Atherosclerotic Plaque Instability and Rupture: Recommended Mouse Models to Empower Clinically Relevant Discoveries, Diagnostics and Therapeutics

Abstract

Clinical Problem:

Atherosclerotic plaque instability/rupture are the major drivers of myocardial infarction and stroke - the leading causes of cardiovascular morbidity/mortality. However, the mechanisms leading to plaque rupture are poorly understood. This limits our ability to establish sensitive and diagnostic tools to identify plaques that are prone to rupture and to develop much-needed plaque-stabilizing therapies.

Recommendations:

The diagnostic identification and therapeutic stabilization of unstable plaques are considered the "holy grail" of cardiovascular medicine, holding the potential to significantly reduce cardiovascular morbidity/mortality. To achieve this, it is vital that preclinical models reflect plaque instability/rupture as observed in patients. This will allow mechanistic discoveries, the development of diagnostic tools, and treatment options to identify and stabilize rupture-prone, unstable atherosclerotic plaques. This can be achieved using appropriate, research question-dependent, but currently underutilized mouse models with direct translational relevance.

Summary of strengths and weaknesses of mouse models for atherosclerosis:

Conventional mouse models of atherosclerosis, LDLR^-/- and ApoE^-/- mice fed a high-fat diet, do not develop unstable atherosclerosis and plaque rupture as observed in patients with myocardial infarction. Modification of these mice with additional gene mutations (e.g., in SRB1 and Fbn1^C1039G+/-) induce the development of unstable plaques and plaque rupture. However, some genetic approaches pose challenges as they can generate additional phenotypes and comorbidities and may not be commercially available or simple to breed. In contrast, surgically induced models of plaque instability/rupture (e.g., carotid Tandem Stenosis (TS) or Transverse Aortic Constriction (TAC)) can easily be used in any athero-susceptible mouse in which a single gene mutation increases atherogenic lipids or can be combined with newer atherosclerosis-inducing approaches (e.g., AAV-PCSK9) to cause unstable atherosclerotic disease. Such increasingly used surgical approaches are suitable for detailed mechanistic studies as they reflect most characteristics of human plaque instability/rupture and can be adapted to many different experimental conditions and research questions.

Introduction

Atherosclerotic plaque rupture, erosion, and the formation of calcific nodules are the three main pathological mechanisms leading to luminal thrombosis, which triggers most major cardiovascular events. Of these, plaque rupture is the primary cause of myocardial infarctions (MIs) and a major contributor to ischemic strokes, underpinning cardiovascular diseases as the leading cause of death globally. Although systematic histopathological studies in patients are limited, they have been instrumental in identifying the characteristics of rupture-prone plaques. These include thin fibrous caps, high leukocyte and low smooth muscle cell numbers, high proteolytic activity, large lipid/necrotic cores, and significant intraplaque hemorrhage.^1–8 In contrast, stable plaques – even if advanced in age or size – would typically not exhibit the combination of these important features. In the research context, prototypical models of atherosclerosis, including ApoE^-/-, LDLR^-/- and AAV-PCSK9 mice on prolonged high fat diets overall develop large stable atherosclerotic plaques but not vulnerable, rupture-prone plaques. It is thus important that the field now advances beyond such models to focus on the clinically relevant pathophysiology, plaque instability and rupture, as triggers of MI and stroke.

Clinical characteristics of plaque instability and rupture

Hemodynamics:

Wall shear stress (WSS) is a major hemodynamic risk factor of atherogenesis.⁹ Physiological WSS (1–2.5 Pa) promotes endothelial cell (EC) release of nitric oxide (NO), exerting strong anti-apoptotic, anti-inflammatory, anti-platelet, and adhesion inhibitory effects. In contrast, curved vessels and bifurcations experience low WSS (< 1 Pa), resulting in lower NO production, making ECs more susceptible to injury and atheroma development. Turbulent flow patterns at the distal end of stenotic plaques result in disturbed and low WSS, which has been associated with the frequency and severity of high-risk plaques, and the probability of acute coronary syndrome.¹⁰ Also, prolonged high WSS at the proximal end correlates with the development of thin-fibrous caps, calcification, intraplaque hemorrhage, plaque destabilization, and MI.¹¹

Lipid accumulation:

Lipid accumulation within macrophages and vascular smooth muscle cells, alongside extracellular lipid and necrotic lipid cores, is critical for destabilizing plaques, with lipid core size linked to clinical events.^12,13 Accumulated plaque lipids exert pleiotropic pro-inflammatory effects, including causing endothelial and smooth muscle cell dysfunction. Beyond the stroma, lipids (and metabolic dysfunction in general) play a vital role in driving inflammatory leukocyte responses within the plaque.¹⁴

Inflammation and Immunity:

Inflammation, predominantly mediated by the immune system, is an implicated driver, diagnostic marker, and potential therapeutic target of plaque instability. Plaque rupture is often directly associated with increased immune cell infiltration, indicating a link between immunity and cardiovascular events.^6,8,15–20 Beyond this, strong clinical evidence of causative immune involvement in plaque rupture has come from the CANTOS trial, which demonstrated that reducing inflammation by blocking IL-1β can prevent rupture-related cardiovascular events.²¹ However, few studies have directly explored how immune-driven inflammation might cause plaque rupture, with most immunological studies utilizing mouse models of stable disease supplemented by relatively few, though highly insightful, studies that apply high-dimensional technologies to characterize the heterogeneity of immune cells within human plaques. As such, this remains a critical area for ongoing investigations. The immunology of atherosclerosis is both critical and complex, incorporating protective and pathogenic immune contributions with a likely autoimmune component.^{18,19,22–24} One of the many mechanisms by which immune responses may cause plaque instability is via inflammation-induced production of matrix metalloproteinases (MMPs), which can degrade the fibrous cap of atherosclerotic plaques.^6,15 Proteomic analysis of unstable versus stable human and mouse plaques, for example, show strong links between proteolytic degradation, inflammation and plaque rupture.^25,26

Cell death:

Necrotic cores can occupy >25% of total ruptured plaque area, leading to a strong association between plaque instability and cell death. Advances in fundamental understandings indicate that many forms of cell death are active and likely important to atherogenesis and plaque rupture.^27,28

Thin fibrous caps & cap rupture:

Fibrous cap thinning is implicated as a key pathway towards plaque rupture, with clinical imaging data and post-mortem histology both supporting the critical influence of plaque rupture on cardiovascular mortality/morbidity.²⁹ In 800 cases of sudden coronary death, for example, 55–60% were attributed to atherosclerotic plaque rupture, with the remaining deaths linked to plaque erosion (30–35%) or calcified nodules (2–7%).³ Moreover, optical coherence tomography (OCT) identified plaque rupture caused 68.6% of ST elevated MIs.³⁰ The mechanisms responsible for cap thinning are largely unknown, as are the events that cause rupture in ‘vulnerable’ plaques versus similar plaques that do not rupture.¹⁷

Biomechanics of cap rupture:

Plaque rupture occurs when the soft tissue in the atheroma cap is torn, ripped, or fissured because the peak circumferential stress (PCS) exceeds the cap tissue ultimate strength, ~545kPa.³¹ The ultimate cap strength is 5 orders of magnitude higher than the physiological WSS, meaning WSS cannot be responsible for the actual rupture of the plaque. The main determinant of PCS is cap thickness. While on its own PCS is also insufficient to fully explain plaque rupture, several other risk factors are known to contribute, including lipid core size, location and shape, tissue composition, remodeling index, and cap microcalcifications.³² Microcalcifications, for example, behave as stress concentrators and can increase the cap stresses by a factor 2–7x depending on their size, location, shape, and the proximity between multiple calcification sites.³³ It is therefore the combination of these biomechanic ‘stressors’ that may drive rupture.

Intraplaque hemorrhage:

Intraplaque hemorrhage (IPH) originates from plaque micro-ruptures or from leaky immature intraplaque vessels formed to vascularize enlarging plaques. IPH is predictor of plaque instability and rupture.³⁴ IPH may trigger rupture via the accumulation of erythrocyte membranes, production and retention of cholesterol crystals, erythrophagocytosis, and enlarging necrotic cores.^5,35,36 It also promotes oxidative and proteolytic activity in plaques, as well as leukocyte recruitment.³⁷ A further consequence of IPH is iron accumulation within plaques, which can drive oxidative stress, lipid peroxidation, inflammation, and immune dysfunction.¹⁴ The importance of IPH for plaque instability is further supported by the discovery that near-infrared autofluorescence (NIRAF) derived from heme degradation products is associated with unstable, rupture-prone plaques in both human and mouse models.³⁸

Mouse models focused on plaque instability and rupture

Both genetic and surgical mouse models of atherosclerotic plaque rupture have been developed to understand rupture mechanisms and assess the efficacy of novel plaque stabilizing therapies (see accompanying Table). Here, we have focused on those we believe to be of greatest translational relevance.

Table:

Mouse models of unstable atherosclerosis and plaque rupture.

	Model	Confirmed Sites of Plaque Vulnerability	Confirmed Immune Infiltrates	Key Instability/Vulnerability Characteristics and End Points
Recommended Surgical Models	Tandem Stenosis Surgery in hyperlipidaemic mice (Chen et al.,60)	Carotid Artery	Monocytes/Macrophages Neutrophils T cells	Plaque rupture & luminal thrombosis, Disruption of thin fibrous caps, Intraplaque hemorrhage
	Transverse Aortic Constriction Surgery in hyperlipidaemic mice (Marino et. al.,61)	Coronary Arteries	Monocytes/Macrophages	Plaque rupture & luminal thrombosis, Disruption of thin fibrous caps, MI (74%) following stress testing
Recommended Genetic Models	ApoE-/-Fbn1C1039G+/- (Van Herck et. al.,51 Van der Donckt et. al.,39)	Aorta & Brachiocephalic Artery	Monocytes/Macrophages T cells	Plaque rupture & luminal thrombosis, Disruption of thin fibrous caps, Intraplaque hemorrhage, Spontaneous death (70%), cerebral hypoxia (~64–73%); coronary plaques & MI (96%)
	LDLR-/- SRB1ΔCT/ΔCT (Shamsuzzaman et. al.,55) Note that there is also an ApoE-/- SRB1-/- model, but this experiences 100% mortality within 8 weeks. (Braun et. al.,66)	Coronary Arteries & Carotid Artery	Monocytes/Macrophages B cells T cells	Plaque rupture & luminal thrombosis, Disruption of thin fibrous caps, Intraplaque hemorrhage, High mortality rates after administration of high fat diet (81% at 26 weeks, with MI in 75% and stroke in 38% of animals)
	ApoESA/SA (Chen et al.,56)	Coronary Arteries	Monocytes/Macrophages Neutrophils	Plaque rupture & luminal thrombosis, Disruption of thin fibrous caps, Intraplaque hemorrhage, High mortality rates (up to 36% of males and 78% of females) by 8 weeks in the context of hypercholesterolemia and hypertension. MI, cardiac dysfunction, and heart failure.
Other models	Perivascular collar + intravascular Ad-p53 Injection +/- phenylephrine in hyperlipidaemic mice (von der Thusen et. al.,67)	Carotid Artery	Monocytes/Macrophages	Mainly disruption of thin fibrous caps, Intraplaque hemorrhage
	Ligation induced flow cessation + collar in hyperlipidaemic mice (Sasaki et. al.,68)	Carotid Artery	Leukocytes, Neutrophils	Rapid induction of rupture
	Partial ligation of left carotid & renal artery in hyperlipidaemic mice (Shu-xuan et. al.,69)	Carotid Artery	Monocytes/Macrophages	Plaque rupture & luminal thrombosis
	ApoE-/- mice with mixed C567BL/6 + 129SvJ genetic background (Johnson & Jackson et. al.,70 Williams et.al.71 Johnson et. al.,72)	Brachiocephalic Artery	N.D.	Infrequent plaque rupture & disruption of thin fibrous caps, MIs in apex of heart; sudden death (65%) that was not attributed to atherosclerosis
	ApoE-/- Akt1-/- (Fernandez-Hernando et. al.,73,74)	Coronary Arteries, Innominate Artery, Aortic Root	Monocytes/Macrophages	Evidence of fibrous cap disruption, Spontaneous death & MI
	ApoE-/- TM-/- (Borissoff et.al.,75)	Carotid Artery	Monocytes/Macrophages, Neutrophils	Plaque rupture & luminal thrombosis,
	ApoE-/- SR-uPA+/0 (Cozen et.al.,76)	Coronary Arteries	N.D.	Coronary occlusion but without obvious plaque rupture, MI present with anticipated 100% mortality by 30 weeks. Cardiac arrythmias identified as likely cause of death.
	Injection of active MMP-9 overexpressing macrophages into hyperlipidaemic mice (Gough et.al.,77)	Brachiocephalic Artery & Aorta	Monocytes/Macrophages	Thrombosis, Disruption of thin fibrous caps, Intraplaque hemorrhage, Sudden death (20%) with unclear relationship to rupture
	Injection of Urokinase-type Plasminogen activator overexpressing macrophages into hyperlipidaemic mice (Hong et.al., 78)	Brachiocephalic Artery & Aorta	N.D.	Thrombosis, Disruption of thin fibrous caps, Intraplaque hemorrhage

MI = Myocardial Infarction

Genetically induced unstable atherosclerosis and plaque rupture:

Three genetic models of plaque rupture have been described that appear superior to others due to their specific phenotype and level of characterization to-date. The first is the ApoE^-/-Fbn1^{C1039G +/−} mouse strain which has been validated across several labs and studies.^39–51 These animals possess a heterozygous mutation in the Fibrillin-1 (Fbn1) gene that promotes elastin fragmentation. In these mice atherosclerotic plaques exhibit large necrotic cores, extensive neovascularization, intraplaque hemorrhage, fibrous cap disruption and luminal thrombosis. These mice are highly prone to MI, ischemic stroke and sudden cardiovascular death. This mutation has also been generated on a LDLR^-/- background, creating unstable plaques but without exhibiting spontaneous cardiovascular events.⁵² It must be acknowledged that the consequences of Fbn1 mutation go beyond atherosclerosis, also leading to abdominal aortic aneurysms and non-vascular comorbidities.^53,54 Thus, while powerful, there is the caveat that these mice present with a complex array of potentially confounding co-morbidities.

Another model is the recently developed LDLR^-/- SRB1^∆CT/∆CT mouse, where the C-terminus of the scavenger receptor class B type 1 (SRB1) is mutated.⁵⁵ These mice develop highly unstable plaques in both the coronaries and right carotid artery, exhibiting frequent MI and spontaneous cardiovascular death; ~30% of the mice exhibit evidence of plaque rupture. ApoE^SA/SA mice, which have liver-biased SRB1 knockdown combined with genetically controlled doxycycline-inducible Angiotensin II expression reflect further innovation.⁵⁶ While perhaps more genetically complex, these animals also consider the interface of both hypertension (Ang II induced) and hypercholesterolemia as key risk factors of plaque rupture and MI, and appear to have a similar phenotype to LDLR^-/- SRB1^∆CT/∆CT under these conditions. LDLR^-/-SRB1^∆CT/∆CT mice are advantageously available from Jackson Laboratories, whereas ApoE^-/-Fbn1^C1039G+/− would need to be generated from founder strains. The commercial availability of ApoE^SA/SA is currently unclear. In contrast to Fbn1 mutated mice, SRB1 mutants do not appear to possess additional disease phenotypes beyond atherosclerosis, which we consider a crucial advantage. However, overall, both Fbn1 and SRB1 mutant mice recapitulate histological features and clinical outcomes of plaque instability/rupture. This is highly advantageous for intervention and/or diagnostics-focused studies. However, these mice may be less suitable for studying the mechanisms of plaque rupture.

Surgically induced unstable atherosclerosis and plaque rupture:

Surgical models of atherosclerosis were first used to study vascular damage and remodeling (e.g., neointimal hyperplasia).^57–59 However, more recently, surgically altering arterial hemodynamic forces in animal models has been used to model plaque instability and rupture, - typically in athero-susceptible mice including ApoE^-/-, LDLR^-/-, and mice treated with the hypercholesterolemia-inducing adenovirus AAV8-PCSK9. The latter especially enables rapid leveraging of the many advanced genetic mouse models available for manipulating specific biological pathways globally and/or in specific cell types, without requiring complex crossbreeding with athero-susceptible mouse strains. These surgical approaches are thus highly suited for mechanistic discoveries but also for the development and testing of novel diagnostics and therapeutics. In our opinion, the two most attractive approaches are the tandem stenosis (TS) model,^57–60 and transverse aortic constriction (TAC) surgery.⁶¹

The TS model involves two partial ligations of the right common carotid artery of hypercholesterolemia mice (LDLR^-/-, ApoE^-/-, or AAV-PCSK9-treated mice).⁶⁰ Within 7 weeks, unstable atherosclerotic lesions with large necrotic cores, immune infiltrates, neovascularization, vascular remodeling, intraplaque hemorrhage, and thin fibrous caps including cap rupture and luminal thrombosis form proximal to the stenoses. Importantly, stable plaques can be found and assessed in the aortic arch and descending aorta of the same mice, providing a “two-for-one” model. The TS model has been utilized in diagnostic, therapeutic, and mechanistic studies, including in combination with key comorbidities such as diabetes and stroke, collectively supporting its strong translational relevance.^60,62–65 The widespread use of this model emphasizes its robustness and broad applicability.

The TAC approach involves a partial ligation between the brachiocephalic artery and left common carotid artery and is typically used as a model of pressure-overload heart failure. However, in athero-susceptible mice, TAC results in unstable atherosclerosis in the coronary arteries with thin fibrous caps that rupture or erode, leading to occlusive thrombi and MI with a high mortality rate.⁶¹ The main potential complications are that 1) TAC induces systolic dysfunction and cardiac fibrosis, which may also influence coronary plaque development and rupture, creating a compounded disease model and 2) murine coronary arteries are very small (diameter ~ 150 µm), making them difficult to isolate and limiting precise analyses beyond histological assessments. We have validated this approach in-house, confirming that TAC drives coronary atherosclerosis and plaque rupture (unpublished). Our recommendation here would be that, if coronary atherosclerosis is paramount, the TAC model would be superior. However, if the research question focuses on plaque instability as a general phenomenon, carotid disease, or requires analyses beyond histology (e.g., flow cytometry, bulk or single-cell omics) the TS model is more appropriate given the ability to directly isolate the carotid arteries and assess these using the wealth of available cellular, molecular and imaging technologies.

General limitations and recommendations

Firstly, the flexibility of surgical approaches like the TS and TAC models allow for combining additional comorbidities or, indeed, clinical events to better reflect human disease. For example, Cao et al. recently combined the TS model with a model of stroke to discover that stroke-induced inflammasome activation leads to exacerbated plaque instability.⁶² However, in respect to limitations, these models do require a reasonably high degree of surgical skill. In contrast, genetic models overcome this need but come with their own caveats, including co-morbidities and logistical challenges related to disease courses being unique to each animal. More generally, we would highlight that no mouse model of atherosclerosis fully reflects the heterogeneity or etiology of human atherosclerotic disease. Most models do not cause atherosclerosis in the coronary arteries and, even when they do, there are significant technical limitations due to the small size of murine coronary arteries. There are often significant differences in lipid profiles, total vessel area, fibrous cap thickness and the composition of extracellular matrix proteins – including calcification, between mice, humans and the different mouse models. However, this in no way detracts from the importance of plaque rupture as the major cause of cardiovascular events, nor the absolute need to apply models of instability and rupture to better understand – and treat – atherosclerosis. We thus strongly recommend that investigators consider the specific features of instability and/or rupture most relevant to their research question – whether this be inflammation, intraplaque hemorrhage, anatomical location etc., – and select their model accordingly. Above all, we would emphasize that atherosclerosis research should rely less on the traditional ApoE^-/- and LDLR^-/- models that have vastly improved our understanding of atherosclerosis biology but remain poorly reflective of clinically relevant plaque phenotypes, such as plaque instability and rupture. We further suggest that human biopsy specimens are used wherever possible to validate the relevance of specific biological pathways or therapies studied. Ultimately, the availability of plaque instability/rupture models alongside the advent of powerful new technologies for high-parameter cellular, molecular, and spatial analysis provides unprecedented opportunities to finally understand the causes of MI and stroke.

Conclusions

Atherosclerotic plaque rupture is the main cause of acute coronary syndrome, leading to most cardiovascular disease morbidity and mortality. Despite this, we know relatively little about the mechanisms that cause plaque rupture. Mouse models are a vital tool for pre-clinical investigations and, if used effectively, should provide significant insights to overcome critical gaps in understanding the underlying mechanisms of unstable atherosclerotic disease. However, conventional mouse models of atherosclerosis are mainly suitable for studies of atherosclerosis development as they typically develop only stable atherosclerotic plaques. It is widely acknowledged that the success of translation from preclinical atherosclerosis models to date has been disappointing, particularly given the significant expense of cardiovascular event-focused clinical trials, often requiring large numbers of patients. Plaque rupture causes cardiovascular events, and as such, preclinical research needs to focus directly on plaque destabilization mechanisms. Fortunately, several mouse models of plaque instability are now available, primarily utilizing surgical and/or genetic manipulations. We believe that combining the AAV8-PCSK9 approach with such surgical manipulations is the most amenable and widely applicable pathway to elucidating the critical biological mechanisms that drive plaque instability, with likely equal applicability of key genetic models when it comes to testing diagnostic and therapeutic approaches in athero-susceptible strains. Ultimately, a shift towards the use of these mouse models will improve our understanding of what drives plaque rupture as the major cause of cardiovascular mortality/morbidity and will help in identifying/detecting/diagnosing unstable, rupture-prone plaques before they cause MI. Most importantly, such approaches should underpin our ability to develop and test plaque-stabilizing drugs targeting robustly validated mechanisms specifically involved in plaque destabilization and rupture.