Mouse models of atherosclerosis in translational research

A similar lipid profile and disease pathology are important considerations for selecting mouse model of atherosclerosis for translational research.Apoe−/− mice, Ldlr−/− mice, and APOE3-Leiden.CETP mice are important preclinical models that have been used to validate FDA-approved lipid-lowering drugs.AAV8-Pcsk9-D377Y injection can induce atherosclerotic plaque formation in C57BL/6J mice without crossbreeding with atherosusceptible mouse strains.Partial carotid ligation surgery in Apoe−/− mice or Ldlr−/− mice creates flow disturbance and emerges as a new model to examine the role of mechanosensitive factors in atherogenesis. Atherosclerotic cardiovascular disease (CVD), the major cause of premature human mortality, is a chronic and progressive metabolic and inflammatory disease in large- and medium-sized arteries. Mouse models are widely used to gain mechanistic insights into the pathogenesis of atherosclerosis and have facilitated the discovery of anti-atherosclerotic drugs. Despite promising preclinical studies, many drug candidates have not translated to clinical use because of the complexity of disease patho-mechanisms including lipid metabolic traits and inflammatory, genetic, and hemodynamic factors. We review the current preclinical utility and translation potential of traditional [apolipoprotein E (APOE)- and low-density lipoprotein (LDL) receptor (LDLR)-deficient mice] and emerging mouse models that include partial carotid ligation and AAV8-Pcsk9-D377Y injection in atherosclerosis research and drug discovery. This article represents an important resource in atherosclerosis research. Atherosclerotic cardiovascular disease (CVD), the major cause of premature human mortality, is a chronic and progressive metabolic and inflammatory disease in large- and medium-sized arteries. Mouse models are widely used to gain mechanistic insights into the pathogenesis of atherosclerosis and have facilitated the discovery of anti-atherosclerotic drugs. Despite promising preclinical studies, many drug candidates have not translated to clinical use because of the complexity of disease patho-mechanisms including lipid metabolic traits and inflammatory, genetic, and hemodynamic factors. We review the current preclinical utility and translation potential of traditional [apolipoprotein E (APOE)- and low-density lipoprotein (LDL) receptor (LDLR)-deficient mice] and emerging mouse models that include partial carotid ligation and AAV8-Pcsk9-D377Y injection in atherosclerosis research and drug discovery. This article represents an important resource in atherosclerosis research. Atherosclerosis (see Glossary) and its clinical sequelae are the main underlying cause of most CVD, including angina, heart attack, heart failure, and stroke. Atherosclerosis is characterized by chronic inflammation, dysfunction of the endothelial lining of blood vessels, and the retention of pro-atherogenic lipoproteins in the arterial wall, all of which contribute to the formation of plaques (Figure 1) [1.Xu S. et al.Endothelial dysfunction in atherosclerotic cardiovascular diseases and beyond: from mechanism to pharmacotherapies.Pharmacol. Rev. 2021; 73: 924-967Crossref PubMed Google Scholar,2.Libby P. et al.Atherosclerosis.Nat. Rev. Dis. Primers. 2019; 5: 56Crossref PubMed Scopus (763) Google Scholar]. The build-up of 'plaque' increases the thickness and stiffness of arteries, resulting in reduced blood flow and oxygen delivery to vital organs and tissues. Atherosclerotic plaque formation in humans typically occurs over decades. However, the sudden rupture of vulnerable atherosclerotic plaques – those characterized by large necrotic cores, thin fibrous caps, calcification, foam cells, and intraplaque hemorrhage – is an acute event precipitating the clinical manifestations of CVD [3.Vergallo R. Crea F. Atherosclerotic plaque healing.N. Engl. J. Med. 2020; 383: 846-857Crossref PubMed Google Scholar,4.Björkegren J.L.M. Lusis A.J. Atherosclerosis: recent developments.Cell. 2022; 185: 1630-1645Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. There is an urgent need for novel therapies for atherosclerosis and CVD. In this regard, preclinical animal models, mouse models in particular, have been useful in validating the anti-atherosclerotic effects of many lipid-lowering drugs as well as anti-inflammation-based pharmacotherapies. In this review we discuss the current preclinical utility and translational potential of mice deficient in apolipoprotein E (APOE) and low-density lipoprotein (LDL) receptor (LDLR) (Apoe−/− and Ldlr−/− mice, respectively) and emerging mouse models that incorporate partial carotid ligation and injection of adeno-associated viral vector serotype 8 (AAV8) expressing the D377Y mutant of proprotein convertase subtilisin/kexin type 9 (PCSK9) (AAV8-Pcsk9-D377Y), in atherosclerosis research and drug discovery. We also discuss factors to consider in utilizing mouse models in atherosclerosis research and offer our perspective on evaluating the use of the various models. In the area of experimental atherosclerosis, mouse models are currently the cornerstone of preclinical research to define the mechanisms of disease etiology upon which new therapeutic candidates can be tested (Figure 2). Most mouse models of atherosclerosis are based on manipulation of genes that play key roles in lipid metabolism, such as Apoe or Ldlr, or surgical interventions coupled with atherogenic feeding strategies. Analysis of the number of publications focusing on the use of different animal models in atherosclerosis research from PubMed revealed that Apoe−/− mice are the most frequently used model, followed by Ldlr−/− mice and others (Figure 3 and Table S1 in the supplemental material online). To achieve a systematic and balanced comparison of different mouse models, we summarize the mode of atherosclerosis development, advantages, limitations, and applications of different mouse models used in atherosclerosis research in Table 1. Because atherosclerotic CVD is the major comorbidity of many types of disease, including diabetes, obesity, fatty liver, and heart failure, we have also reviewed compound disease models of atherosclerosis (Box 1).Figure 3Yearly publications of different animal models used in atherosclerosis research.Show full captionPublication numbers were retrieved from the PubMed database using the key subject terms: 'atherosclerosis AND apolipoprotein E knockout mice OR Apoe knockout mice OR apolipoprotein E deficient mice OR Apoe deficient mice OR apolipoprotein E null mice OR Apoe null mice'. The same search strategy was used to determine publication numbers for other animal models from 2000 to 2021, as of 5/29/2022. Over the years analyzed, Apoe−/− mice was the most frequently used model with a steady increase, followed by Ldlr−/− mice and others. More details are given in Table S1 in the supplemental information online.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1A comparative study of mouse models of atherosclerosisaAbbreviations: APOE, apolipoprotein E; AS, atherosclerosis; ASO, antisense oligonucleotide; CETP, cholesterol ester transfer protein; d-flow, disturbed flow; Fbn1, fibrillin 1; HFD, high-fat diet; LDLR, low-density lipoprotein (LDL) receptor; PCL, partial carotid ligation; PCSK9, proprotein convertase subtilisin/kexin type 9; TC, total cholesterol; VLDL, very low density lipoprotein.Mouse modelMode of AS developmentTC (mg/dl)AdvantagesLimitationsApplicationsRefsApoe−/− miceChow diet~600Develop hyperlipidemia and complex lesions on regular chow diet and extensive atherosclerosis on HFDHigher total cholesterol levels than Ldlr−/− miceAbsence of a human-like lipid profile, rare plaque rupture, no thrombus or coronary lesion formationAltered inflammation plays a role in plaque developmentSuitable to study mechanism of atherosclerosis progressionFacilitate preclinical studies of therapeutic agents[11.Li Y. et al.Progression of atherosclerosis in ApoE-knockout mice fed on a high-fat diet.Eur. Rev. Med. Pharmacol. Sci. 2016; 20: 3863-3867PubMed Google Scholar,12.Oppi S. et al.Mouse models for stherosclerosis research – which is my line?.Front. Cardiovasc. Med. 2019; 6: 46Crossref PubMed Scopus (66) Google Scholar]HFD>1000APOE3-Leiden.CETP miceHFD>1000Functional Apoe, no effect on inflammationHuman-like response to lipid-lowering drugsDietary manipulations necessary for lesion formationNo plaque rupture or thrombus formationSuitable to study the protective effects of anti-atherosclerotic drugs[28.Westerterp M. et al.Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (170) Google Scholar,29.Paalvast Y. et al.Male apoE*3-Leiden.CETP mice on high-fat high-cholesterol diet exhibit a biphasic dyslipidemic response, mimicking the changes in plasma lipids observed through life in men.Physiol. Rep. 2017; 5e13376Crossref PubMed Scopus (13) Google Scholar]APOE2.KI miceChow diet and HFD~1400Develop robust hypercholesterolemia with human APOE2 expressionDevelop human-like type III hyperlipoproteinemiaDefective in removing circulating VLDL residuesNo plaque rupture or thrombus formationSuitable to study the protective effects of anti-atherosclerotic compounds[35.Sullivan P.M. et al.Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2.J. Clin. Invest. 1998; 102: 130-135Crossref PubMed Google Scholar,37.Hennuyer N. et al.The novel selective PPARα modulator (SPPARMα) pemafibrate improves dyslipidemia, enhances reverse cholesterol transport and decreases inflammation and atherosclerosis.Atherosclerosis. 2016; 249: 200-208Abstract Full Text Full Text PDF PubMed Google Scholar]Ldlr−/− miceChow diet~200–300Lipoprotein pattern similar to humansDevelop site-specific lesions in a time-dependent mannerLDLR mutations are common in patients with familial hypercholesterolemiaRequire HFD for atherosclerosis inductionNo plaque rupture; no thrombus or coronary lesion formationGood model to study familial hypercholesterolemiaFacilitate preclinical studies of therapeutic agents[40.Ishibashi S. et al.Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice.J. Clin. Invest. 1994; 93: 1885-1893Crossref PubMed Google Scholar,41.Rajamannan N.M. Atorvastatin attenuates bone loss and aortic valve atheroma in LDLR mice.Cardiology. 2015; 132: 11-15Crossref PubMed Scopus (15) Google Scholar]HFD>1500Ldlr−/− ApoB100 miceChow diet~300Develop atherosclerosis on a regular chow dietDevelop more human-like hypercholesterolemiaAbsence of spontaneous plaque ruptures; no thrombus or coronary lesion formationSuitable model to study atherosclerosis and to test the effects of new potential drugs and therapies[45.Véniant M.M. et al.Lipoprotein clearance mechanisms in LDL receptor-deficient 'Apo-B48-only' and 'Apo-B100-only' mice.J. Clin. Invest. 1998; 102: 1559-1568Crossref PubMed Google Scholar,46.Hellberg S. et al.Effects of atorvastatin and diet interventions on atherosclerotic plaque inflammation and [18F]FDG uptake in Ldlr−/−Apob100/100 mice.Atherosclerosis. 2017; 263: 369-376Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]Ldlr−/− Apoe−/− Fbn1C1039G+/− miceHFD>1000Strong resemblance to human-like complicationsDevelop exacerbated atherosclerosis with plaque instability and spontaneous plaque rupturesLong-term Western-type diet feedingHas not yet been used for testing the efficacy of potential therapeutic drugsSuitable to study end-stage atherosclerosis and to study the patho-mechanisms of the rupture of vulnerable plaques[47.Van Herck J.L. et al.Impaired fibrillin-1 function promotes features of plaque instability in apolipoprotein E-deficient mice.Circulation. 2009; 120: 2478-2487Crossref PubMed Scopus (68) Google Scholar, 48.Van der Donckt C. et al.Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.Eur. Heart J. 2015; 36: 1049-1058Crossref PubMed Scopus (106) Google Scholar, 49.Wang X. et al.Establishment of a novel mouse model for atherosclerotic vulnerable plaque.Front. Cardiovasc. Med. 2021; 8642751Google Scholar]Apoe−/− mice+ PCLSurgery and HFD~700Rapidly induce endothelial dysfunction and carotid atherosclerosis via d-flowAbsence of a human-like lipid profileNo plaque rupture or thrombus formationSuitable to investigate different genes and therapeutic drugs involved in d-flow-dependent atherosclerosis[50.Nam D. et al.Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.Am. J. Physiol. Heart Circ. Physiol. 2009; 297: H1535-H1543Crossref PubMed Scopus (329) Google Scholar,51.Mitra R. et al.The comparative effects of high fat diet or disturbed blood flow on glycocalyx integrity and vascular inflammation.Transl. Med. Commun. 2018; 3: 10Crossref PubMed Google Scholar]AAV8-Pcsk9-D377Y injected miceAAV8-Pcsk9 injection and HFD>1000No germline genetic manipulation is neededRapid, easy, and cost-effective approach to develop persistent and advanced atherosclerosisSuitable to study plaque calcificationAbsence of spontaneous plaque ruptures and thrombus formationPCSK9 non-specific binding and cleavage of other substrates may lead to LDLR-independent effectsSuitable to rapidly induce atherosclerosis and to study atherosclerosis regression and therapeutic interventions by avoiding multiple rounds of backcrossing[62.Bjørklund M.M. et al.Induction of atherosclerosis in mice and hamsters without germline genetic engineering.Circ. Res. 2014; 114: 1684-1689Crossref PubMed Scopus (138) Google Scholar,63.Kumar S. et al.Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation.Lab. Investig. 2017; 97: 935-945Crossref PubMed Scopus (40) Google Scholar]Ldlr-ASO miceASO and HFD~400–800Develop inducible and reversible atherosclerosis without genetically altering lipoprotein metabolismAbsence of spontaneous plaque ruptures; no thrombus or coronary lesion formationMore direct, time-efficient, and obviates the need of extensive breedingSuitable to study atherosclerosis regression[74.Basu D. et al.Novel reversible model of atherosclerosis and regression using oligonucleotide regulation of the LDL receptor.Circ. Res. 2018; 122: 560-567Crossref PubMed Scopus (34) Google Scholar]a Abbreviations: APOE, apolipoprotein E; AS, atherosclerosis; ASO, antisense oligonucleotide; CETP, cholesterol ester transfer protein; d-flow, disturbed flow; Fbn1, fibrillin 1; HFD, high-fat diet; LDLR, low-density lipoprotein (LDL) receptor; PCL, partial carotid ligation; PCSK9, proprotein convertase subtilisin/kexin type 9; TC, total cholesterol; VLDL, very low density lipoprotein. Open table in a new tab Publication numbers were retrieved from the PubMed database using the key subject terms: 'atherosclerosis AND apolipoprotein E knockout mice OR Apoe knockout mice OR apolipoprotein E deficient mice OR Apoe deficient mice OR apolipoprotein E null mice OR Apoe null mice'. The same search strategy was used to determine publication numbers for other animal models from 2000 to 2021, as of 5/29/2022. Over the years analyzed, Apoe−/− mice was the most frequently used model with a steady increase, followed by Ldlr−/− mice and others. More details are given in Table S1 in the supplemental information online. Given the complexities and chronic nature of the pathogenesis of atherosclerosis, many factors need to be considered when choosing the appropriate mouse model for the study of atherosclerosis or disease-modifying drugs [5.Daugherty A. et al.Recommendation on design, execution, and reporting of animal atherosclerosis studies: a scientific statement from the American Heart Association.Arterioscler. Thromb. Vasc. Biol. 2017; 37: e131-e157Crossref PubMed Scopus (186) Google Scholar]. Such factors include (but are not limited to) strain, gender, diet and duration, circadian rhythm, age, ease of maintenance, and comparability to the relevant human condition (Figure 4) [5.Daugherty A. et al.Recommendation on design, execution, and reporting of animal atherosclerosis studies: a scientific statement from the American Heart Association.Arterioscler. Thromb. Vasc. Biol. 2017; 37: e131-e157Crossref PubMed Scopus (186) Google Scholar]. Because plaque development in atherosusceptible mouse strains is accelerated after feeding with a western-type diet (WD) containing high fat and cholesterol, we have listed all types of routinely used atherogenic diets and their composition (Table 2). This information is important for investigators commencing atherosclerosis research because multiple formulations of WD are available and dietary differences may represent a key confounding issue for reproducibility. In the following sections we overview traditional and emerging new mouse models of atherosclerosis and discuss their use in the validation of disease-modifying medicines.Table 2Choice of diet for inducing atherosclerosis in miceType of dietDiet compositionVendor and catalog numberPaigen diet15% saturated fat, 1.25% cholesterol, 0.5% sodium cholateaSodium cholate or cholic acid induces inflammation and reduces cholesterol disposal via bile acid synthesis.Envigo, TD88051Western-type diet (WD) or high-fat diet (HFD)bThe type of diet most widely used to induce atherosclerosis.21% total fat, 60% saturated fatty acids, 34% sucrose, 0.2% cholesterolEnvigo, TD8813741% fat, 43% carbohydrate, 17% proteinResearch diet, D12079BHigh-cholesterol diet (HCD)1.25% cholesterol, 40% fatResearch diet, D12108C1.25% cholesterol, 21% milk fat, 0.5% sodium cholateaSodium cholate or cholic acid induces inflammation and reduces cholesterol disposal via bile acid synthesis.Envigo, TD02028Amylin liver NASH (AMLN) diet40% kcal fat (80% trans-fat), 22% fructose, 2% cholesterolResearch diet, D09100301a Sodium cholate or cholic acid induces inflammation and reduces cholesterol disposal via bile acid synthesis.b The type of diet most widely used to induce atherosclerosis. Open table in a new tab APOE is a 34 kDa glycoprotein and is involved in the distribution/redistribution of lipids among different tissues/cell types. It has antioxidant, anti-inflammatory, and anti-atherogenic properties [6.Huang Y. Mahley R.W. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer's diseases.Neurobiol. Dis. 2014; 72: 3-12Crossref PubMed Scopus (344) Google Scholar]. The major function of APOE protein is to mediate the binding of lipoproteins or lipid complexes in plasma or interstitial fluids to specific cell-surface receptors (Figure 1). Genetic deficiency of Apoe in mice can impair the clearance of very low density lipoprotein (VLDL) remnants and chylomicrons. In 1992 the first strain of Apoe−/− mice was successfully produced simultaneously in two laboratories by manipulating the Apoe gene in embryonic stem cells via homologous recombination [7.Piedrahita J.A. et al.Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4471-4475Crossref PubMed Scopus (747) Google Scholar,8.Plump A.S. et al.Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1858) Google Scholar]. Apoe−/− mice show an appreciable increase of total plasma cholesterol level compared to wild-type mice [9.Emini Veseli B. et al.Animal models of atherosclerosis.Eur. J. Pharmacol. 2017; 816: 3-13Crossref PubMed Scopus (267) Google Scholar]. Apoe−/− mice on a regular chow diet accumulate VLDL remnant lipoproteins and chylomicrons, and develop human-like type III hyperlipidemia, supporting their clinical relevance for atherosclerotic research and drug discovery/repurposing efforts. An important feature of Apoe−/− mice is that their cholesterol metabolism is impaired even when raised on a regular chow diet, and their mean circulating cholesterol level was 600 mg/dl [10.Nakashima Y. et al.ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.Arterioscler. Thromb. 1994; 14: 133-140Crossref PubMed Google Scholar]. Upon high-fat diet (HFD) or WD feeding, the level of total cholesterol of Apoe−/− mice further increases and can exceed 1000 mg/dl. After HFD challenge, Apoe−/− mice develop atherosclerosis more rapidly and extensively throughout the vascular tree [11.Li Y. et al.Progression of atherosclerosis in ApoE-knockout mice fed on a high-fat diet.Eur. Rev. Med. Pharmacol. Sci. 2016; 20: 3863-3867PubMed Google Scholar]. In Apoe−/− mice, atherosclerosis development typically starts in areas exposed to disturbed blood flow (d-flow) [9.Emini Veseli B. et al.Animal models of atherosclerosis.Eur. J. Pharmacol. 2017; 816: 3-13Crossref PubMed Scopus (267) Google Scholar] and the distribution of lesions is also somewhat similar to that in humans [9.Emini Veseli B. et al.Animal models of atherosclerosis.Eur. J. Pharmacol. 2017; 816: 3-13Crossref PubMed Scopus (267) Google Scholar,12.Oppi S. et al.Mouse models for stherosclerosis research – which is my line?.Front. Cardiovasc. Med. 2019; 6: 46Crossref PubMed Scopus (66) Google Scholar]. However, a drawback of using Apoe−/− mice is that the lipoprotein profile is different from that in human. In Apoe−/− mice most plasma cholesterol is transported by VLDL and chylomicrons, whereas in humans it is mostly carried by LDL [12.Oppi S. et al.Mouse models for stherosclerosis research – which is my line?.Front. Cardiovasc. Med. 2019; 6: 46Crossref PubMed Scopus (66) Google Scholar]. Another potential drawback of this mouse model compared to humans is the rare and different nature of plaque rupture [12.Oppi S. et al.Mouse models for stherosclerosis research – which is my line?.Front. Cardiovasc. Med. 2019; 6: 46Crossref PubMed Scopus (66) Google Scholar]. It is important to note that Apoe deficiency, per se, may have other effects that are independent of lipoprotein metabolism, including a role in vascular function [13.Vasquez E.C. et al.Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse.J. Biomed. Sci. 2012; 19: 22Crossref PubMed Scopus (60) Google Scholar], blood–brain barrier [14.Methia N. et al.ApoE deficiency compromises the blood brain barrier especially after injury.Mol. Med. 2001; 7: 810-815Crossref PubMed Google Scholar], and most importantly in macrophage biology [15.Pepe M.G. Curtiss L.K. Apolipoprotein E is a biologically active constituent of the normal immunoregulatory lipoprotein, LDL-In.J. Immunol. 1986; 136: 3716-3723Crossref PubMed Google Scholar]. This is due to the fact that Apoe is also expressed in hematopoietic cells (even though the majority of APOE in plasma is of hepatic origin), which can partly protect against atherosclerosis development. Apoe expression in hematopoietic cells regulates their proliferative capacity, and Apoe deficiency can thus lead to increased proliferation, disrupted cholesterol efflux, and systemic leukocytosis [16.Murphy A.J. et al.ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice.J. Clin. Invest. 2011; 121: 4138-4149Crossref PubMed Scopus (351) Google Scholar]. Notably, transplantation of bone marrow from wild-type mice into Apoe−/− mice has been shown to protect Apoe−/− mice from diet-induced atherosclerosis in a dosage-dependent manner [17.Linton M.F. et al.Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation.Science. 1995; 267: 1034-1037Crossref PubMed Google Scholar,18.Van Eck M. et al.Bone marrow transplantation in apolipoprotein E-deficient mice. Effect of ApoE gene dosage on serum lipid concentrations, (beta)VLDL catabolism, and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3117-3126Crossref PubMed Google Scholar]. This phenomenon, however, is not seen in Ldlr−/− mice because LDLR derived from bone marrow cells does not have a large impact on atherosclerosis or lipids level [19.Getz G.S. Reardon C.A. Animal models of atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1104-1115Crossref PubMed Scopus (395) Google Scholar]. The Apoe−/− mouse model, as the most popular animal model in atherosclerosis research, has been used to evaluate the potential anti-atherosclerotic effects of drugs already approved by the FDA for other conditions (Figure 2). For example, atorvastatin, a lipid-lowering drug, was reported to inhibit plaque development in Apoe−/− mice independently of lipid-lowering effects [20.Bot I. et al.Atorvastatin inhibits plaque development and adventitial neovascularization in ApoE deficient mice independent of plasma cholesterol levels.Atherosclerosis. 2011; 214: 295-300Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. In addition, Apoe−/− mice have been used to validate the anti-atherosclerotic effects of some antidiabetic medications. For example, glucagon-like peptide 1 receptor agonists (GLP-1 RAs) are antidiabetic medications that act by decreasing glucagon secretion, slowing gastric emptying, and increasing insulin secretion and satiety. Liraglutide and semaglutide, two widely used GLP-1 RAs, have been reported to reduce atherosclerosis in Apoe−/− mice through anti-inflammatory mechanisms [21.Rakipovski G. et al.The GLP-1 analogs liraglutide and semaglutide reduce atherosclerosis in ApoE−/− and LDLr−/− mice by a mechanism that includes inflammatory pathways.JACC Basic Transl. Sci. 2018; 3: 844-857Crossref PubMed Scopus (0) Google Scholar]. Furthermore, other types of antidiabetic drugs, such as dapagliflozin [22.Chen Y.C. et al.Sodium-glucose co-transporter 2 (SGLT2) inhibitor dapagliflozin stabilizes diabetes-induced atherosclerotic plaque instability.J. Am. Heart Assoc. 2022; 11e022761Crossref Scopus (1) Google Scholar] and empagliflozin [23.Ganbaatar B. et al.Empagliflozin ameliorates endothelial dysfunction and suppresses atherogenesis in diabetic apolipoprotein E-deficient mice.Eur. J. Pharmacol. 2020; 875173040Crossref PubMed Scopus (34) Google Scholar] which block glucose reabsorption in the kidney by inhibiting sodium/glucose cotransporter 2 (SGLT2), have also been shown to reduce atherosclerosis in diabetic Apoe−/− mice. FDA-approved anti-inflammatory drugs have also been studied for their anti-atherosclerotic potential using Apoe−/− mice. For example, colchicine prevents atherosclerosis in Apoe−/− mice by inhibiting the activation of NLRP3 (NLR family pyrin domain-containing 3) inflammasomes and the expression of proinflammatory cytokines including interleukin 1β (IL-1β) [24.Spartalis M. et al.The beneficial therapy with colchicine for atherosclerosis via anti-inflammation and decrease in hypertriglyceridemia.Cardiovasc. Hematol. Agents Med. Chem. 2018; 16: 74-80Crossref PubMed Scopus (4) Google Scholar]. In addition, targeting IL-1β in Apoe−/− mice by using canakinumab dampens atherosclerosis progression through reduced production of inflammatory leucocytes [25.Hettwer J. et al.Interleukin-1β suppression dampens inflammatory leukocyte production and uptake in atherosclerosis.Cardiovasc. Res. 2021; (Published online October 28, 2021)https://doi.org/10.1093/cvr/cvab337Crossref PubMed Google Scholar]. Importantly, the anti-atherosclerotic effects of canakinumab successfully translated to the clinical setting, as evidenced by a significantly lower incidence of recurrent cardiovascular events in canakinumab-treated patients (at a dose of 150 mg every 3 months) with atherosclerotic CVD [26.Ridker P.M. et al.Antiinflammatory therapy with canakinumab for atherosclerotic disease.N. Engl. J. Med. 2017; 377: 1119-1131Crossref PubMed Scopus (4269) Google Scholar]. Overall, the Apoe−/− mouse model is the most frequently used animal model of atherosclerosis (Figure 3) and it represents a valuable model to discover new therapeutic drugs and drug targets for atherosclerosis. One of the major limitations of studies involving mice is the relatively rapid clearance of triglyceride-rich lipoproteins (TRLs) in mice as compared to humans [27.van Vlijmen B.J. et al.Apolipoprotein E*3-Leiden transgenic mice as a test model for hypolipidaemic drugs.Arzneimittelforschung. 1998; 48: 396-402PubMed Google Scholar]. Mice lack cholesterol ester transfer protein (CETP), which promotes the redistribution of cholesteryl esters from anti-atherogenic high-density lipoproteins (HDLs) to pro-atherogenic TRLs in humans (Figure 1). The lack of this enzyme limits pharmaceutical evaluation of the effects of CETP inhibitors (a category of HDL-raising drugs) on lipid metabolism and atherosclerosis in mice. To address this issue, humanized apolipoprotein E3 (APOE3)-Leiden.CETP mice have been developed by crossbreeding APOE3-Leiden mice with transgenic human CETP mice [28.Westerterp M. et al.Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (170) Google Scholar]. In this mouse model, the APOE3-Leiden transgene reduces the clearance of TRLs (i.e., VLDL remnants and chylomicrons) while the CETP transgene (with HFD feeding) further moves the cholesterol profile closer to that of human [28.Westerterp M