Translational atherosclerosis research: From experimental models to coronary artery disease in humans

Atherosclerosis 248 (2016) 110e116

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Review article

Translational atherosclerosis research: From experimental models to coronary artery disease in humans Christian A. Gleissner Department of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 November 2015 Received in revised form 9 February 2016 Accepted 8 March 2016 Available online 14 March 2016

Atherosclerosis is the leading cause of death worldwide. Research on the pathophysiological mechanisms of atherogenesis has made tremendous progress over the past two decades. However, despite great advances there is still a lack of therapies that reduce adverse cardiovascular events to an acceptable degree. This review addresses successes, but also questions, challenges, and chances regarding the translation of basic science results into clinical practice, i.e. the capability to apply the results of basic and/or clinical research in order to design therapies suitable to improve patient outcome. Specifically, it discusses problems in translating findings from the most broadly used murine models of atherosclerosis into clinically feasible therapies and strategies potentially improving the results of clinical trials. Most likely, the key to success will be a multimodal approach employing novel imaging methods as well as large scale screening toolsesummarized as “omics” approach. Using individually tailored therapies, plaque stabilization and regression could prevent adverse cardiovascular events thereby improving outcome of a large number of patients. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Atherosclerosis Coronary artery disease Research Clinical trials Therapy Personalized medicine

1. Atherosclerosis Despite great advances in basic and clinical research, atherosclerosis still represents the major cause of death worldwide [1,2]. In addition, non-fatal myocardial infarction and stroke induce a large burden of morbidity with all its social and economic consequences [3e5]. Atherogenesis is a multifactorial process promoted by a plethora of risk factors [6]. Very briefly, one can differentiate between non-modifiable risk factors (such as gender, age, and genetic predisposition), and modifiable risk factors such as arterial hypertension, hyperlipidemia, diabetes mellitus, obesity, or tobacco consumption. The long-standing history of research elucidating the mechanisms of atherogenesis starts with the early works of Rudolph v. Virchow in 1856, who identified the atherosclerotic plaque as analogue of the abscess, thereby revealing the crucial role of inflammation for atherogenesis [7]. Gerrity et al. could demonstrate the relevance of monocyte-derived macrophages for atherogenesis [8,9]. The crucial role of modified lipoproteins could be confirmed in many studies [10]. Williams and Tabas have summarized the work of many investigators in the response-to-retention

E-mail address: [email protected]. http://dx.doi.org/10.1016/j.atherosclerosis.2016.03.013 0021-9150/© 2016 Elsevier Ireland Ltd. All rights reserved.

hypothesis [11], which claims subendothelial retention of atherogenic lipoproteins to be the central pathogenic process in atherogenesis. They conclude that other contributory processes are either not individually necessary or are not sufficient [11]. Thus, all inflammatory processes resulting from the presence of lipoproteins are considered a consequence. In fact, clinical studies have shown that lowering cholesterol reduces the likelihood of future adverse cardiovascular events (multiple statin trials [12], IMPROVE-IT [13]), while a number of trials specifically focussing on anti-inflammatory therapeutics have failed (ARISE [14], STABILITY [15], SOLID TIMI 52 [16]). Also, while statins have been shown effective in reducing cardiovascular events in patients with increased high-sensitivity C-reactive protein (CRP), Mendelian randomization could not confirm a causal involvement of CRP in cardiovascular disease [17]. Thus, the evidence that reducing inflammation reduces adverse cardiovascular events has not yet been given by randomized clinical trials. Currently, several studies specifically investigating the effects of anti-inflammatory drugs such as low-dose methotrexate or anti TNF antibodies are under way. Results of the CIRT [18] and the CANTOS [19] trials are expected in 2016 and 2017 and may change our understanding of the clinical relevance of inflammation in human atherosclerotic disease. Cardiovascular medicine has lead to significantly improved

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outcome of patients suffering from cardiovascular events. E.g. in Germany, over the past two decades, mortality from coronary artery disease could be reduced by 28%. Mortality of acute myocardial infarction even decreased by 40% (“Heart Report 2014”, German Heart Foundation). However, even with optimal interventional and medical therapy, 20% of patients suffer from recurrent acute coronary syndrome (ACS) within three years [20,21]. Considering the high prevalence of ACS, these numbers are unacceptably high and there is still need for improvement. In the following paragraphs, I will try to summarize some of the most important questions and challenges we are currently facing in atherosclerosis research. Furthermore, I will discuss some of the chances, which may be opened by novel approaches to understand and treat the fateful disease. 2. Successes in atherosclerosis research The role of lipoproteins as triggers of atherogenesis has had tremendous impact on the way the disease is treated today. Native and modified lipoproteins affect atherogenesis at various levels. Thus, they facilitate monocyte attachment to and transmigration through the vascular endothelium [22]. Furthermore, lipid uptake by monocyte-derived macrophages induces foam cell formation, which is associated with the induction of various pro-inflammatory mechanisms and mediators [23,24]. Accordingly, based on the current international guidelines, lowering cholesterol is one of the main goals when treating patients with coronary artery disease [25,26]. These recommendations are based on a multitude of clinical studies, in most of which lowering LDL has been demonstrated to efficiently reduce the cardiovascular event rate [27]. Most of these studies investigated the role of statins as lipidlowering agents. Statins were first described in 1976 [28]. Mechanistically, they inhibit the HMG-CoA reductase, which is the ratelimiting enzyme of cholesterol synthesis [29]. First clinical trials were conducted in 1980 with Iovastatin being the first statin marketed in the U.S. in 1987 [30]. While lipid-lowering seems to be the major effect through which statins prevent adverse cardiovascular events, numerous pleitropic effects have been postulated, many of which could be confirmed in vitro or in animal models [31]. Additional approaches to lower cholesterol have been developed over the past fifteen years: Ezetimibe is an inhibitor of intestinal cholesterol resorption, which may be prescribed in patients who do not tolerate statins due to side effects. In 2015, the IMPROVE-IT trial could demonstrate that addition of significantly reduces adverse cardiovascular events (notably without reducing all-cause mortality) [32]. As a very recent addition, PCSK9 antibodies have been introduced into clinical practice. Briefly, blocking PCSK9 using monoclonal antibodies prevents degradation of the LDL receptor thereby leading to fast and significant reduction of plasma LDL [33]. The role of PCSK9 was identified through a mutation associated with familial hypercholesterolemia [34]. It took about one decade after identification of the therapeutic target until clinical tools became available targeting PCSK9 [33]. Data proving that PCSK9 inhibition improves cardiovascular outcome are expected by 2017/18. Taken together, the development of lipid-lowering drugs is one of the milestones, which have lead to substantial improvement of patient outcome. 3. Questions in atherosclerosis research A large body of atherosclerosis research relies on murine disease models. In 1992, the Apoe / mouse was simultaneously described by Piedrahita et al. and Plump [35,36]. Apoe / mice spontaneously develop atherosclerosis on a standard chow diet; furthermore,

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atherogenesis and vascular wall inflammation are increased in these mice when fed a high-fat, Western-type diet. Apoe / mice are an excellent model to study human atherosclerosis as the plaques developing in these mice (especially in the brachiocephalic trunk) are very similar to those found in humans. Even though, they do not develop plaque rupture on a regular basis, Apoe / mice have become a broadly used model to study the mechanisms of plaque development in vivo. One year later, Ishibashi et al. described the Ldlr / mouse, which has since then become another generally accepted mouse model of atherosclerosis [37]. While Apoe / mice spontaneously develop atherosclerotic lesions, Ldlr / mice only do so after being fed a high fat diet. Furthermore, lesion development in Ldlr / mice takes much longer. When searching for the terms “atherosclerosis” and “Apoe mouse” (or “atherosclerosis” and “Ldlr mouse”) on PubMed, we get 4127 (1011) hits (as of 03 February 2016). Since 2010, 300 to 400 papers dealing with the Apoe / mouse model and atherosclerosis have been published per year. It is virtually impossible to refer all studies employing murine atherosclerosis models published during the last 22 years. A decade ago, Meir and Leitersdorf summarized the results of atherosclerosis research in the murine Apoe / mouse [38]. In 2007, Zadelaar et al. have summarized studies addressing the ability of pharmacological compounds to ameliorate atherosclerosis in mouse models including Apoe / , Ldlr / , and Apoe3 Leiden mice [39]. Several recent review articles summarize our current understanding of the role of murine models of atherosclerosis, plaque progression, and plaque rupture [40e42]. There is a large number of papers investigating the role of specific cell types, exogeneous agents, or certain genes (either knocked out or overexpressed) in the different murine models. Many of them have given valuable insight in disease mechanisms: E.g. the important role of M-CSF, the major growth factor promoting monocyte macrophage differentiation, could be identified using the Apoe / mouse model [43]. Similarly, the importance of specific epitopes of oxidized LDL and antibodies directed against these epitopes was shown in the Apoe / mouse model [44]. Furthermore, a good example is a very elegant study identifying the important role of activated platelets for the disease process by repeated injection of activated platelets into Apoe / mice [45]. Another very interesting recently published paper could demonstrate that lesional macrophage accumulation in Apoe / mice is largely the consequence of local proliferation rather than increased monocyte recruitment [46]. A potentially useful model of plaque rupture that employs Apoe / Fbn1C1039G ± mice has recently beend described. These mice have a mutation in the fibrillin-1 gene leading to elastin fragmentation, which in turn results in a highly unstable plaque phenotype displaying intra-plaque hemorrhage, plaque neovascularization, and plaque rupture [47]. Other murine models of plaque destabilization involving arterial ligation or cast placement around specifically defined arteries have recently been compared demonstrating model-specific differences [48]. Taken together, we have gained valuable insight into basic mechanisms of atherogenesis by using murine models of atherosclerosis. One could add dozens of other findings, comprehensively summarized in several recent review articles [6,49]. By contrast, animal models may also lead to confusion: E.g. the role of the scavenger receptors SR-A and CD36 has been controversially discussed. Deficiency of CD36 or SRA-A or both have been associated with reduced atherogenesis in Apoe / mice [50,51], however, there was no additive effect on lesion development when both were knocked out [52]. In some of these studies, scavenger receptor deficiency was associated with reduction of foam cell formation or lesion complexity without affecting lesion size [53,54]. These different findings may be explained by differences in genetic backgrounds, time points assessed, and different methods

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used to measure atherosclerosis [55]. They illustrate the difficulties associated with research exclusively relying on murine models. When looking at the current guidelines to treat stable coronary artery disease (CAD), the options include a large number of symptomatic therapies such as nitrates, beta blockers, calcium channel blockers, molsidomin, ivabradin or ranolazine [56]. We also find a much smaller number of options that potentially slow down the disease progression and thereby improve prognosis. As mentioned above, these include lipid-lowering agents, but aso antiplatelet therapies and blockers of the renin angiotensin aldosterone system [56]. Thus, despite having elucidated important mechanisms of atherogenesis in murine models, only very few of these experiments have actually contributed to clinically feasible therapeutic approaches. What is the reason for the difficulties in bringing findings from animal models into clinical practice? e There are several reasons: Firstly, our murine models of atherosclerosis are somewhat artificial. The most widely used models are the Apoe / mouse [35,36,38], the Ldlr / mouse [37,39], and the Apoe3 Leiden mouse [57,58]. These models basically mimic a human disease called familial hypercholesterolemia (FH) and are characterized by excessive blood cholesterol levels when fed a high fat diet (in Ldlr / mice, this high fat diet is indispensible for the induction of atherogenesis and lesion development is much slower than in Apoe / mice). In fact, patients with FH are at high risk for developing cardiovascular disease, but most patients suffering acute coronary syndromes display LDL levels way below what is seen in FH patients [59]. Secondly, one has to bear in mind that lipid metabolism in mice differs significantly from what is known in humans. Thus, in mice the majority of lipoproteins is found in the HDL fraction, whereas in humans LDL and VLDL represent the majority of lipoproteins. Considering the important role attributed to LDL and HDL during atherogeneosis in general and specifically macrophage foam cell formation [23,24], these differences may be of fundamental importance when translating results from murine experiments to the clinical setting. Thirdly, atherosclerotic lesions in Apoe / , Ldlr / , or Apoe3 Leiden mice differ from what is seen in humans. Even when considering the differences in life expectancy between mice and humans, the time frame during which murine atherosclerotic lesions develop is much shorter than in humans. Furthermore, in most cases they do not display plaque rupture [60]. However, rupture of the culprit lesion with subsequent atherothrombosis presents the key event leading to acute coronary syndrome. While narrowing of coronary arteries due to plaque development may result in angina, it is in most cases not the cause of fatal cardiovascular events. In fact, most plaque ruptures leading to ACS occur in plaques with low grade stenosis [61]. The underlying causes are not entirely clear. In this context, mouse models may enhance our understanding of the events leading to plaque destabilization and subsequent atherothrombosis. E.g. one interesting study by Caligiuri showed that in Apoe / mice mental stress or hypoxia led to acute ischemia with clinical, electrogardiographic and biochemical signs of acute myocardial infarction [62]. Another recent paper by Dutta et al. could demonstrate that in Apoe / mice myocardial infarction in turn leads to further lesion progression, most likely due to increased monocyte recruitment [63]. It remains to be tested whether these findings can be confirmed in humans. Fourthly, considering that plaque development is largely mediated by the immune system leading to local inflammation, the differences between the murine and human immune systems should also be recognized as potential confounders of mouse experiments. Many inflammatory processes depend on the genetic background of the mouse model used, i.e. different strains may

display different findings when studying the same molecule [64]. Furthermore, at the transcriptomic level, there are significant differences between murine and human monocyte subsets [65]. Similarly, basic mechanisms of inflammation significantly differ between mice and humans [66]. Accordingly, we cannot expect atherosclerotic plaques in murine aortas to exactly mimic atherosclerotic lesions in human coronary arteries. Based on these considerations, a key question is: Can we improve our mouse models so that they better reflect human pathology? e Of course, one approach could be improving currently used mouse models in order to obtain plaques that show signs of instability and rupture. In fact, there have been many efforts to mimic plaque rupture in mice. These approaches have been comprehensively reviewed recently [42]. One of the murine models that are able to reproduce plaque rupture and atherothrombosis is the brachiocephalic artery in Apoe / mice fed a high-fat diet, with or without angiotensin II infusion [67,68]. Another model, that requires more efforts utilizes induction of endogenous renovascular hypertension by ligation of the left renal artery and the left common carotid artery in 8 week old Apoe / mice [69]. In 50% of these mice, this results in formation of a left common artery lumen thrombus combined with severe atherosclerosis; histological analyses confirm features of unstable plaques in these mice. Very recently, murine models of plaque rupture have been systematically compared demonstrating the difficulties in mimicking this process of human atherosclerosis. Thus, while placing a cast around the left common carotid artery may induce plaque destabilization very similar to what is seen in human plaques, it lacks the feature of intra-plaque hemorrhage which may be seen in other models [70]. Accordingly, one can conclude that none of the currently used murine models are suitable to mimic all stages of human plaque development and that additional efforts or other approaches are necessary if we want to translate findings obtained with these models into our patients. What other alternative options do we have? Basically, there are several options to complement murine in vivo models: In vitro models working with human cell types relevant to atherosclerosis such as monocyte-derived macrophages [71]. However, while these models overcome the problem with differences of immunity in mouse and human, they are not suitable to study complex interactions between various cell types present in advanced atherosclerotic lesions. Therefore, ex vivo models of human carotid plaques may be employed which allow studying pro-atherogenic mechanisms within the complex environment of an explanted carotid plaque [72]. Again, the major limitation of these models is that they do not allow observation of plaques over a longer time course. 4. Challenges Translational medicine can be defined as “area of research that aims to improve human health and longevity by determining the relevance to human disease of novel discoveries in the biological sciences” (Encyclopaedia Britannica). When thinking of translational research, two approaches are possible: Firstly, mutations associated with increased or decreased risk for cardiovascular disease may be identified. The mechanisms underlying these observations need to be confirmed in vitro and in vivo, allowing the development of specifically designed therapies, which in turn need to be tested in randomized clinical trials (e.g. PCSK9 antibodies). Secondly, experiments in vitro or in animal models may lead to the discovery of a potential therapeutic target, which is subsequently validated in pre-clinical and clinical studies eventually leading to novel therapies (e.g. anti-platelet therapies). Thirdly, therapeutic targets and agents may be identified by chance. Even though, this is approach cannot be considered optimal, it has been anecdotally

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reported (e.g. the discovery of penicillin by Fleming). Taking into account these considerations, it seems reasonable to search for additional or alternative strategies in atherosclerosis research. Such strategies should definitely involve human individuals. However, clinical trials with stable CAD patients are challenging: Thus, when trying to translate results from bench to bedside, it oftentimes turns out that mechanisms that have successfully been targeted in animal models do not improve cardiocvascular outcome in human patients. A good example for this phenomenon is Acyl-CoA-cholesterol acytransferase (ACAT) inhibition, which has significantly reduced atherosclerosis in mice and rabbits. By contrast, these findings could not be confirmed in human patients [73e75]. One explanation could be the fact that in most animal models, development of atherosclerotic lesions is studied, while in most patients lesions are already established. Thus, we actually need therapies that induce to lesion stabilization or even regression. Another challenge is that the majority of patients we see have stable CAD [2]. Myocardial infarction is in most cases caused by rupture of unstable plaques resulting in occlusion of the vessel caused by atherothrombosis. This means that the rate of adverse events such as unstable angina, or myocardial infarction (both Non ST-elevation and ST-elevation myocardial infarction) is fairly low. Clinical or animal models to study atherothrombosis are scarce. Also, atherothrombosis is an active process, thus in vivo or post mortem analyses are difficult. Thus, to study the effects of a specific treatment that aims at reducing adverse events in stable CAD patients, large patient numbers and long follow-up periods are needed. This in turn means that these trials become extremely expensive and industry sponsorship becomes less likely. Using surrogate markers as end points is probably not the best way to solve this problem. As we have seen in many trials in the past, a drug may well affect a surrogate marker without improving outcome: E.g. cholesterol ester transfer protein (CETP) inhibition could be demonstrated to significantly increase HDL levels without lowering adverse cardiovascular events [76,77]. Recent data indicate that this may be the cause of HDL functionality that differs between healthy individuals and patients with atherosclerosis: Thus, it could be demonstrated that HDL isolated from individuals with atherosclerosis not only lacks anti-inflammatory effects on myeloid cells in vitro, but even may be pro-inflammatory [78]. Another example for a potentially misleading surrogate marker is the controversy regarding the role of high sensitivity C-reactive protein (CRP): While some studies suggest that treating CRP levels may ameliorate cardiovascular outcome, data from human Mendelian randomization studies do not support a causal role of CRP in cardiovascular disease [79]. Accordingly, we need better surrogate parameters. Alternatively, large well-powered studies with long follow-up periods are indispensible to find out whether a specific therapeutic approach may be suitable to reduce the risk of cardiovascular events in stable CAD. Unless at least partially governmental funding will be available, it is unlikely that such trials will be performed in large numbers. 5. Chances Despite the challenges mentioned above, atherosclerosis research has great potential. Successful development of novel therapeutic strategies may be of great value to societies, as these strategies apply to many individuals and at the same time may prevent a large number of disabling or even fatal events [2]. While animal models usually focus on the development of atherosclerotic lesions, strategies in human CAD patients should rather aim at stabilizing atherosclerotic plaques or inducing plaque

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regression. Plaque stabilization may thereby actively prevent plaque rupture with subsequent atherothrombosis. To develop therapies that stabilize plaques, our basic understanding of plaque development needs to improve. One approach to analyze the mechanics of plaque development and destabilization could be computational studies [80]. Such an approach may help to better understand biomechanical and morphological changes leading to plaque rupture. These efforts need to be complemented by results from imaging studies that give insight into the human coronary plaque morphology [81]. Suitable approaches include intravascular ultrasound [82], optical coherence tomography [83], and coronary computed tomography angiography [81]. These imaging tools may allow studying plaque progression an destabilization in human individuals in vivo and allow for thorough characterization of human atherosclerotic lesions. Furthermore, they may help to evaluate both the natural course of human atherosclerotic plaques and also the effects of therapeutic interventions [61]. Provided that imaging tools recognize unstable plaques with sufficient accuracy, imaging trials may potentially be used to deliver novel more reliable surrogate markers in clinical trials with stable CAD patients. Another rapidly developing field covers large scale analysis technologies summarized as “omics”. These strategies represent a global, unbiased, non-targeted approach based on high throughput technologies that analyze genetic and epigenetic characteristics, changes of gene and protein expression, metabolic or lipidomic features associated with specific diseases and thereby allow identification of thus far unknown disease mechanisms and biomarkers [84]. Also, they allow identifying cellular networks by integrating large data samples (e.g. derived from genomics, proteomics, metabolomics screens), which bares the potential to improve our understanding of complex biological processes. Of course, the use of “omics” technologies does not mean that confirmatory in vitro and in vivo experiments have or will become unnecessary. Thus, a careful evaluation of pathomechanisms and therapeutic targets identified by “omics” approaches remains indispensable. The quality of “omcis” data strongly depends on proper selection of samples and appropriate controls. One approach to ascertain proper controls is to study tissue that can be easily obtained from healthy individuals as well as from patients suffering from atherosclerotic disease (e.g. blood [85]). Another approach may be the use of diseased tissues and healthy control tissues from the same individuals (e.g. the use of endothelial cells derived from atherosclerotic plaques and endothelial cells from plaque-free areas of the same vessel [86]). In summary, these approaches may not only reveal novel diagnostic or therapeutic targets, but they may also allow personalized strategies specifically taylored for the individual patient. Finally, novel ways to deliver drugs specifically to the plaque where they should unfold their therapeutic effect are being developed. These include viral therapies [87] as well as nanomedicine based strategies [88]. Viral therapies have significantly improved and now allow delivery of DNA to a specific organ with sufficient specificity and transduction efficiency. Similarly, liposome-based drug delivery may allow both non-targeted and specifically targeted application of therapeutic agents. This overcomes the problems associated with side effects due to off-target action of drugs. Thus, in an ideal future world a patient would undergo cardiovascular imaging to identify unstable plaques. A combination of clinical parameters, imaging results and potentially genetic or epigenetic characteristics would allow assessing the individual risk of this patient. Based on these data, a specific individualized therapy would be designed that would involve delivery of an antiinflammatory drug (or nucleic acid) directly to the unstable plaque, which then induces plaque stabilization and regression of the

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lesion. Side effects would be minimal or (ideally) absent. 6. Summary and conclusions Treating coronary artery disease remains an important goal, which may reduce both mortality and morbidity worldwide. Primary and secondary prevention can address modifiable risk factors such as arterial hypertension, hyperlipidemia, or diabetes mellitusewith the adaption of Western life style, the metabolic syndrome is becoming an increasingly prevalent in Asian countries. While research relying on murine models has generated a large body of data relevant to atherogenesis, the focus should be shifted to plaque stabilization and regression. Clinical trials that successfully translate results from mouse studies into clinical practice are scarce. Accordingly, research employing novel animal models and even more importantly novel imaging technologies are needed to specifically study mechanisms of plaque destabilization and rupture. These research efforts should be complemented by identification of novel disease markers and pathways derived from large scale “omics” studies. Based on these results, innovative personalized drug delivery techniques may allow a tailored plaquestabilizing therapy allowing a concept of “personalized medicine” that takes into account individual patient characteristics.

[14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

References [22] [1] A.S. Go, D. Mozaffarian, V.L. Roger, E.J. Benjamin, J.D. Berry, M.J. Blaha, S. Dai, E.S. Ford, C.S. Fox, S. Franco, H.J. Fullerton, C. Gillespie, S.M. Hailpern, J.A. Heit, V.J. Howard, M.D. Huffman, S.E. Judd, B.M. Kissela, S.J. Kittner, D.T. Lackland, J.H. Lichtman, L.D. Lisabeth, R.H. Mackey, D.J. Magid, G.M. Marcus, A. Marelli, D.B. Matchar, D.K. McGuire, E.R. Mohler, C.S. Moy, M.E. Mussolino, R.W. Neumar, G. Nichol, D.K. Pandey, N.P. Paynter, M.J. Reeves, P.D. Sorlie, J. Stein, A. Towfighi, T.N. Turan, S.S. Virani, N.D. Wong, D. Woo, M.B. Turner, Executive summary: heart disease and stroke statisticsd2014 update: a report from the American heart association, Circulation 129 (3) (2014) 399e410. [2] V.L. Roger, A.S. Go, D.M. Lloyd-Jones, R.J. Adams, J.D. Berry, T.M. Brown, M.R. Carnethon, S. Dai, G. de Simone, E.S. Ford, C.S. Fox, H.J. Fullerton, C. Gillespie, K.J. Greenlund, S.M. Hailpern, J.A. Heit, P.M. Ho, V.J. Howard, B.M. Kissela, S.J. Kittner, D.T. Lackland, J.H. Lichtman, L.D. Lisabeth, D.M. Makuc, G.M. Marcus, A. Marelli, D.B. Matchar, M.M. McDermott, J.B. Meigs, C.S. Moy, D. Mozaffarian, M.E. Mussolino, G. Nichol, N.P. Paynter, W.D. Rosamond, P.D. Sorlie, R.S. Stafford, T.N. Turan, M.B. Turner, N.D. Wong, J. Wylie-Rosett, o. b. o. t. A. H. A. S. Committee, Stroke Statistics Subcommittee, O. b. o. t. A. H. A. H, Disease and stroke statistics writing group: heart disease and stroke statisticse2011 update: a report from the American heart association, Circulation 123 (4) (2011) e18e209. [3] U.K. Sampson, S. Fazio, M.F. Linton, Residual cardiovascular risk despite optimal LDL cholesterol reduction with statins: the evidence, etiology, and therapeutic challenges, Curr. Atheroscler. Rep. 14 (1) (2012) 1e10. [4] P.M. Ridker, Moving beyond JUPITER: will inhibiting inflammation reduce vascular event rates? Curr. Atheroscler. Rep. 15 (1) (2013) 295. [5] G. Sirimarco, J. Labreuche, E. Bruckert, L.B. Goldstein, K.M. Fox, P.M. Rothwell, P. Amarenco, Perform, S. Investigators and Committees, Atherogenic dyslipidemia and residual cardiovascular risk in statin-treated patients, Stroke 45 (5) (2014) 1429e1436. [6] E. Galkina, K. Ley, Immune and inflammatory mechanisms of atherosclerosis (*), Annu. Rev. Immunol. 27 (2009) 165e197. €se Prozess der Arterien, Wien. Med. Wochenschr [7] R. Virchow, Der atheromato 51 (1856) 809e812. [8] R.G. Gerrity, The role of the monocyte in atherogenesis: II. Migration of foam cells from atherosclerotic lesions, Am. J. Pathol. 103 (2) (1981) 191e200. [9] R.G. Gerrity, The role of the monocyte in atherogenesis: I. Transition of bloodborne monocytes into foam cells in fatty lesions, Am. J. Pathol. 103 (2) (1981) 181e190. [10] W. Palinski, M.E. Rosenfeld, S. Yla-Herttuala, G.C. Gurtner, S.S. Socher, S.W. Butler, S. Parthasarathy, T.E. Carew, D. Steinberg, J.L. Witztum, Low density lipoprotein undergoes oxidative modification in vivo, Proc. Natl. Acad. Sci. U. S. A. 86 (4) (1989) 1372e1376. [11] K.J. Williams, I. Tabas, The response-to-retention hypothesis of early atherogenesis, Arterioscler. Thromb. Vasc. Biol. 15 (5) (1995) 551e561. [12] P.M. Ridker, E. Danielson, F.A.H. Fonseca, J. Genest, A.M. Gotto, J.J.P. Kastelein, W. Koenig, P. Libby, A.J. Lorenzatti, J.G. MacFadyen, B.G. Nordestgaard, J. Shepherd, J.T. Willerson, R.J. Glynn, Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein, N. Engl. J. Med. 359 (21) (2008) 2195e2207. [13] E.A. Bohula, R.P. Giugliano, C.P. Cannon, J. Zhou, S.A. Murphy, J.A. White,

[23] [24]

[25]

[26]

[27] [28]

[29] [30] [31]

[32]

A.M. Tershakovec, M.A. Blazing, E. Braunwald, Achievement of dual lowdensity lipoprotein cholesterol and high-sensitivity C-reactive protein targets more frequent with the addition of Ezetimibe to Simvastatin and associated with better outcomes in IMPROVE-IT, Circulation 132 (13) (2015) 1224e1233. J.-C. Tardif, J. J. V. McMurray, E. Klug, R. Small, J. Schumi, J. Choi, J. Cooper, R. Scott, E. F. Lewis, P. L. L'Allier and M. A. Pfeffer: Effects of succinobucol (AGI1067) after an acute coronary syndrome: a randomised, double-blind, placebo-controlled trial. Lancet, 371(9626), 1761e1768. Darapladib for preventing ischemic events in stable coronary heart disease, N. Engl. J. Med. 370 (18) (2014) 1702e1711. M.L. O'Donoghue, E. Braunwald, H.D. White, et al., Effect of darapladib on major coronary events after an acute coronary syndrome: the solid-timi 52 randomized clinical trial, JAMA 312 (10) (2014) 1006e1015. P. Elliott, J.C. Chambers, W. Zhang, et al., GEnetic loci associated with creactive protein levels and risk of coronary heart disease, JAMA 302 (1) (2009) 37e48. B.M. Everett, A.D. Pradhan, D.H. Solomon, N. Paynter, J. MacFadyen, E. Zaharris, M. Gupta, M. Clearfield, P. Libby, A.A.K. Hasan, R.J. Glynn, P.M. Ridker, Rationale and design of the cardiovascular inflammation reduction trial (CIRT): A test of the inflammatory hypothesis of atherothrombosis, Am. heart J. 166 (2) (2013), 199e207.e15. P.M. Ridker, T. Thuren, A. Zalewski, P. Libby, Interleukin-1b inhibition and the prevention of recurrent cardiovascular events: rationale and design of the canakinumab anti-inflammatory thrombosis outcomes study (CANTOS), Am. Heart J. 162 (4) (2011) 597e605. G.W. Stone, A. Maehara, A.J. Lansky, B. de Bruyne, E. Cristea, G.S. Mintz, R. Mehran, J. McPherson, N. Farhat, S.P. Marso, H. Parise, B. Templin, R. White, Z. Zhang, P.W. Serruys, A prospective natural-history study of coronary atherosclerosis, N. Engl. J. Med. 364 (3) (2011) 226e235. C.P. Cannon, S.A. Murphy, E. Braunwald, Intensive lipid lowering with atorvastatin in coronary disease, N. Engl. J. Med. 353 (1) (2005) 93e96 author reply 93e6. C.A. Gleissner, N. Leitinger, K. Ley, Effects of native and modified low-density lipoproteins on monocyte recruitment in atherosclerosis, Hypertension 50 (2) (2007) 276e283. P. Shashkin, B. Dragulev, K. Ley, Macrophage differentiation to foam cells, Curr. Pharm. Des. 11 (23) (2005) 3061e3072. H.J. Cho, P. Shashkin, C.A. Gleissner, D. Dunson, N. Jain, J.K. Lee, Y. Miller, K. Ley, Induction of dendritic cell-like phenotype in macrophages during foam cell formation, Physiol. Genom. 29 (2) (2007) 149e160. M. Task Force, G. Montalescot, U. Sechtem, S. Achenbach, F. Andreotti, C. Arden, A. Budaj, R. Bugiardini, F. Crea, T. Cuisset, C. Di Mario, J.R. Ferreira, B.J. Gersh, A.K. Gitt, J.S. Hulot, N. Marx, L.H. Opie, M. Pfisterer, E. Prescott, F. Ruschitzka, M. Sabate, R. Senior, D.P. Taggart, E.E. van der Wall, C.J. Vrints, E. S. C. C. f. P. Guidelines, J.L. Zamorano, S. Achenbach, H. Baumgartner, J.J. Bax, H. Bueno, V. Dean, C. Deaton, C. Erol, R. Fagard, R. Ferrari, D. Hasdai, A.W. Hoes, P. Kirchhof, J. Knuuti, P. Kolh, P. Lancellotti, A. Linhart, P. Nihoyannopoulos, M.F. Piepoli, P. Ponikowski, P.A. Sirnes, J.L. Tamargo, M. Tendera, A. Torbicki, W. Wijns, S. Windecker, R. Document, J. Knuuti, M. Valgimigli, H. Bueno, M.J. Claeys, N. Donner-Banzhoff, C. Erol, H. Frank, C. Funck-Brentano, O. Gaemperli, J.R. Gonzalez-Juanatey, M. Hamilos, D. Hasdai, S. Husted, S.K. James, K. Kervinen, P. Kolh, S.D. Kristensen, P. Lancellotti, A.P. Maggioni, M.F. Piepoli, A.R. Pries, F. Romeo, L. Ryden, M.L. Simoons, P.A. Sirnes, P.G. Steg, A. Timmis, W. Wijns, S. Windecker, A. Yildirir, J.L. Zamorano, 2013 ESC guidelines on the management of stable coronary artery disease: the task force on the management of stable coronary artery disease of the European society of cardiology, Eur. Heart J. 34 (38) (2013) 2949e3003. J. Perk, G. De Backer, H. Gohlke, I. Graham, Z. Reiner, M. Verschuren, C. Albus, P. Benlian, G. Boysen, R. Cifkova, C. Deaton, S. Ebrahim, M. Fisher, G. Germano, R. Hobbs, A. Hoes, S. Karadeniz, A. Mezzani, E. Prescott, L. Ryden, M. Scherer, M. Syvanne, W.J. Scholte op Reimer, C. Vrints, D. Wood, J.L. Zamorano, F. Zannad, P. European Association for Cardiovascular, Rehabilitation and E. S. C. C. f. P. Guidelines, European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The fifth joint task force of the European society of cardiology and other societies on cardiovascular disease prevention in clinical practice (constituted by representatives of nine societies and by invited experts), Eur. Heart J. 33 (13) (2012) 1635e1701. A.P. Agouridis, M.S. Elisaf, D.R. Nair, D.P. Mikhailidis, All for statins and statins for all; an update, Curr. Pharm. Des. 22 (1) (2015) 18e27. A. Endo, M. Kuroda, K. Tanzawa, Competitive inhibition of 3-hydroxy-3methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity, FEBS Lett. 72 (2) (1976) 323e326. S.M. Grundy, HMG-CoA reductase inhibitors for treatment of hypercholesterolemia, N. Engl. J. Med. 319 (1) (1988) 24e33. J.A. Tobert, Lovastatin and beyond: the history of the HMG-CoA reductase inhibitors, Nat. Rev. Drug Discov. 2 (7) (2003) 517e526. M. Satoh, Y. Takahashi, T. Tabuchi, Y. Minami, M. Tamada, K. Takahashi, T. Itoh, Y. Morino, M. Nakamura, Cellular and molecular mechanisms of statins: an update on pleiotropic effects, Clin. Sci. (Lond) 129 (2) (2015) 93e105. C.P. Cannon, M.A. Blazing, R.P. Giugliano, A. McCagg, J.A. White, P. Theroux, H. Darius, B.S. Lewis, T.O. Ophuis, J.W. Jukema, G.M. De Ferrari, W. Ruzyllo, P. De Lucca, K. Im, E.A. Bohula, C. Reist, S.D. Wiviott, A.M. Tershakovec, T.A. Musliner, E. Braunwald, R.M. Califf, I.-I. Investigators, Ezetimibe added to

C.A. Gleissner / Atherosclerosis 248 (2016) 110e116

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51]

[52]

[53]

[54]

statin therapy after acute coronary syndromes, N. Engl. J. Med. 372 (25) (2015) 2387e2397. Y.J. Shimada, C.P. Cannon, PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future, Eur. Heart J. 36 (36) (2015) 2415e2424. M. Abifadel, M. Varret, J.P. Rabes, D. Allard, K. Ouguerram, M. Devillers, C. Cruaud, S. Benjannet, L. Wickham, D. Erlich, A. Derre, L. Villeger, M. Farnier, I. Beucler, E. Bruckert, J. Chambaz, B. Chanu, J.M. Lecerf, G. Luc, P. Moulin, J. Weissenbach, A. Prat, M. Krempf, C. Junien, N.G. Seidah, C. Boileau, Mutations in PCSK9 cause autosomal dominant hypercholesterolemia, Nat. Genet. 34 (2) (2003) 154e156. J.A. Piedrahita, S.H. Zhang, J.R. Hagaman, P.M. Oliver, N. Maeda, Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells, Proc. Natl. Acad. Sci. U. S. A. 89 (10) (1992) 4471e4475. A.S. Plump, J.D. Smith, T. Hayek, K. Aalto-Setala, A. Walsh, J.G. Verstuyft, E.M. Rubin, J.L. Breslow, Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells, Cell 71 (2) (1992) 343e353. S. Ishibashi, M.S. Brown, J.L. Goldstein, R.D. Gerard, R.E. Hammer, J. Herz, Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery, J. Clin. Invest. 92 (2) (1993) 883e893. K.S. Meir, E. Leitersdorf, Atherosclerosis in the apolipoprotein-E-deficient mouse: a decade of progress, Arterioscler. Thromb. Vasc. Biol. 24 (6) (2004) 1006e1014. S. Zadelaar, R. Kleemann, L. Verschuren, J. de Vries-Van der Weij, J. van der Hoorn, H.M. Princen, T. Kooistra, Mouse models for atherosclerosis and pharmaceutical modifiers, Arterioscler. Thromb. Vasc. Biol. 27 (8) (2007) 1706e1721. F.R. Kapourchali, G. Surendiran, L. Chen, E. Uitz, B. Bahadori, M.H. Moghadasian, Animal models of atherosclerosis, World J. Clin. Cases 2 (5) (2014) 126e132. A. Millon, E. Canet-Soulas, L. Boussel, Z. Fayad, P. Douek, Animal models of atherosclerosis and magnetic resonance imaging for monitoring plaque progression, Vascular 22 (3) (2014 Jun) 221e237. T. Matoba, K. Sato, K. Egashira, Mouse models of plaque rupture, Curr. Opin. Lipidol. 24 (5) (2013) 419e425. J.D. Smith, E. Trogan, M. Ginsberg, C. Grigaux, J. Tian, M. Miyata, Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E, Proc. Natl. Acad. Sci. U. S. A. 92 (18) (1995) 8264e8268. S. Horkko, E. Miller, E. Dudl, P. Reaven, L.K. Curtiss, N.J. Zvaifler, R. Terkeltaub, S.S. Pierangeli, D.W. Branch, W. Palinski, J.L. Witztum, Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein, J. Clin. Invest. 98 (3) (1996) 815e825. Y. Huo, A. Schober, S.B. Forlow, D.F. Smith, M.C. Hyman, S. Jung, D.R. Littman, C. Weber, K. Ley, Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E, Nat. Med. 9 (1) (2003) 61e67. C.S. Robbins, I. Hilgendorf, G.F. Weber, I. Theurl, Y. Iwamoto, J.L. Figueiredo, R. Gorbatov, G.K. Sukhova, L.M. Gerhardt, D. Smyth, C.C. Zavitz, E.A. Shikatani, M. Parsons, N. van Rooijen, H.Y. Lin, M. Husain, P. Libby, M. Nahrendorf, R. Weissleder, F.K. Swirski, Local proliferation dominates lesional macrophage accumulation in atherosclerosis, Nat. Med. 19 (9) (2013) 1166e1172. C. Van der Donckt, J.L. Van Herck, D.M. Schrijvers, G. Vanhoutte, M. Verhoye, I. Blockx, A. Van Der Linden, D. Bauters, H.R. Lijnen, J.C. Sluimer, L. Roth, C.E. Van Hove, P. Fransen, M.W. Knaapen, A.S. Hervent, G.W. De Keulenaer, H. Bult, W. Martinet, A.G. Herman, G.R. De Meyer, Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death, Eur. Heart J. 36 (17) (2015 May 1) 1049e1058. H. Hartwig, M. Drechsler, D. Lievens, B. Kramp, P. von Hundelshausen, E. Lutgens, C. Weber, Y. Doring, O. Soehnlein, Platelet-derived PF4 reduces neutrophil apoptosis following arterial occlusion, Thromb. Haemost. 111 (3) (2014) 562e564. C. Weber, H. Noels, Atherosclerosis: current pathogenesis and therapeutic options, Nat. Med. 17 (11) (2011) 1410e1422. V.R. Babaev, L.A. Gleaves, K.J. Carter, H. Suzuki, T. Kodama, S. Fazio, M.F. Linton, Reduced atherosclerotic lesions in mice deficient for total or macrophagespecific expression of scavenger receptor-A, Arterioscler. Thromb. Vasc. Biol. 20 (12) (2000) 2593e2599. M. Febbraio, E.A. Podrez, J.D. Smith, D.P. Hajjar, S.L. Hazen, H.F. Hoff, K. Sharma, R.L. Silverstein, Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice, J. Clin. Invest. 105 (8) (2000) 1049e1056. S. Kuchibhotla, D. Vanegas, D.J. Kennedy, E. Guy, G. Nimako, R.E. Morton, M. Febbraio, Absence of CD36 protects against atherosclerosis in ApoE knockout mice with no additional protection provided by absence of scavenger receptor A I/II, Cardiovasc. Res. 78 (1) (2008) 185e196. K.J. Moore, V.V. Kunjathoor, S.L. Koehn, J.J. Manning, A.A. Tseng, J.M. Silver, M. McKee, M.W. Freeman, Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice, J. Clin. Invest. 115 (8) (2005) 2192e2201. J.J. Manning-Tobin, K.J. Moore, T.A. Seimon, S.A. Bell, M. Sharuk, J.I. AlvarezLeite, M.P. de Winther, I. Tabas, M.W. Freeman, Loss of SR-A and CD36 activity

[55] [56]

[57]

[58]

[59] [60] [61]

[62]

[63]

[64]

[65]

[66]

[67] [68]

[69]

[70]

[71]

115

reduces atherosclerotic lesion complexity without abrogating foam cell formation in hyperlipidemic mice, Arterioscler. Thromb. Vasc. Biol. 29 (1) (2008) 19e26. J.L. Witztum, You are right too!, J. Clin. Invest. 115 (8) (2005) 2072e2075. P. Kolh, S. Windecker, F. Alfonso, J.-P. Collet, J. Cremer, V. Falk, G. Filippatos, C. Hamm, S.J. Head, P. Jüni, A.P. Kappetein, A. Kastrati, J. Knuuti, U. Landmesser, G. Laufer, F.-J. Neumann, D.J. Richter, P. Schauerte, M. Sousa Uva, G.G. Stefanini, D.P. Taggart, L. Torracca, M. Valgimigli, W. Wijns, A. Witkowski, J.L. Zamorano, S. Achenbach, H. Baumgartner, J.J. Bax, H. Bueno, V. Dean, C. Deaton, Ç. Erol, R. Fagard, R. Ferrari, D. Hasdai, A.W. Hoes, P. Kirchhof, J. Knuuti, P. Kolh, P. Lancellotti, A. Linhart, P. Nihoyannopoulos, M.F. Piepoli, P. Ponikowski, P.A. Sirnes, J.L. Tamargo, M. Tendera, A. Torbicki, W. Wijns, S. Windecker, M. Sousa Uva, S. Achenbach, J. Pepper, A. Anyanwu, L. Badimon, J. Bauersachs, A. Baumbach, F. Beygui, N. Bonaros, M. De Carlo, C. Deaton, D. Dobrev, J. Dunning, E. Eeckhout, S. Gielen, D. Hasdai, P. Kirchhof, H. Luckraz, H. Mahrholdt, G. Montalescot, D. Paparella, A.J. Rastan, M. Sanmartin, P. Sergeant, S. Silber, J. Tamargo, J. ten Berg, H. Thiele, R.-J. van Geuns, H.-O. Wagner, S. Wassmann, O. Wendler, J.L. Zamorano, 2014 ESC/ EACTS guidelines on myocardial revascularization: the task force on myocardial revascularization of the european society of cardiology (ESC) and the European association for cardio-thoracic surgery (EACTS) developed with the special contribution of the European association of percutaneous cardiovascular interventions (EAPCI), Eur. J. Cardio-Thoracic Surg. 46 (4) (2014) 517e592. B.J. van Vlijmen, A.M. van den Maagdenberg, M.J. Gijbels, H. van der Boom, H. HogenEsch, R.R. Frants, M.H. Hofker, L.M. Havekes, Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice, J. Clin. Invest 93 (4) (1994) 1403e1410. A.M. van den Maagdenberg, M.H. Hofker, P.J. Krimpenfort, I. de Bruijn, B. van Vlijmen, H. van der Boom, L.M. Havekes, R.R. Frants, Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia, J. Biol. Chem. 268 (14) (1993) 10540e10545. B. Pitt, J. Loscalzo, J. Ycas, J.S. Raichlen, Lipid levels after acute coronary syndromes, J. Am. Coll. Cardiol. 51 (15) (2008) 1440e1445. J.F. Bentzon, E. Falk, Atherosclerotic lesions in mouse and man: is it the same disease? Curr. Opin. Lipidol. 21 (5) (2010) 434e440. G.W. Stone, A. Maehara, A.J. Lansky, B. de Bruyne, E. Cristea, G.S. Mintz, R. Mehran, J. McPherson, N. Farhat, S.P. Marso, H. Parise, B. Templin, R. White, Z. Zhang, P.W. Serruys, P. Investigators, A prospective natural-history study of coronary atherosclerosis, N. Engl. J. Med. 364 (3) (2011) 226e235. G. Caligiuri, B. Levy, J. Pernow, P. Thoren, G.K. Hansson, Myocardial infarction mediated by endothelin receptor signaling in hypercholesterolemic mice, Proc. Natl. Acad. Sci. U. S. A. 96 (12) (1999) 6920e6924. P. Dutta, G. Courties, Y. Wei, F. Leuschner, R. Gorbatov, C.S. Robbins, Y. Iwamoto, B. Thompson, A.L. Carlson, T. Heidt, M.D. Majmudar, F. Lasitschka, M. Etzrodt, P. Waterman, M.T. Waring, A.T. Chicoine, A.M. van der Laan, H.W. Niessen, J.J. Piek, B.B. Rubin, J. Butany, J.R. Stone, H.A. Katus, S.A. Murphy, D.A. Morrow, M.S. Sabatine, C. Vinegoni, M.A. Moskowitz, M.J. Pittet, P. Libby, C.P. Lin, F.K. Swirski, R. Weissleder, M. Nahrendorf, Myocardial infarction accelerates atherosclerosis, Nature 487 (7407) (2012) 325e329. A.R. Schenkel, T.W. Chew, W.A. Muller, Platelet endothelial cell adhesion molecule deficiency or blockade significantly reduces leukocyte emigration in a majority of mouse strains, J. Immunol. 173 (10) (2004) 6403e6408. M.A. Ingersoll, R. Spanbroek, C. Lottaz, E.L. Gautier, M. Frankenberger, R. Hoffmann, R. Lang, M. Haniffa, M. Collin, F. Tacke, A.J. Habenicht, L. ZieglerHeitbrock, G.J. Randolph, Comparison of gene expression profiles between human and mouse monocyte subsets, Blood 115 (3) (2010) e10e9. J. Seok, H.S. Warren, A.G. Cuenca, M.N. Mindrinos, H.V. Baker, W. Xu, D.R. Richards, G.P. McDonald-Smith, H. Gao, L. Hennessy, C.C. Finnerty,  pez, S. Honari, E.E. Moore, J.P. Minei, J. Cuschieri, P.E. Bankey, C.M. Lo J.L. Johnson, J. Sperry, A.B. Nathens, T.R. Billiar, M.A. West, M.G. Jeschke, M.B. Klein, R.L. Gamelli, N.S. Gibran, B.H. Brownstein, C. Miller-Graziano, S.E. Calvano, P.H. Mason, J.P. Cobb, L.G. Rahme, S.F. Lowry, R.V. Maier, L.L. Moldawer, D.N. Herndon, R.W. Davis, W. Xiao, R.G. Tompkins, Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly mimic human inflammatory diseases, Proc. Natl. Acad. Sci. U S A. 110 (9) (2013 Feb 26) 3507e3512. J.L. Johnson, C.L. Jackson, Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse, Atherosclerosis 154 (2) (2001) 399e406. J. Johnson, K. Carson, H. Williams, S. Karanam, A. Newby, G. Angelini, S. George, C. Jackson, Plaque rupture after short periods of fat feeding in the apolipoprotein E-knockout mouse: model characterization and effects of pravastatin treatment, Circulation 111 (11) (2005) 1422e1430. S.X. Jin, L.H. Shen, P. Nie, W. Yuan, L.H. Hu, D.D. Li, X.J. Chen, X.K. Zhang, B. He, Endogenous renovascular hypertension combined with low shear stress induces plaque rupture in apolipoprotein E-deficient mice, Arterioscler. Thromb. Vasc. Biol. 32 (10) (2012) 2372e2379. H. Hartwig, C. Silvestre-Roig, J. Hendrikse, L. Beckers, N. Paulin, K. Van der Heiden, Q. Braster, M. Drechsler, M.J. Daemen, E. Lutgens, O. Soehnlein, Atherosclerotic plaque destabilization in mice: a comparative study, PLoS One 10 (10) (2015) e0141019. C. Erbel, G. Rupp, C.M. Helmes, M. Tyka, F. Linden, A.O. Doesch, H.A. Katus, C.A. Gleissner, An in vitro model to study heterogeneity of human macrophage differentiation and polarization, J. Vis. Exp. 76 (2013) e50332.

116

C.A. Gleissner / Atherosclerosis 248 (2016) 110e116

[72] C. Erbel, D. Okuyucu, M. Akhavanpoor, L. Zhao, S. Wangler, M. Hakimi, A. Doesch, T.J. Dengler, H.A. Katus, C.A. Gleissner, A human ex vivo atherosclerotic plaque model to study lesion biology, J. Vis. Exp. 87 (2014). [73] N. Terasaka, A. Miyazaki, N. Kasanuki, K. Ito, N. Ubukata, T. Koieyama, K. Kitayama, T. Tanimoto, N. Maeda, T. Inaba, ACAT inhibitor pactimibe sulfate (CS-505) reduces and stabilizes atherosclerotic lesions by cholesterollowering and direct effects in apolipoprotein E-deficient mice, Atherosclerosis 190 (2) (2007) 239e247. [74] J.C. Tardif, J.J. McMurray, E. Klug, R. Small, J. Schumi, J. Choi, J. Cooper, R. Scott, E.F. Lewis, P.L. L'Allier, M.A. Pfeffer, I. Aggressive Reduction of Inflammation Stops Events Trial, Effects of succinobucol (AGI-1067) after an acute coronary syndrome: a randomised, double-blind, placebo-controlled trial, Lancet 371 (9626) (2008) 1761e1768. [75] K. Kitayama, T. Koga, N. Maeda, T. Inaba, T. Fujioka, Pactimibe stabilizes atherosclerotic plaque through macrophage acyl-CoA:cholesterol acyltransferase inhibition in WHHL rabbits, Eur. J. Pharmacol. 539 (1e2) (2006) 81e88. [76] P.J. Barter, M. Caulfield, M. Eriksson, S.M. Grundy, J.J.P. Kastelein, M. Komajda, J. Lopez-Sendon, L. Mosca, J.-C. Tardif, D.D. Waters, C.L. Shear, J.H. Revkin, K.A. Buhr, M.R. Fisher, A.R. Tall, B. Brewer, Effects of torcetrapib in patients at high risk for coronary events, N. Engl. J. Med. 357 (21) (2007) 2109e2122. [77] G.G. Schwartz, A.G. Olsson, M. Abt, C.M. Ballantyne, P.J. Barter, J. Brumm, B.R. Chaitman, I.M. Holme, D. Kallend, L.A. Leiter, E. Leitersdorf, J.J.V. McMurray, H. Mundl, S.J. Nicholls, P.K. Shah, J.-C. Tardif, R.S. Wright, Effects of dalcetrapib in patients with a recent acute coronary syndrome, N. Engl. J. Med. 367 (22) (2012) 2089e2099. [78] C. Besler, K. Heinrich, L. Rohrer, C. Doerries, M. Riwanto, D.M. Shih, A. Chroni, K. Yonekawa, S. Stein, N. Schaefer, M. Mueller, A. Akhmedov, G. Daniil, C. Manes, C. Templin, C. Wyss, W. Maier, F.C. Tanner, C.M. Matter, R. Corti, C. Furlong, A.J. Lusis, A. von Eckardstein, A.M. Fogelman, T.F. Luscher, U. Landmesser, Mechanisms underlying adverse effects of HDL on eNOSactivating pathways in patients with coronary artery disease, J. Clin. Invest 121 (7) (2011) 2693e2708.

[79] W. Koenig, High-sensitivity C-reactive protein and atherosclerotic disease: from improved risk prediction to risk-guided therapy, Int. J. Cardiol. 168 (6) (2013) 5126e5134. [80] G.A. Holzapfel, J.J. Mulvihill, E.M. Cunnane, M.T. Walsh, Computational approaches for analyzing the mechanics of atherosclerotic plaques: a review, J. Biomech. 47 (4) (2014) 859e869. [81] G. Korosoglou, S. Giusca, G. Gitsioudis, C. Erbel, H.A. Katus, Cardiac magnetic resonance and computed tomography angiography for clinical imaging of stable coronary artery disease. Diagnostic classification and risk stratification, Front. Physiol. 5 (2014) 291. [82] G.S. Mintz, Clinical utility of intravascular imaging and physiology in coronary artery disease, J. Am. Coll. Cardiol. 64 (2) (2014) 207e222. [83] S.K. Nadkarni, B.E. Bouma, J. de Boer, G.J. Tearney, Evaluation of collagen in atherosclerotic plaques: the use of two coherent laser-based imaging methods, Lasers Med. Sci. 24 (3) (2009) 439e445. [84] R. Puri, S.E. Nissen, The complementary roles of imaging and 'omics' for future anti-atherosclerotic drug development, Curr. Pharm. Des. 19 (33) (2013) 5963e5971. [85] P.R. Sinnaeve, M.P. Donahue, P. Grass, D. Seo, J. Vonderscher, S.D. Chibout, W.E. Kraus, M. Sketch Jr., C. Nelson, G.S. Ginsburg, P.J. Goldschmidt-Clermont, C.B. Granger, Gene expression patterns in peripheral blood correlate with the extent of coronary artery disease, PLoS One 4 (9) (2009) e7037. [86] O.L. Volger, J.O. Fledderus, N. Kisters, R.D. Fontijn, P.D. Moerland, J. Kuiper, T.J. van Berkel, A.-P.J.J. Bijnens, M.J.A.P. Daemen, H. Pannekoek, A.J.G. Horrevoets, Distinctive expression of chemokines and transforming growth factor-b Signaling in human arterial endothelium during atherosclerosis, Am. J. Pathol. 171 (1) (2007) 326e337. [87] O.J. Muller, H.A. Katus, R. Bekeredjian, Targeting the heart with gene therapyoptimized gene delivery methods, Cardiovasc. Res. 73 (3) (2007) 453e462. [88] M. Schiener, M. Hossann, J.R. Viola, A. Ortega-Gomez, C. Weber, K. Lauber, L.H. Lindner, O. Soehnlein, Nanomedicine-based strategies for treatment of atherosclerosis, Trends Mol. Med. 20 (5) (2014) 271e281.