republic of China; zhangyun{at}


Studies of the pathophysiological mechanism of both acute coronary syndromes and plaque stabilising treatment are driving the development of animal models of vulnerable plaque. In contrast to advances in human studies of pathology, the definition, criteria and classification of vulnerable and ruptured plaques in animal models are still in dispute. Many approaches to increasing the intrinsic vulnerability of plaques or extrinsic forces on plaques have been reported. However, an ideal animal model mimicking human plaque rupture is still lacking, and the exact cellular and molecular mechanisms of plaque progression are not fully understood. This review summarises current progress in animal model studies related to plaque destabilisation and disruption and the possible mechanisms involved.

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Coronary artery disease is the major cause of mortality and morbidity in developed countries and some developing countries.1 Pathological and angioscopic studies have demonstrated that both plaque rupture and erosion leading to thrombosis are the most common causes of acute coronary syndrome (ACS).2,3,4 Investigation of the pathophysiological mechanism of ACS and possible treatment for plaque stabilisation is driving the development of animal models of vulnerable plaque. In terms of pathology, in an ideal animal model, the atherosclerotic process should be identical to that in humans, plaque should feature the same vulnerability in the animal model as in its human counterpart, and plaque rupture should at least in some cases be accompanied by the formation of platelet-rich thrombi.5 Despite tremendous research efforts during the past decade, such an ideal animal model is still lacking. Furthermore, regardless of expert consensus on the definition, classification and criteria of vulnerable plaque in human pathology,1 results from animal studies of vulnerable plaque are highly contradictory.6,7,8 As a result, our understanding of the cellular and molecular mechanisms of vulnerable plaque is far from being complete despite that the discovered and new therapies for the stabilisation of plaques are emerging.

Definition and criteria of plaque destabilisation and disruption in humans

In clinical practice, the term “vulnerable plaque” commonly refers to thrombosis-prone plaques and the plaque has a high probability of undergoing rapid progression to thrombosis.1 The classic histopathological features of vulnerable plaques in humans include a large necrotic core (occupying 25% of plaque area), a thin fibrous cap (<65 μm), depleted of extracellular matrix (collagen and proteoglycans) and smooth muscle cells (SMCs), infiltration by macrophages and T-cells with outward positive remodelling and increased plaque vascularity.9

A large and eccentric lipid core may contribute to plaque vulnerability by increasing the circumferential stress in the shoulder regions of the plaque, where plaque rupture tends to occur.10 Such a plaque with lipid core often contains oxidised lipids and macrophage-derived tissue factor, which makes the plaque highly thrombogenic when the lipid content is exposed to blood. A thin fibrous cap in vulnerable plaque contains fewer type I collagen fibrils and SMCs than those in stable plaques, thereby weakening the mechanical resistance of the plaque. Active inflammation is a major determinant of plaque vulnerability in which monocytes/macrophages are the predominant inflammatory cells recruited by adhesion molecules and chemokines or derived from adventitial neovasculature.9 Plaques with positive remodelling have a greater concentration of stress on the surface than those with negative remodelling, which renders plaques prone to rupture.11 Increased neovascularity provides a source of inflammatory cells into the plaque. Moreover, thin-walled vasa are prone to rupture, which results in intraplaque haemorrhage. In addition to the typical plaque rupture, plaque erosion and calcified nodules are the other two less common types of vulnerable plaque.1

In autopsy studies of coronary arteries, plaque rupture was originally defined as fibroatheroma with cap disruption and luminal thrombus communicating with the underlying necrotic core.12 Fibrous cap rupture leads to thrombosis by exposure of the thrombogenic contents of the plaque and subsequent activation of the clotting cascade, as well as platelet adhesion, activation and aggregation. Autopsy studies revealed that 70% to 80% of the coronary thrombi occurred at sites of fibrous cap rupture and extended into the plaque and the lumen.9 In addition, 20% to 30% of the coronary thrombi were formed on the surface of plaques with endothelium erosion and calcified nodules, and an enhanced thrombogenic state was proposed to explain this pathological consequence.9

Although plaque rupture causes symptoms in most cases of ACS, plaque rupture may also occur without the consequence of symptoms in some people.13 Morphological studies suggest that plaque progression beyond 40% to 50% cross-sectional luminal narrowing occurs secondary to repeated ruptures that are potentially clinically silent. The thrombus is usually small and heals with smooth muscle cell infiltration and proteoglycan deposition. According to Mann and Davies,14 the incidence of healed plaque ruptures was 16% in plaques with 0% to 20% diameter stenosis, 19% with 21% to 50% stenosis and 73% with >50% narrowing. For these reasons, a recent expert report has redefined plaque rupture as a structural defect (a gap) in the fibrous cap that separates the necrotic core of an atherosclerotic plaque from the lumen, thus resulting in exposure of the necrotic core to blood via the gap in the cap.15

Definition and criteria of plaque destabilisation and disruption in animal models

In contrast to the advances in studies of human pathology, the definition and criteria of vulnerable plaque in animal models are still in dispute. Since an animal model of vulnerable plaque simulating human abnormality is not yet available, data are imperfect on which plaques are at high risk of rupture, thrombosis and becoming culprit plaques. Thus, Falk et al suggested that anatomically meaningful terms other than vulnerable plaque should be used for animal studies.16 By analogy to the human pathology of plaques, the histological markers for vulnerable plaques in animals are considered a large lipid core, a thin fibrous cap, increased macrophages and inflammatory mediator molecules and decreased extracellular matrix. However, because the arterial structures of humans and animals differ markedly, some qualitative and quantitative criteria for detecting vulnerable plaques in humans cannot be used for the study of animals. For example, a thin fibrous cap is defined as <65 μm thick in humans, but in mice the fibrous cap is difficult to measure because it consists of thin lamellae of loosely organised connective tissue.8

Likewise, the criteria for defining plaque rupture in animals are not well established.7 A luminal thrombus communicating with a gap in the fibrous cap is generally accepted as the most reliable sign of plaque rupture in animal models.6,8 In our study, luminal thrombosis combined with plaque cap disruption was found in more than 80% of rabbits fed a high-fat diet and undergoing local p53 gene transfection and pharmacological triggering with procoagulant Russel’s viper venom (RVV) and vasopressor histamine.17 In contrast, this pathological feature was surprisingly uncommon in apolipoprotein-E knockout (apoE−/−) mice, possibly because of a low level of plasma plasminogen activator inhibitor-1 and thrombin-activated fibrinolysis inhibitor in mice.18,19,20 The fibinolytic function seems to be more pronounced in mice than in humans, and for this reason, Jackson et al proposed that in the mice model, the presence of luminal thrombosis should not be regarded as a defining characteristic of plaque rupture.6

Taking into account the phenotype variations of arteriothrombosis in different animals, we believe that a recently proposed definition of plaque rupture for humans can be used for animal studies.15 According to this definition, neither luminal thrombus nor plaque/intraplaque haemorrhage is required; however, these pathological features are considered by experts to be important in excluding artificial plaque disruption. Under this definition, some pathological findings in animals, such as plaque fissures, endothelial denudation and intraplaque haemorrhage secondary to leaking vasa vasorum, should not be considered plaque rupture because these lesions do not expose a necrotic core to the lumen.

Intraplaque haemorrhage is a common feature of complex lesions preceding acute ischaemic events and was considered the characteristic of plaque rupture in some studies.21,22 There are two different views for the source of intraplaque haemorrhage: in most cases, it is secondary to rupture of the neovessels growing into the plaque, and in some cases, it may be derived from cap disruption directly so erythrocytes may intrude from the lumen into the plaque after cap disruption and be trapped below the cap even in the presence of potent thrombolysis. Therefore, multiple serial sectioning is necessary to demonstrate a communication between haemorrhage and cap disruption or neovessel rupture. Regardless of the source of haemorrhage, the relation between intraplaque haemorrhage and plaque vulnerability is positive. Our laboratory23 and other investigators24,25 have shown that accumulation of erythrocytes released from haemorrhage masses within the plaque may increase local oxidative stress and cholesterol concentration leading to increased plaque rupture.

Another hotly debated sign of plaque rupture is “buried caps,” which represent a multilayered structure in the intima of advanced murine lesions. Buried caps have been observed in human atherosclerotic lesions and are highly suggestive of remnants of previous ruptured caps.26,27,28 Burke et al demonstrated that the appearance of layers in plaques is probably the consequence of previous clinically silent ruptures; patients who died with acute plaque rupture and those with healed myocardial infarction showed the highest frequency of healed plaque ruptures (75% and 80%, respectively).13 Buried caps were frequently observed in murine atheromas that may be prone to erosion or rupture.6 In brachiocephalic artery lesions of apoE−/− mice, buried caps formed only at the sites where plaque ruptures occurred and were markedly associated with fibrin deposition.29 However, the interpretation of buried caps as indirect evidence for previous plaque rupture has been criticised.8 Schwartz and colleagues pointed out that buried caps could also occur by episodes of rapid lipid deposition, macrophage efflux and SMC recruitment without invoking fibrous cap rupture and repair.8 In our study, buried caps were found to be more common in apoE−/− mice treated with mental stress and lipopolysaccharide stimulation than in mice without any stimulation.30 Observation of serial cross-sections consistently revealed cap disruption at the end of the buried caps in most plaques with buried caps. Therefore, buried caps in connection with cap disruption should be an indicator of plaque rupture.

Large-animal models of plaque destabilisation and disruption

Great efforts have been made to establish an ideal animal model with vulnerable plaque and plaque rupture. The available models are summarised in table 1.

Table 1

Animal models of plaque destabilisation and disruption

Atherosclerotic lesions in some large animals, such as primates and pigs, are similar to those in humans.31,32,33,34,35 Kusumi et al reported that aortic and coronary plaques with many features of advanced human atherosclerotic lesions, including a fibrous cap and a necrotic core, developed in a female rhesus monkey with a genetic deficiency in low-density lipoprotein receptor (LDLR−/−) and hypercholesterolaemia.31 Pigs, when fed a high-cholesterol diet, showed elevated serum lipid levels and atherosclerotic lesions similar to those in patients with coronary artery disease. Prescott et al found serum low-density lipoprotein (LDL) levels increased and plaque haemorrhage and rupture common in the major coronary arteries of pigs with inherited hypercholesterolaemia at age 3 years.34 Recently, Granada et al established a pig model of complex atherosclerotic lesions by injection of cholesteryl linoleate into the vessel wall.35

Although primates and pigs may serve as ideal animal models of vulnerable plaques, the high expense, restricted availability and ethical issues have limited their use in this field. By far the most useful large-animal models are restricted to rabbits. Typical aortic plaques similar to humans can be induced in rabbits by high-cholesterol diet and endothelial balloon injury. To promote plaque rupture, pharmaceutical triggers have been used. More than 40 years ago, Constantinides et al established an atherosclerotic plaque model of rupture and thrombosis by intraperitoneal injection of procoagulant RVV followed by intravenous injection of vasopressor histamine in rabbits fed a high-fat diet.36 This model was later reproduced by Abela et al, and the plaque disruption and overlying platelet-rich thrombus induced was similar to that observed in patients with ACS.37 Similarly, Nakamura et al induced acute myocardial infarction in Watanabe rabbits with heritable hyperlipidaemia challenged with RVV combined with serotonin or angiotensin II (AngII) which promoted aortic thrombosis associated with segmental medial necrosis and intimal disruption.38 Our laboratory developed a rabbit model with a high rate of plaque rupture and thrombosis by first locally delivering adenovirus-mediated p53 gene to plaques and then using RVV and histamine as triggers; excessive SMC apoptosis and active inflammation in the fibrous cap induced by overexpressed p53 was considered the major mechanism of plaque destabilisation.17 The predominant feature of this model is that the frequently observed luminal thrombi accompanied by plaque rupture resembled human lesions, whereas the limitation of the model is that the triggering process is not physiological and differs from spontaneous plaque rupture in humans.

Some studies of animal models have induced direct mechanical injury to plaques, by inflating an angioplasty balloon embedded into the plaque39 or by directly squeezing the plaque with forceps.40 In New Zealand White rabbits fed a high-fat diet, perivascular electrical injury at the common carotid artery produced bulky, macrophage-rich and lipid-rich plaques.41 Radiation of the iliac artery lesions of New Zealand White rabbits enhanced plaque vulnerability, most probably because of intensified oxidative stress and inflammation.42,43 Although these animals exhibited some features of vulnerable plaques, the stimulation procedure is not physiological and differs from the pathological process in humans.

Erythrocytes released from intraplaque haemorrhage have been related to plaque vulnerability.25,44 Kolodgie et al injected washed autologous erythrocytes (25–50 μl) into established atherosclerotic plaques in rabbits; atheromas resulting from injected erythrocytes had more extensive macrophage infiltration and lipid content than control lesions.25 Rabbit lesions with erythrocyte-induced intramural haemorrhage consistently showed cholesterol crystals with erythrocyte fragments, foam cells and iron deposits. We injected two doses of autologous erythrocytes or cholesterol into rabbit plaques and found plaques from the erythrocyte or cholesterol groups with, dose-dependently, more macrophage infiltration, more superoxide and lipid content, thinner plaque fibrous cap, higher mRNA level of monocyte chemoattractant protein 1 (MCP-1), interleukin 1 (IL-1) or interferon gamma and higher vulnerability index than controls; plaque vulnerability index and rupture rate were highest in the group receiving the largest erythrocyte dose.23 Thus, erythrocyte treatment can dose-dependently induce vulnerability of plaques. Accumulation of lipid content and augmentation of oxidative stress and inflammation in the plaques are the probable pathological mechanisms.

Small-animal models of plaque destabilisation and disruption

A major step forward in the development of small-animal models of atherosclerosis was the development of genetically engineered mice lacking important genes in lipid metabolism, such as apoE−/− and LDLR−/− mice.45 Although atherosclerotic lesions develop spontaneously in these mice, most plaques thus formed are stable, and various approaches to increase the intrinsic vulnerability of plaques or impose extrinsic forces on plaques have been advocated to induce plaque destabilisation and disruption.

Plaque rupture induced by intrinsic vulnerability

Interleukin-18 (IL-18) is a proinflammatory mediator that has received considerable attention in plaque development, rupture and thrombosis.46 De Nooijer et al transferred the IL-18 gene into the apoE−/− mice fed a high-fat diet and induced a marked decrease in the intimal collagen content and a phenotype of vulnerable plaque and intraplaque haemorrhage; the mechanism of plaque vulnerability induced by IL-18 overexpression was attributed to matrix degradation by enhancing the collagenolytic activity of vascular SMCs.47 Matrix metalloproteinase 9 (MMP-9) is expressed in late atherosclerotic lesions in humans and has been suggested to mediate plaque vulnerability.48,49 Adenovirus-mediated MMP-9 transfection in carotid lesions of apoE−/− mice increased the prevalence of intraplaque haemorrhage. CD31 staining showed that the presence of neoangiogenesis was the source of MMP9-induced intraplaque haemorrhage.50 In addition, expression of an autoactivating form of MMP-9 in macrophages in vitro was found to greatly enhance elastin degradation and induce significant plaque disruption when overexpressed by macrophages in advanced atherosclerotic lesions of apoE−/− mice in vivo.51 SMCs, as the main cells in fibrous caps, are responsible for plaque integrity, and any intervention to reduce SMCs would weaken fibrous caps and plaque stability. Zadelaar et al reported that adenoviral-mediated transfection of the FasL gene in the carotid lesions of apoE−/− mice enhanced plaque vulnerability by increasing apoptosis rate and decreasing the number of cap cells after gene transfer.52

A previous study showed that a high level of endogenous AngII caused by clipping the kidney artery in hypertensive apoE−/− mice induced vulnerable plaques compared with plaques in hypertensive ApoE−/− mice with normal AngII levels; enhanced inflammation with increased macrophage accumulation and a skewed T-helper type 1-like lymphocyte profile was the major mechanism of this model.53 Continuously administering AngII into six-month-old apoE−/− mice by a subcutaneous osmotic minipump for 4 weeks led to plaques with increased intraplaque neovasculature and haemorrhage; levels of active MMP-2 and inflammatory mediators, including MCP-1 and vascular cell adhesion molecule, were all increased after AngII treatment.54

Plaque rupture induced by extrinsic forces

Multiple approaches in mice have been proposed to promote plaque destabilisation and disruption. Von der Thusen et al first transfected carotid plaques in apoE−/− mice with the p53 gene locally to enhance plaque vulnerability by increasing SMC apoptosis in the fibrous caps, then phenylephrine (α1-adrenocepter agonist) was administered to trigger plaque rupture.21 Compared with a rabbit model treated with p53 gene transfection and pharmacological triggers,17 mice treated in the same way had a high frequency of plaque disruption but a low incidence of arterial thrombosis.

Physiological stressors have been proposed to establish more clinically relevant models of plaque rupture. Hemdahl et al reported that acute myocardial infarction, as determined by elevated ST-segment and serum levels of troponin T, was induced by hypoxic or mental stress in 7-month-old mice fed a high-fat diet; myocardial infarction developed in 50% of the mentally stressed mice and 56% of the hypoxia-exposed mice.55,56 However, the experiment was very long, and the mortality rate was excessively high.

A recent study by Cheng et al found that shear stress of blood flow affected not only atherosclerotic lesion size but also plaque vulnerability.57 Low shear stress may contribute to the development of atherosclerotic lesions, whereas high shear stress may cause regression of intimal lesions owing to the loss of SMCs and matrix.58 However, in the presence of established plaques, an abrupt increase in shear stress might be the predominant mechanical factor to trigger plaque rupture, which often occurs at the shoulder of the fibrous caps upstream of the stenoses.59

We have simulated in mice the major risk factors of plaque rupture in humans and established an animal model of hyperlipidaemia (induced by high-fat diet), high level of inflammation (induced by lipopolysaccharide injection) and hyperhaemodynamics (induced by electric foot-shock and noise stimulation) in apoE−/− mice.30 Carotid plaque of this model showed a vulnerable plaque phenotype (that is, a thin fibrous cap, a large lipid core, loss of collagen and extracellular matrix and active inflammation). Plaque rupture occurred in more than 60% of the mice. Nonetheless, thrombosis accompanied by plaque rupture was uncommon. The dissimilarity in arterial thrombosis between mice and humans is probably due to an enhanced fibrinolytic function in mice. Several studies demonstrated that plasma levels of plasminogen activator inhibitor 1 and thrombin-activatable fibrinolysis inhibitor were low in mice.18,19,20 In contrast, 50% of transgenic mice overexpressing human plasminogen activator inhibitor 1 (PAI-1) gene exhibited spontaneous coronary thrombosis and subendocardial myocardial infarction.60 Therefore, further studies aimed at enhancing the coagulation system of mice are required to establish thrombosis-prone vulnerable plaque models.

Mouse models of spontaneous plaque rupture

Calara et al reported on aortic plaque rupture and/or thrombi in older apoE−/− or LDLR−/− mice (age 9–20 months); however, the incidence of plaque rupture was considerably lower.61 Unlike the genetically modified mice, the aorta is not a frequent site of clinically significant lesions in humans. Moreover, aortic lesion responses to treatment do not invariably reflect the situation elsewhere in the vascular tree.

Vulnerable plaque has been found to develop spontaneously in the innominate (brachiocephalic) artery of apoE−/− mice, a small vessel connecting the aortic arch to the right subclavian and right carotid artery. Rosenfeld et al described a high frequency of intraplaque haemorrhage and a fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap in the innominate artery of apoE−/− mice aged 42–54 weeks fed a rodent chow diet; the disruption appeared to occur predominantly within the lateral xanthoma and was consistent with similar plaque rupture along the lateral margins of plaque in humans.62 Johnson and Jackson observed luminal thrombi associated with ruptured plaques in the brachiocephalic artery of apoE−/− mice (37–59 weeks old) that died spontaneously after 46 (3) weeks of high-fat diet feeding; the ruptured plaques were characterised by fragmentation and loss of elastin in the fibrous caps of relatively small and lipid-rich plaques overlying large complex lesions, with intraplaque haemorrhage.63 Williams et al studied 98 apoE−/− mice fed a high-fat diet for 5–59 weeks and found 51 with acute ruptured plaque in the brachiocephalic artery; the number of buried caps within the lesion was significantly higher in ruptured than in intact lesions.64 More recently, this model has been studied further by Johnson et al, who found that spontaneous plaque rupture in the brachiocephalic arteries sharply increased from 3% after 7 weeks to 62% after 8 weeks of a high-fat diet; these acute plaque ruptures then appeared to heal and form buried fibrous caps.29 Although encouraging, these findings are still debated in terms of their causal relation to plaque vulnerability.8,16

Future directions

Despite the tremendous research efforts made in the field of plaque vulnerability in the past decade, an ideal animal model of plaque rupture is still lacking and the exact cellular and molecular mechanisms of plaque progression remain unclear. Further studies should develop a more efficient and reproducible animal model of atherosclerosis that exhibits the salient features of spontaneous human plaque rupture. Such a model would greatly facilitate elucidation of the basic mechanisms, detection of vulnerable plaques and development of plaque-stabilising therapies. Discovery of novel genes involved in human plaque rupture may provide a more focused roadmap for developing new animal models.



  • Funding This work was supported by the National 973 Basic Research Program of China (No 2006CB503803), the National High-tech Research and Development Program of China (No 2006AA02A406), the Program of Introducing Talents of Discipline to Universities (No B07035) and the State Key Program of National Natural Science of China (No 60831003).

  • Competing interests None.

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