SR-B1-/-ApoE-R61h/h Mice Mimic Human Coronary Heart Disease

Abstract

Cardiovascular diseases are the leading cause of death in the modern world. Atherosclerosis underlies the majority of these pathologies and may result in sudden life-threatening events such as myocardial infarction or stroke. Current concepts consider a rupture (resp. erosion) of “unstable/vulnerable” atherosclerotic plaques as a primary cause leading to thrombus formation and subsequent occlusion of the artery lumen finally triggering an acute clinical event. We and others described SR-B1-/-ApoE-R61h/h mice mimicking clinical coronary heart disease in all major aspects: from coronary atherosclerosis through vulnerable plaque ruptures leading to thrombus formation/coronary artery occlusion, finally resulting in myocardial infarction/ischemia. SR-B1-/-ApoE-R61h/h mouse provides a valuable model to study vulnerable/occlusive plaques, to evaluate bioactive compounds as well as new anti-inflammatory and “anti-rupture” drugs, and to test new technologies in experimental cardiovascular medicine. This review summarizes and discuss our knowledge about SR-B1-/-ApoE-R61h/h mouse model based on recent publications and experimental observations from the lab.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10557-023-07475-8.

Atherosclerosis and Vulnerable Plaques

Cardiovascular diseases are the leading cause of death in the modern world. The major underlying cause of these pathologies (mainly coronary heart disease and stroke) is atherosclerosis—a chronic inflammatory disease of the vascular vessel wall. Current concepts consider a rupture (resp. erosion) of “unstable/vulnerable” atherosclerotic plaques as a primary cause leading to thrombus formation and subsequent occlusion of the artery lumen finally triggering an acute clinical event. Vulnerable plaques (terminology used in the field also includes the names “thin-cap fibroatheroma, unstable-, rupture-prone-, high-risk-, or thrombosis-prone plaque”) are advanced atherosclerotic lesions. They consist of a large necrotic core, are full-blown by lipids and inflammatory cells, only possess a very thin or none fibrous cap, and present with extracellular matrix (reviewed in [1, 2]). Nowadays, the classical description of a vulnerable plaque is basically still restricted to the size of its necrotic core and thickness of the fibrous cap (reviewed in [3]). Additionally, other characteristics are proposed for plaque vulnerability. For example, neovessels can be a major source for recruitment of inflammatory cells [4], but they can also trigger intraplaque hemorrhage (bleeding) leading to thrombosis in situ [5].

Murine Models of Coronary Heart Disease

Currently, studies of coronary vulnerable plaques are basically restricted to clinical/autopsy retrospective studies. Numerous studies of murine and non-murine animal models of atherosclerosis, vulnerable (non/coronary) plaques, myocardial infarctions (MI: spontaneous/induced) as well as various spieces comparative studies of coronary arteries have been described in research articles, summarized in textbooks, and reviewed elsewhere, exemplarily [6–23]. A major limitation for pathophysiological studies on vulnerable plaques and plaque ruptures is the lack of widely accepted murine model(s) developing spontaneous plaque rupture associated with thrombosis and acute (clinical) events. Potentially relevant murine models displaying features of human coronary heart disease (CHD) have been generated (as discussed further and see Suppl. Table S1). However, only few CHD models have been characterized with respect to the presence/vulnerability of coronary plaques and associated acute events.

The presence of atherosclerotic plaques in murine coronary arteries per se (with possible deep plaque disruptions), particularly in ApoE-deficient mice, has only been rarely observed and/or described [14–16]. Severe stenotic and occluded coronary atherosclerotic plaques were observed in older (more than 7 months) hypercholesterolemic ApoE-/-LDLR-/- and ApoE-/- mice. Increased mortality was associated with MI caused by occlusions found mainly in proximal branches of coronary arteries. Development of acute ischemia with subsequent infarction was shown to be pronounced under the stress (hypoxic conditions) and prevented by treatment using a blocker of endothelin type A receptor [24]. It was also reported that more than 50% of mice deficient in all nitric oxide synthase isoforms (neuronal/inducible/endothelial) spontaneously died due to MI. The authors described that areas of infarctions were associated with severe atherosclerosis although typical atherosclerotic lesions were rarely noticed. These mice were hypertonic and dyslipidemic, but they also showed other metabolic syndromes like visceral obesity or impaired glucose tolerance [25]. Typical hallmarks of CHD like coronary atherosclerosis, MI, and spontaneous death were described in several mouse models generated in Prof. Monty Krieger’s lab: mice deficient in scavenger receptor class B type 1 (SR-B1) and apolipoprotein E (ApoE) [26]; mice deficient in SR-B1 and hypomorphic for mutated ApoE-R61 (SR-B1-/-ApoE-R61h/h) [27]; or double knockout for ApoE and PDZK1—a four PDZ domain protein [28]. SR-B1-/-ApoE-/- mouse exhibits severe CHD and occlusive coronary atherosclerosis even under standard chow [26]. An early death within 5–8 weeks of age of SR-B1-/-ApoE-/- mice however remains a challenge for experimental setups. Zhang et al. generated the SR-B1-/-ApoE-R61h/h mouse [27] as a more suitable model for studying causes, consequences, and eventually pharmacological interventions of CHD, rapidly induced by the high-fat/high-cholesterol (HFC) and cholate-containing (Paigen) diet. Another model, a double-knockout mouse for PDZK-1 and ApoE develops CHD (coronary artery occlussions, MI/cardiac fibrosis) within 3 months on Paigen diet as well, although cardiac dysfunction, statistically significant death rate, and atherothrombosis were not observed in this report [28]. In contrast, the vulnerability/thrombosis of atherosclerotic plaques in coronary arteries has been noticed in SR-B1-/-ApoE-R61h/h mice [29, 30]. In our study, we described that SR-B1-/-ApoE-R61h/h mouse phenotype (MI) is associated with spontaneous coronary plaque ruptures leading to atherothrombosis. The latter study investigated hyperglycemic SR-B1-/-ApoE-R61h/h mice displaying atherothrombosis and MI. In SR-BIΔCT model [31], Ala/STOP mutation producing a truncated receptor (SR-BIΔCT) was introduced into SR-BI gene. This carboxy-terminal deletion of the HDL receptor (SR-B1) caused hypercholesterolemia, and in SR-BIΔCT/apoE KO mice (fed a standard chow diet, 9 weeks old when histologically analyzed) also partial or complete occlusive coronary atherosclerotic lesions and fatal heart disease. The authors suggest that a combined SR-BIΔCT/ApoeR61h/h model might be even better model (due to female fertility [31, 32]) to study CHD.

Significance of SR-B1 and ApoE in Atherosclerosis and CHD

Scavenger receptor class B type 1 (SR-B1, SR-BI, Scarb1) is an 82-kDa glycoprotein on cell membranes. It was originally identified as a homolog of CD-36, another member of the class B scavenger receptors [33] and later as a receptor for high-density lipoprotein (HDL) [34]. It also acts as a receptor for other lipoproteins such as low-/very-low-density lipoprotein (LDL/VLDL), and it additionally participates in the regulation of steroid metabolism (reviewed in [35]). Most importantly, SR-B1 mediates reverse cholesterol transport (RCT) through the selective uptake of cholesteryl esters from HDL [36]. SR-B1 is primarily expressed in the liver, steroidogenic tissues (adrenal glands, ovaries, testes), and macrophages ([34] and reviewed in [35]). In the vessel wall, SR-B1 expression was confirmed on endothelial cells, vascular smooth muscle cells, and monocytes/macrophages [37, 38]. Cell-specific expression of SR-B1 partially mediating lipid turnover in atherosclerotic plaques differs according to plaque stage—vascular smooth muscle cells expressing SR-B1 are mostly involved in early lesions, whereas macrophages expressing SR-B1 are involved in advanced lesions [38]. SR-B1 is also expressed by platelets [39]. Adoptive transfer of bone marrow (BM) cells from SR-B1-/-ApoE-/- and SR-B1+/+ApoE-/- mice to recipient ApoE-/- mice leads to more severe atherosclerosis in ApoE-/- mice lacking SR-B1 in comparison to ApoE-/- mice expressing SR-B1. This indicates an atheroprotective role of SR-B1 specifically expressed on macrophages [40]. Similarly, LDLR-/- recipient mice develop larger atherosclerotic lesions after transplantation of ABCA1 (ATP-binding cassette transporter A1 mediating cholesterol efflux) and SR-B1-deficient BM-derived cells [41]. SR-B1 is protective against early development of atherosclerosis by regulation of cholesterylester uptake and by maintenance of expression patterns of inflammatory molecules on the endothelium [32, 42]. Interestingly, also attenuated expression in SR-B1 hypomorphic mice leads to increased atherosclerosis, which can be even accelerated by hepatocyte-specific knockdown (hypomSR-B1liver-/-), however to a lesser extent than in SR-B1 knockout [43]. Although the majority of studies describes the protective character of SR-B1 in atherogenesis, its role is much more complex. SR-B1 expression can affect BM derived cells (e.g., monocytes/macrophages) also in the opposite way: a short-term fat diet for 4 weeks results in promotion of fatty streak lesions [44]. From studies in mice deficient in SR-B1 (itself or in combination with other deleted genes), we know that SR-B1 deficiency impairs the development and function of various blood cells like lymphocytes, erythrocytes, or platelets. SR-B1 deficiency results in age-dependent development of autoimmune disorders accompanied with the presence of circulating autoantibodies, imbalanced expansion of T and B lymphocytes, and their increased activation as well as proliferation [45]. Red blood cells of hypercholesterolemic SR-B1-/- mice and SR-B1-/-ApoE-/- mice have an irregular shape, a high cholesterol content, and large intracellular phagolysosomes [46]. SR-B1-deficient mice also show thrombocytopenia, thrombomegalia, increased platelet activation, and greater susceptibility of platelets to aggregate in vivo [47]. HFC diet even pronounces these effects of SR-B1 deficiency, e.g., on erythrocyte and platelet counts, morphology, and function. Taken together, SR-B1 is a multifunctional player in atherosclerosis regulating lipid/cholesterol metabolism and in maintenance of homeostasis of various cell types and their proper functions. Humans carrying SR-BI (SR-B1) polymorphisms [48–55] and ApoE4 variant [56–58] are under greater risk of atherosclerosis and CHD as well as severe COVID-19/related pathologies (suppl. references). Several studies with respect to SR-B1 and ApoE4 genetic variants seem to be partially controversial. One has to consider various factors for the best possible interpretation: from race/gender/age to clinical status/epigenetics (environment-gene interactions)/lifestyle of respective patients/population. Importantly, a population size, single vs. multiparameter analyses, and disease factor (biological) interaction vs. statistical interaction should be taken into account [59].

Apolipoprotein E (ApoE) is a glycoprotein which is synthesized mainly not only by hepatocytes, but also by macrophages and other cell types. ApoE plays a crucial role in metabolism of chylomicrons and very-low-density lipoprotein remnants (reviewed in [56]). There are 3 common ApoE isoforms in humans: ApoE2, ApoE3, and ApoE4 (encoded by ε2, ε3, and ε4 alleles). The two latter isoforms differ by single amino acid substitution at position 112—ApoE3 has a cysteine, whereas ApoE4 has an arginine. The frequency of the six possible phenotypes (E2/E2, E3/E3, E4/E4, E3/E2, E4/E2, E4/E3) varies among the population depending, for example, on the geographical status and the race. Numerous studies refer that the carriers of at least one ε4 allele do have a greater risk of CHD (reviewed/analyzed in [56–58]). Northern scandinavian populations of middle age, specifically Finnish men—living in the east—who possess ε4 allele are under greater risk of CHD [60, 61]. Similarly, a population study in Argentina shows that ε4 allele is associated with an age-dependent and increased risk of atherosclerosis in men [62]. Environmental factors (and epigenetic changes) play more important role as the population gets older. Such genotype-environmental interactions can counteract also some results dependent on gender. CHD risk among the ε4 carriers might be also directly associated with the incidence of Alzheimer disease and is probably dependent on sex [63, 64]. Thus, further research should show whether ApoE4 will become a relevant therapeutic target (reviewed in [65]). More studies are also needed to reveal whether ε4 is a risk factor (together with others) for ischemic stroke worldwide or just in certain regions [66]. In mice, wild-type ApoE displays the properties of the human ApoE3 isoform. In order to mimic the human ApoE4 isoform, Raffai et al. generated a hypomorphic mouse model with arginine at position 61 as an equivalent to human arginine at position 112. The hypomorphic allele was created by homologous recombination of a gene targeting vector with the ApoE locus. The insertion of a neo-cassette into the third intron of the wild-type ApoE allele caused a point mutation (Thr-61→Arg-61) of ApoE. An exchange of Arg-61 for Thr-61 resulted in a domain interaction (between Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain) responsible for preferential binding of ApoE to low-density lipoproteins [67, 68]. Interestingly, the substitution of arginine for threonine resulted in higher susceptibility to atherosclerosis in mice expressing hypomorphic forms of ApoE, although non-hypomorphic mice with single amino acid substitution of ApoE were resistant to diet-induced atherosclerosis [69]. This study again stressed the importance of the domain interaction as a mechanism of proatherogenic nature of Arg-61 hypomorphic ApoE. Hypomorphic ApoE mice express reduced levels of ApoE—approximately 5% of the level of normal ApoE mRNA—in all tissues. Importantly, the lipoprotein cholesterol levels in hypomorphic mice under standard chow are nearly normal. Once the mice are challenged by a diet, they develop hypercholesterolemia which can be reverted by a feeding switch back to standard chow for 3 weeks and by Cre-mediated gene repair [67, 68]. Conditional gene repair—macrophage specific—was also shown to reduce the hypercholesterolemia and atherosclerosis in hypomorphic mice due to re-increased levels of ApoE expression up to 40% [70]. Another investigation of an ApoE hypomorphic mouse model involving Cre-mediated recombination of the hypomorphic allele showed the capacity of ApoE to regulate the turnover of neutral lipids in atherosclerotic plaques in addition to its lowering effect on plasma cholesterol levels [71]. A comparative study of hyperlipidemic LDLR-/- mice with reduced/no ApoE expression revealed a decreased number of leukocytes in the circulation as well as a decreased expression of some adhesion molecules on leukocytes and on the endothelium of the aortic arch in ApoEh/h/LDLR-/- mice in contrast to ApoE-/-LDLR-/- mice [72]. These findings suggest an atheroprotective anti-inflammatory role of ApoE under hyperlipidemic conditions. Taken together, studies in mouse models hypomorphic for ApoE indicate a proatherogenic function of the murine ApoE4-like isoform. Additionally, studies conducted in experimental models expressing reduced levels of ApoE (mutated and wild type) also contribute to the knowledge of antiatherogenic function of ApoE in respective models.

SR-B1-/-ApoE-R61h/h Mouse Model: Experimental Evidence and Discussion

A mouse model deficient in SR-B1 and expressing reduced levels of a hypomorphic mutant form of ApoE, ApoE-R61(h/h), has been introduced in 2005 [27]. Based on the current knowledge of the role of SR-B1 and hypomorphic ApoE-R61 in atherosclerosis, CHD described in this transgenic mouse model is very likely a consequence of interconnected internal and external triggers at various levels as illustrated in Fig. 1. Up to date, original studies on this model are summarized in Suppl. Table S2.

Fig. 1.

Scheme of interconnected internal and external triggers contributing to coronary heart disease in SR-B1-/-ApoE-R61h/h mouse model. CHD is trigerred by HFC diet. Changes at the cellular and metabolic levels are primarily driven by deficiency for SR-B1 and by reduced levels of ApoE-R61. Modification in the diet further changes lipids/cholesterol metabolism and causes the accumulation of lipids/cholesterol in various cell types. Subsequently, those cells possess even more affected morphology and function, further contributing (more or less) to CHD

Atherosclerosis in Mice Fed a Normal Chow Diet

In our experimental setups, we investigated a model deficient in SR-B1 and hypomorphic for ApoE-R61 under a normal (standard) chow diet, high-fat high-cholesterol diet (HFC), and combined HFC diet with a pharmacological intervention [29]. The normal chow diet can cause the development of atherosclerosis. More studies also noticed coronary and aortic atherosclerotic lesions in SR-B1-/-ApoE-R61h/h mice fed a normal diet [29, 73, 74]. Macroscopically, we did not notice atherosclerotic lesions in aortas under standard chow diet, although Nakaoka et al. [73] did. However, we observed atherosclerotic plaques in coronary arteries (Fig. 2). The majority of plaques were classified as low degree stenotic (see also Suppl. Fig. 5 in [29]). We did not observe any intraluminal thrombi in coronary arteries in standard chow fed mice of any age, although few local hemorrhages were noticed possibly indicating vulnerable/healed plaque: Fig. 2, D1–D3: darker red areas associated with cholesterol (also see [5, 75, 76]). Occasionally, we observed small fibrotic areas associated with high-grade stenotic plaques in papillary muscles, but not in other locations. This suggests that standard chow-fed mice, especially aged ones and those with high-grade stenotic and/or occlusive plaques, may suffer from MI/chronic ischemic disease. In this regard, ¹⁸F-FDG-PET imaging (Fig. 3) might be limited as ischemic conditions may lead to an increased uptake of glucose (reviewed in [77, 78]). Ischemic states can be temporarily hidden by rupture-healing cycles and myocardium recovery. This concept is supported by the study showing the effects of diet shortening [73]. Moreover, the authors observed that very few HypoE mice (for nomenclature, see Suppl. Table S2) on the normal chow had died during the experimental period. Their investigation revealed that these mice died due to MI/heart failure rather than from cerebral infarctions [73]. Accordingly, when we analyzed some brain tissues from both normal and HFC groups, there was no sign of lesions/infarctions. In conclusion, the presence of atherosclerotic plaques in mice under standard chow diet raised the possibility that some individuals in experimental setups might be predisposed to more rapid development of CHD after the onset of HFC diet. Future studies may also reveal whether plaque ruptures/thrombosis repetitively occur in mice kept on normal diet. When we analyzed the hearts of SR-B1+/-ApoE-R61h/h mice (at age 30 weeks) on standard chow, no atherosclerotic plaque was found in coronary arteries. This suggests that partial expression of SR-B1 is protective against the development of coronary atherosclerotic plaques in agreement with the studies elucidating the role of attenuated SR-B1 expression in atherosclerosis progression in the aorta [43] or the protective effect of partial SR-B1 expression on atherosclerosis in coronary arteries of SR-B1+/-ApoE-/- mice (mentioned in the study by [79]).

Fig. 2.

Coronary atherosclerosis in standard diet fed SR-B1-/-ApoE-R61h/h mice. Four exemplary atherosclerotic coronary arteries: A, B, C, and D (with atherosclerotic plaques found in septal heart regions) are shown in 4 rows. For each artery, 3 serial sections (H&E stainings on paraffin sections, first to third columns) from serial sectioning along the artery are depicted. Scale corresponds 100 μm for A–C; 10× objective magnification for D (black letter). Histological images (software-based magnification) with detailed characteristics: necrotic core/cholesterol clefts (black #), macrophages (black §), and hemorrhages (black *) are shown in the 5th row (white letter D)

Fig. 3.

HFC diet triggers myocardial infarctions. Short-axis ¹⁸F-FDG-PET images (first and third columns, scale bar: cps/ml) with corresponding Masson Goldner-stained paraffin cross sections (second and fourth columns) of the basal, mid-ventricular, and apical heart regions from 2 exemplary SR-B1-/-ApoE-R61h/h mice under standard diet (left) versus HFC diet (right). Masson Goldner stainings differentiate between viable (orange-red) and post-ischemic/fibrotic (green) myocardium. These areas are clearly matched with regional ¹⁸F-FDG uptake. Physiological ¹⁸F-FDG uptake (1st column) in standard diet fed mouse correlates with histological images (second column, orange-red heart tissue). Areas of decreased ¹⁸F-FDG uptake (third column) in HFC-fed mouse correlate with post-ischemic myocardial damage (green areas in heart sections, fourth column) observed throughout the basal (septal part), midventricular (septal part), and apical regions. The scales refer to all samples in respective column

Atherosclerosis in Mice Fed a High-Fat/High-Cholesterol Diet

SR-B1-/-ApoE-R61h/h mice were reported to develop CHD associated with occlusive coronary atherosclerosis, cardiac dysfunction, and spontaneous death when fed HFC diet [27]. After onset of HFC diet, SR-B1-/-ApoE-R61h/h mice died within 7 weeks. All mice under standard chow survived in this study [27] as well as in our experimental setups, although other authors reported some death cases in the control—normal diet group [73]. The HFC diet resulted in extensive atherosclerosis in the aortic root and coronary arteries [27] and Fig. 4. Atherosclerosis in aortas showed typical hallmarks of advanced lesions with necrotic cores, cholesterol crystals, and foam cells/macrophages (Fig. 4A–E). Furthermore, we observed fused macrophages (see Fig. 4C, D) forming multinucleated giant cells, very likely due to cholate presence in the HFC diet [80]. SR-B1-/-ApoE-R61h/h mice are protected against diet-induced atherosclerosis by restoration of SR-B1 expression in bone marrow (BM)-derived cells. This approach results in attenuated myocardial fibrosis, reduced plasma levels of some inflammatory markers, and reduced monocyte recruitment into atherosclerotic plaques [74]. SR-B1-/-ApoE-R61h/h mice (called modified HypoE mice), in which HFC diet was sustained for limited time (7 days), were proposed to study ischemic cardiomyopathy caused by multiple MI [73]. These mice display cardiac dysfunction and increased survival (36 days as median survival period). The authors also appeal that the appropriate duration of the Paigen diet should be determined each time HypoE mice are imported into a new breeding laboratory (new environmental conditions). Supporting observations were reported also by another group investigating mice fed by Paigen diet for various time points [81]. Furthermore, social isolation of HFC-fed SR-B1-/-ApoE-R61h/h mice led to their decreased survival. In our lab, we also investigated further diet interventions. Two groups of mice were put on HFC diet. The first group was switched back to standard chow 1 week after HFC onset and the second group 2 weeks after HFC onset. The survival of both groups increased when compared to the survival of mice permanently fed by HFC diet. After 28 days from the beginning of experiments, there were 90% survivors in the first group and 45% survivors in the second group. After 60 days from the start of experiments, there were 80% survivors in the first group and 10% survivors in the second group. Interestingly, these data also indicate that—considering a short-term 1-month interval—a pharmacological treatment (aspirin used in our study [29]) set immediately from the beginning of HFC feeding seems to be more efficient than the diet change, especially 2 weeks of HFC feeding appear to be critical. Taken together, our data confirm that HFC diet results in rapid progression of atherosclerosis and CHD. As expected, Western-type diet results in atherosclerotic plaques in coronary arteries in these mice as well (our lab data).

Fig. 4.

HFC diet triggers atherosclerosis in the aorta and coronary arteries in SR-B1-/-ApoE-R61h/h mice. A Representative photo of explanted aorta. B Representative cryosection of the whole aorta stained for lipids: red-colored, oil-red counterstained with haematoxylin, scale corresponds 1 mm. Cryosections of aortic atherosclerotic lesions stained for C lipids: red colored, oil-red counterstained with haematoxylin, scale 100 μm; D macrophages: brown colored, Mac-3 staining, scale 100 μm; and E morphology: H&E staining, scale 100 μm. Yellow circles in C and D indicate multinucleated giant cells. Representative heart cryosections with F semi- and G totally (thrombosed) occluded atherosclerotic coronary arteries stained for lipids: red-colored, oil-red counterstained with haematoxylin, scale corresponds 10 μm

Heart Pathology and Vulnerable Plaques

HFC-fed SR-B1-/-ApoE-R61h/h mice displayed MI and heart dysfunction which was confirmed by histology and echocardiography [27, 29]. Lipid analysis revealed structural abnormalities of some lipoprotein particles in the plasma of hypercholesterolemic HFC-fed SR-B1-/-ApoE-R61h/h mice, but not in HFC-fed control groups [27]. Microvascular dysfunction was shown to contribute to ischemic cardiomyopathy in SR-B1-/-ApoE-R61h/h mice. SR-B1 deficiency primarily caused impairment of the nitric oxide-mediated dilation of microvessels and conductance [82]. Concerning structural changes, the authors described that hypertrophy of cardiomyocytes in the HFC group contributed to the observed cardiomegaly [27]. Typical signs of heart failure such as increased heart weights (see Suppl. Fig. 3 in [29]) and increased lung weights were observed in our experimental setups. The authors of the original study described that the hypertrophy of cardiomyocytes (as stated, e.g., compared to HFC-fed ApoE-R61h/h mice and other controls) is a contributing factor to the observed increase of heart weights of mice on HFC diet [27]. However, our measurements showed no statistically significant difference in diameters of cardiomyocytes between SR-B1-/-ApoE-R61h/h mice on standard chow and HFC diet (Suppl. Fig. S1). There are several possible reasons for heart enlargement. The most likely one is a compromised heart function (as supported by our echocardiographic data, Suppl. Fig. 2 in [29]) due to MI and subsequent heart enlargement due to interstitial edema, immune cell infiltration, and remodeling processes associated with post-infarcted stages. Other factors may involve ventricular hypertrophy caused by an increase in cardiomyocyte number (hypothetically due to HFC diet), but this will require further investigations. In line with the latter, there are some reports about rapid (and reversible) increase (40% within 2 days) in ventricular muscle mass caused by meal in Burmese pythons [83]. Mechanistically, dysfunctional HDL has been shown to be a key player in cardiac pathology hemodynamically measured in SR-B1-deficient and normal diet-fed mice [84]. Furthermore, it is possible that heart enlargement might be partially caused by greater physical demands due to motion and/or by stress factors [81, 85]. Further studies may also reveal if physiologically more active/moving mice—kept under various diets—are protected from CHD/MI or predisposed to more rapid CHD, and/or eventually recover faster after acute MI events. Regarding other pathologies (associated with SR-B1 deficiency), we observed the same features which have been already described, for example, splenomegaly. Moreover, the spleens of SR-B1-/-ApoE-Rh/h mice on HFC diet were significantly (2.2×) larger (823.15 ± 163.53 mg) comparing to the ones of mice on standard chow (371.62 ± 105.02 mg). Splenomegaly can be explained by affection of the maturation and the lifespan of erythrocytes in SR-B1-deficient mice [46], which increases erythropoiesis resp. reticulocytosis leading to organ enlargement. Another potential factor contributing to spleen enlargement SR-B1-/-ApoE-R61h/h mice might be related to monocyte overproduction in response to MI triggered by the HFC diet. Supporting this hypothesis, it was shown that mobilization of the splenic reservoir of monocytes leads to their recruitment to an infarcted area to promote tissue digestion and repair [86]. MI-related increase of the proliferation of progenitor cells in the spleen has been shown as well [87].

Based on the unpredictability and suddenness of MI in SR-B1-/-ApoE-R61h/h mice on HFC diet, we hypothesized that vulnerable plaques are present, and they rupture, trigger sudden events, and finally result in spontaneous deaths. For this purpose, we employed serial ¹⁸F-FDG-PET imaging over time (author’s note: biomarkers might be employed for similar purposes as well) to detect the first newly occurring acute myocardial events in individual SR-B1-/-ApoE-R61h/h mice (example is shown in Fig. 3 and see [29]). Subsequently, we systematically studied the hearts for the presence of coronary atherosclerotic plaques and intraluminal thrombi. We showed for the first time that MI in HFC-fed SR-B1-/-ApoE-R61h/h mice are associated with intraluminal atherothrombosis resulting from ruptures of vulnerable plaques (Figs. 4F, G and 5 and see Figs. 2 and 4 in [29]). Coronary atherosclerotic plaques presented with typical signs of vulnerable plaques: necrotic cores, inflammatory cell infiltrates, cholesterol crystals, and lipids (HFC diet: Figs. 4F, G and 5). The plaques—depending on the stage/diet course—often also displayed hypo-/a-cellular morphology. Importantly, SMA (marker for smooth muscle cells) positivity was markedly reduced in coronary atherosclerotic plaques indicating further characteristics of vulnerable plaques: a thin fibrous cap and limited smooth muscle cell number as well as their migration within such plaques (Suppl. Fig. S2). Naturally, a high number of occlusive atherosclerotic plaques found in HFC-fed mice [29] provokes the question of chronic ischemia in HFC-fed mice (see also [82] and consider various phenotypes of individuals, ischemic cardiomyopathy with or without MI). However, the sudden drop of ¹⁸F-FDG uptake measured by PET and subsequently confirmed by histology rather indicates a loss of viable myocardial tissue in HFC-fed SR-B1-/-ApoE-R61h/h mice. We consider the high-degree stenotic and/or occlusive plaques as contributing factors to the CHD pathology seen in HFC-fed SR-B1-/-ApoE-R61h/h mice. Indeed, in some cases, the occlusive plaques were present also in the same heart walls as the thrombosed ones. These plaques may be symptomatic, but they might be also asymptomatic, and/or newly forming arterial collaterals can rescue the blood supply in related myocardial regions. Even human autoptic studies of sudden death cases revealed a simultaneous presence of high-degree stenotic and/or occlusive plaques in addition to arteries with thrombi which were the primary cause of MI [88]. Interestingly, the authors also described that thrombi were located relatively closely to the ostia of coronary arteries, regardless whether the arteries originated from the right coronary artery or the left coronary artery. This fact also correlates with our observations in HFC-fed SR-B1-/-ApoE-R61h/h mice. We found the majority of thrombi located in proximal heart regions thus corresponding to large and medium size coronary arteries (Fig. 6). Therefore, the septal location of almost 50% of thrombosed arteries might not be surprising as the coronary arteries supplying septum are rather early proximal branches of the right coronary artery, the left coronary artery, or eventually of the aortic sinus itself (for anatomical variations of arteries—in C57BL/6 mouse strains—see [89]). Furthermore, other contributing factors like local flow disturbances in septal arteries might be responsible for more proximal septal predilection site of atherosclerotic plaques and associated thrombi formation. Interestingly, and supporting our observation, 60-week-old ApoE-/- mice on standard chow displayed the highest number of atherosclerotic lesions in septal coronary arteries as well [16]. Other studies also showed that the atherosclerotic plaques of aged (more than 7 months) ApoE-/-LDLR-/- and ApoE-/- mice were found in proximal branches of coronary arteries. Semi- and occlusive plaques were associated with MI, possible plaque disruptions (in few cases, indicated by recanalization in necrotic cores), and increased mortality in those mice [15, 24].

Fig. 5.

Plaque ruptures and thrombi formation in HFC-fed SR-B1-/-ApoE-R61h/h mice. Examples of thrombi found in various myocardial regions of HFC-fed mice: in septum (A–C), lateral wall (D), and right ventricle (E). First column (A–E): heart sections (scale 1 mm) with white frames indicating the respective location of the thrombotic artery (scale 100 μm). Second to fourth columns (A1–A3 to E1–E3): 3 serial cuts of the thrombotic artery. Symbols are used to label lipid-rich core (white #), thrombus (white *), and perivascular inflammation (white §). H&E stainings were performed on paraffin sections

Fig. 6.

The size of atherosclerotic coronary arteries in HFC-fed SR-B1-/-ApoE-R61h/h mice. The arteries with atherosclerotic plaques and thrombi were categorized according to their size. The value of the outer equivalent diameter (OED) was derived from the measurement of outer areas of atherosclerotic arteries which were considered as the safest quantifiable areas. The value of OED determines the diameter of a circle with the same area as the measured object: Eqdia = sqrt (4 × area / π). The measurements (on H&E paraffin sections) were done for 34 low-grade stenotic (< 50% reduction of original luminal area), 36 high-grade stenotic (> 50% reduction of original luminal area), 30 randomly chosen occlusive, and 29 thrombosed arteries

Pharmacological Interventions

This phenotype—HFC diet triggered CHD in SR-B1-/-ApoE-R61h/h mice—is at least partially reverted by pharmacological intervention using aspirin [29]. We investigated the effects of aspirin (acetylsalicylic acid) treatment on CHD in HFC-fed mice. Importantly, an aspirin intervention suppressed atherothrombosis and prevented HFC-fed mice from premature death (Fig. 5 in [29]), thus supporting the hypothesis of vulnerable plaque rupture in SR-B1-/-ApoE-R61h/h mice. However, aspirin treatment did not prevent coronary atherosclerosis and MI/ischemic events in these mice (Fig. 7). The effects of aspirin on CHD are generally assigned to its potential to inhibit platelets (reviewed in [90]). Indeed, our results (Fig. 5 in [29]) confirmed that the aspirin treatment in SR-B1-/-ApoE-R61h/h mice on HFC diet resulted in suppression of plaque-associated atherothrombosis, which was accompanied by increased survival of aspirin-treated mice, and also by decreased number and slower rate of ¹⁸F-FDG defects as assessed by the PET segmental analysis. When comparing HFC-fed SR-B1-/-ApoE-R61h/h aspirin-treated group to the non-treated group, there were no significant differences in atherosclerotic plaques with respect to number and degree of stenosis (Fig. 5 in [29]). Supporting our findings, it was shown that aspirin (itself or in combination with clopidogrel) reduces atherothrombosis up to 50% in ApoE-/- mice with mechanically induced plaque ruptures in the carotid artery [91], but has no effect on atherosclerosis in the carotid artery and aorta. We also have to consider various explantation time in our experimental setups: the hearts of non-treated HFC-fed group were excised after the first observed defect within different time points, whereas the hearts of treated HFC-fed group were excised after 1-month period. This suggests that aspirin might be also atheroprotective in HFC-fed mice, but further experiments are needed with respect to synchronized explantation time-point and to ideally age-matched siblings’ paired mice for treated and non-treated groups in order to minimize inter-individual heterogeneity. If aspirin affects also the atherosclerosis, progression in HFC-fed SR-B1-/-ApoE-R61h/h can thus only be speculated. The studies investigating the aspirin effects on atherosclerosis progression in mice are contradictive. ApoE-/- mice on standard chow followed long-term with high-dose aspirin (0.5mg/mouse/day) treatment responded variably and time dependently [92]. The study indicates also the potential pro-atherogenic effect of aspirin, implying various mechanisms of drug action. Concerning the usage of low-dose aspirin (5mg/kg/day), it has been reported that the drug in LDLR-/- mice on high-fat diet [93] has anti-inflammatory effects and leads to more stabilizing features of atherosclerotic plaques due to collagen deposition and increased number of smooth muscle cells. Similarly, SMA immunohistochemistry indicates that coronary atherosclerotic plaques might be more stable in aspirin-treated mice (Suppl. Fig. S2). Taken together, despite the finding that SR-B1-/-ApoE-R61h/h mice on HFC diet with aspirin treatment suffered from occlusive coronary atherosclerosis and ischemic/infarction events, our data (survival and ¹⁸F-FDG defects) suggest a milder progression of the disease. Furthermore, except its anti-thrombotic effects, aspirin had beneficial effects at the systemic level in SR-B1-/-ApoE-R61h/h: in comparison to non-treated mice, treated mice did not lose weight as rapidly over time as measured simultaneously before the first and the last PET scans: average mean weight difference {first − last scan}: HFC {1.7 g}, HFC + ASA {0.9 g}. We also showed that ASA treatment significantly attenuated HFC-mediated increase in white blood cells [29]. Similarly, pharmacological interventions using an analogue of sphingosine-1-phosphate FTY720 led to systemic immunosuppression and decreased myocardial inflammation in these mice. Applying FTY720 reduced mortality, improved left ventricle function, and reduced infarct size [94, 95]. This agent had no influence on the extent of coronary atherosclerosis nor hypercholesterolemia in HFC-fed SR-B1-/-ApoE-R61h/h mice. Similarly, we observed that aspirin treatment had no influence on the degree of atheroslerosis in HFC-fed mice [29]. In contrast, administration of oral anticoagulant drug rivaroxaban led also to reduced atherosclerosis. Applying this drug increased survival and suppressed cardiomyopathy, further supporting clinical relevance—a recent acute coronary syndrome—of this model [96]. The model was employed to investigate and/or (re)evaluate beneficial versus adverse effects of bioactive compounds (natural products) used as lipid-lowering substances [97], vitamins C and E [98], and red wine grape pomace [99]. SR-B1-/-ApoE-R61h/h model might be valid to study also hyperglycemia-induced plaque thrombosis and MI. Gonzalez et al. investigated the effect of streptozotocin (STZ)-induced hyperglycemia on CHD [30]. The authors did not observed changes in the extent of atherosclerosis in coronary arteries; however, there was an increased accumulation of platelets in atherosclerotic lesions, increased myocardial fibrosis, and reduced survival in hyperglycemic mice. Interestingly, the mice treated with STZ and maintained on a normal chow diet had smaller aortic sinus atherosclerotic plaque sizes [30]. This observation again points out the heterogeneity among the mice (before starting treatments) as some mice kept on normal diet spontaneously develop atherosclerotic lesions [29, 73, 74]. Taken together, various drugs differ in their effects on the degree of atheroslerosis in HFC-fed SR-B1-/-ApoE-R61h/h mice. This can be explained by several factors: experimental setups, various drug doses used, modes of drug action, drug efficiency, receivers‚ response, and heterogeneity among the individuals/experimental groups (age, gender, spontaneous atherosclerotic lesions present in mice kept on normal diet before pharmacological treatments, lab/mouse facility environment).

Fig. 7.

¹⁸F-FDG-PET and histological findings in HFC-fed SR-B1-/-ApoE-R61h/h mice with aspirin treatment. Short-axis ¹⁸F-FDG-PET images (first column, scale bar: cps/ml) and corresponding Masson Goldner-stained cross sections (second to fourth columns) of the basal, mid-ventricular, and apical heart regions from the exemplary mouse on HFC diet with aspirin treatment are shown. Masson Goldner stainings differentiate between viable (orange-red) and post-ischemic/fibrotic (green) myocardium. The areas of post-ischemic myocardial tissue (light green colored) correlate with decreased uptake of ¹⁸F-FDG. Detailed histological images (yellow frames in second column, third and fourth columns) either depict remodeling processes = formation of collagen-based scar (basal region: first row, green parallel collagen fibers) or post-ischemic necrotized myocardium and tissue debris (mid-ventricular region: second row and apical region: third row, green colored). The scales refer to all samples in respective column

Clinical Relevance

In summary, SR-B1-/-ApoE-R61h/h mouse model represents one of the best known and described models of CHD. These mice mimic clinical MI and ischemic disease in all major aspects: from coronary atherosclerosis through vulnerable plaque ruptures leading to thrombus formation/coronary artery occlusion, finally resulting in MI/ischemia. According to the key features of vulnerable plaques [3], the hypothesis of vulnerable plaques in SR-B1-/-ApoE-R61h/h mice is supported by the following facts: (1) majority of thrombosed atherosclerotic plaques located in arteries of larger size, (2) predominant location of thrombosed plaques in more-proximal regions of the coronary tree, (3) lipid- and cholesterol-rich atherosclerotic plaques, (4) the presence of perivascular inflammation, (5) the presence of thrombi communicating with necrotic core of atherosclerotic plaques, (6) clear association with clinical event: MI and spontaneous death, (7) pharmacological eventually diet interventions are able to at least partially revert the clinical features associated with CHD in SR-B1-/-ApoE-R61h/h mice, and (8) other pathologies (such as hyperglycemia) accelerate atherothrombotic CHD. Employing these mice in future studies of vulnerable plaques/thrombi-related pathologies and other investigations remains a challenge with respect to the appropriate timing of the experimental setup, since there is unpredictability and individual heterogeneity among the mice. Biomarkers and/or imaging-guided follow-up of mice represent valid tools to make new discoveries in HFC-fed mice and to explore the potential as well as limits of current methods. Furthermore, short-/long-term monitoring studies based on in vivo imaging and/or on biomarker detection (CHD phase profiling) in mice kept on standard diet may result in new research concepts and help to resolve discussions on human CHD pathology. Also, further intervention studies will be needed to reveal the potential of current and newly developed drugs used in either prevention or treatment of CHD. Crossing these mice—and possibly pathologically comparable dKO mice: SR-B1 combined either with ApoE [26] or LDLR [100, 101]—with other strains for mechanistic/other studies [102, 103] would be demanding. But once the labs work with these strains and with heterozygots for SR-B1, respectively, an efficient work (suppl. references) may lead to more research investigations. Alternatively, SR-BIΔCT/ApoeR61h/h model awaits to participate on further relevant studies.

Author Contribution

AS wrote and submitted the review (an extended doctoral thesis—submitted in 2012 and defended in 2013—which has been updated by recent publications). AS contributed to the presentation and discussion of experimental data design; data generation and collection; and data analyses. Other contributors are those professionals listed in acknowledgements: Michael Kuhlmann, Michael Schaefers, Sven Hermann, and Bodo Levkau.

Funding

The work (material used for the generation of presented data) and the author were mainly supported by doctoral fellowships and associated grants (both 2 + 1 years) of the NRW Research School “Cell Dynamics and Disease” (AS). This and cited work (ref. 29) was partly supported by the Deutsche Forschungsgemeinschaft, Collaborative Research Center SFB 656 “Cardiovascular Molecular Imaging,” the Interdisciplinary Center for Clinical Research, Münster, Germany; a research grant from Siemens Medical Solution, Erlangen, Germany.

Data Availability

All data generated or analyzed during this study are included in this review.

Code Availability

Not applicable.

Declarations

Ethics Approval

All experiments were in accordance with the German Law on the Care and Use of Laboratory Animals and approved by the local authorizing agency of North Rhine-Westphalia (NRW, Germany). No human studies were carried out by the author for this article.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Conflict of Interest

The author declares no competing interests.