Characterization of Atherosclerosis Formation in a Murine Model of Type IIa Human Familial Hypercholesterolemia

A murine genetic model of LDL-cholesterol- (LDL-C-) driven atherosclerosis, based on complete deficiencies of both the LDL-receptor (Ldlr−/−) and key catalytic component of an apolipoprotein B-edisome complex (Apobec1−/−), which converts apoB-100 to apoB-48, has been extensively characterized. These gene deficiencies allow high levels of apoB-100 to be present and inefficiently cleared, thus leading to very high levels of LDL-C in mice on a normal diet. Many key features of atherosclerotic plaques observed in human familial hypercholesterolemia are found in these mice as they are allowed to age through 72 weeks. The general characteristics include the presence of high levels of LDL-C in plasma and macrophage-related fatty streak formation in the aortic tree, which progressively worsens with age. More specifically, plaque found in the aortic sinuses contains a lipid core with relatively high numbers of macrophages and a smooth muscle cell α-actin- and collagen-containing cap, which thins with age. These critical features of plaque progression suggest that the Ldlr−/−/Apobec1−/− mouse line presents a superior model of LDL-C-driven atherosclerosis.


Introduction
Atherosclerosis is a self-sustaining inflammatory fibroproliferative disease that progresses in discrete stages and involves a number of cell types and effector molecules [1,2]. Lipid metabolic disorders, principally those dyslipidemias that lead to high LDL and triglyceride levels and/or low HDL levels, are heavily involved in the genesis and progression of atherosclerosis in humans, and thrombotic complications substantially contribute to the coronary end-stage arterial disease that sometimes accompanies atherosclerosis. Because of genetic differences in lipid metabolism between mice and humans, LDL and its precursors, VLDLs and IDLs, are rapidly cleared in mice. Thus, differing from humans, the cholesterol present in murine plasma is mostly carried in the artheroprotective HDL fraction. As a result, wild-type (WT) mice are more resistant than humans to dietary-induced elevation of LDL and, thus, useful metabolic murine models of human atherosclerosis were not prevalent until recent advances in in vivo gene targeting methods. Application of this technology then allowed generation of genetic strains of mice that possess some characteristics of lipid metabolism of humans.
While accelerated injury-and transplant-based atherosclerosis, as well as spontaneous genetic models of Type II familial hypercholesterolemia (FH), the most frequent type of FH observed in humans that leads to atherosclerosis, are continually emerging, the most widely studied murine models of FH-mediated human atherosclerosis are the low density lipoprotein receptor-deficient (Ldlr −/− ) [3] and apoE deficient (Apoe −/− ) [4][5][6] mouse lines. In the former case, by allowing LDL elevation in plasma via elimination of the apoB-100 receptors, Ldlr −/− mice provide a potential model of atherosclerosis elevated LDL-containing cholesterol (LDL-C) levels. However, differing from the human disease, these 2 BioMed Research International mice, when placed on a normal chow diet, present with only 2-fold elevated cholesterol concentrations and develop atherosclerosis very slowly [3]. High-fat, high-cholesterol diets are required for development of aortic atherosclerotic lesions in these mice [7] although VLDL-C not LDL-C is extremely elevated in these diets. With regard to the Apoe −/− mouse strain, these mice display very high levels of plasma cholesterol and severe atherosclerosis, which is enhanced by a high-cholesterol diet [5]. However, the cholesterol is mainly associated with the VLDL and IDL lipoprotein fractions, not the LDL fraction, as in the case of humans. Thus, while nonetheless very valuable, neither of these models closely reflects the lipid profiles of the disease in humans. This is, in part, due to the presence of an enzyme in mouse liver, apobec-1, a RNA-specific cytidine deaminase. This enzyme is a catalytic component of an edisome that alters the apoB-100-encoding mRNA to an mRNA coding for apoB-48. Incorporation of apoB-48 into VLDL initiates its rapid clearance by scavenger receptors prior to conversion of VLDL into LDL particles. This editing, while also occurring in human intestines, does not take place in human liver, and its occurrence in mouse liver results in resistance to elevation in LDL in mice.
VLDL-C driven atherosclerotic plaques in Apoe −/− and Ldlr −/− mice are lipid-rich and abundant with the presence of foam cells converted from macrophages [8] and contain thinner fibrous cap and extremely small extracellular matrices [9]. In contrast, mainly LDL-C driven human arteriosclerotic plaques are extracellular matrix-rich and abundant with the presence of smooth muscle cells, and they are erosion-prone and vulnerable [10]. It is not known whether such differences are due to species or cholesterol profile.
Because of the limited utility of the Ldlr −/− and Apoe −/− mouse lines in terms of lipid metabolism, other murine models have been developed that have focused on elevation of apoB-100 levels in mice. Among these models are the apoB-100 transgenic strain ((Tg)APOB100 +/+ ) [11] and mice with a combined (Tg)APOB100 +/+ /Ldlr −/− genotype [12]. While this latter model possesses important advantages in lipid metabolism, it should be emphasized that introducing other gene modifications to this line is difficult because the APOB100 loci are not fixed. A potentially important mouse model of human atherosclerosis with elevated LDL-C is one in which both the Ldlr and Apobec1 genes are deleted (Ldlr −/− /Apobec1 −/− , hereafter referred to as L −/− /A −/− ), since these mice lack the ability to convert apoB-100 to apoB-48 in liver and also are defective in LDL clearance. These doubly deficient mice exhibit high levels of apoB-100-LDL-C, more closely mirroring the plasma lipid profiles in human Type II FH [13], and slowly and progressively present with severe spontaneous atherosclerosis on a normal chow diet. Thus, a good model is potentially available to further test the evolution of the atheroma and its in vivo relationships with specific proteins. While some initial characterizations of this murine model have been published, a thorough analysis of plaque development and progression is warranted. The current manuscript presents results of an investigation in which the nature of the plaque has been characterized over the majority of the lifespan of the L −/− /A −/− mouse.

2.1.
Mice. L −/− /A −/− mice have been initially described [13,14]. These mice were back-crossed to C57Bl6/J mice (Jackson Laboratory, Bar Harbor, ME) for at least seven generations before cross-breeding. Each genotype was determined using PCR analysis with genomic DNA from ear punch biopsy. Male mice were used in all experiments. The mice were maintained on a low-fat diet for 12, 18, 24, 36, 48, 60, and 72 weeks. Mice fasted at least 6 h and sacrificed at each time point using overinhalation of isoflurane, and then their blood was obtained with sodium citrate or heparin as an anticoagulant. Their hearts and whole aortic trees were removed for morphometric analyses after perfusion with isotonic saline. All animal experiments described herein were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Notre Dame and Hamamatsu University School of Medicine.

Lipid
Analysis of Whole Plasma. Plasma was separated from whole citrated blood and used for the measurement of total cholesterol and triglycerides employing the cholesterol CII (Wako Chemicals, Richmond, VA) and GPO TRINDER kits (Sigma Diagnostics, St. Louis, MO), respectively. For the assay, a volume of 2 L of plasma was mixed with 100 L of the assay reagent from each kit and incubated at 37 ∘ C for 30 min. The absorbancies at 500 nm and 540 nm were obtained for total cholesterol and triglyceride, respectively, and calculated using the standards present in each kit. These procedures were as described in our previous report [15].

FPLC Analysis of Whole Plasma.
A volume of 100 L of plasma was analyzed by FPLC, using gel filtration on Superose 6 HR resin (Amersham Pharmacia Biotech, Piscataway, NJ). The samples were eluted at a flow rate of 0.5 mL/min with a column equilibration buffer, namely, 10 mM Tris-HCl/0.15 M NaCl/0.01% (w/v) EDTA, pH 7.4, as previously described [6]. Column volumes (500 L) were collected (36 fractions) and a 50 L aliquot from each tube was added to 100 L of cholesterol CII reagent for the determination of the cholesterol concentration in each fraction. These procedures were as described in our previous report [15].

Analysis of Atherosclerotic Lesions of Whole Aortic Trees.
After perfusion of the mice, the appearance of the aortic arch was photographed, and then the aortas were exposed and cut longitudinally, in situ, exposing the lumen. Whole aortic trees were removed and placed on 150 m gapped glass slides, with the lumen-side up. A glass slide was then placed over the lumens of the aortas to hold them in place during fixation. The aortas were then fixed in 10% normal buffered formalin for 16 hr at room temperature. They were rinsed with H 2 O and stained with Sudan IV (Sigma) solution (38% 2-propanol with supersaturated Sudan IV) for 16 hr at 4 ∘ C. A digital camera was used to capture the whole image and the total number of pixels for whole aortas and plaque areas BioMed Research International 3 were measured using Adobe Photoshop 7.0, thus allowing calculation of the % of the total surface area of plaque in the aortas. These procedures were as described in our previous report [15].

Sections of Hearts.
Hearts were cut at the level of the lower edge of the atrium and the lower region containing the aortic valve was fixed with periodate-lysineparaformaldehyde (PLP) for 16 h at 4 ∘ C. After fixation, some samples were directly embedded in Tissue-Tec OCT compound (Sakura Fine Tec, Torrance, CA), and others were processed and embedded in paraffin. A total of 30 serial sections (from #1 to #30) were obtained at a 4 m thickness from the aortic valve towards the ascending aorta. These procedures were as described in our previous report [15].

Histochemistry for Analysis of Plaque Progression in Aortic
Sinus. Serial sections (#1, #11, and #21) were stained with hematoxylin II and eosin Y (H&E) (Richard Allen Scientific, Kalamazoo, MI) for morphometric analysis. Serial sections (#2, #12, and #22) were stained with Oil Red-O (Sigma) for detecting lipid accumulation in the plaque. For the determination of the size of plaques in aortic sinuses, these images were captured and calculated as above, and the number of pixels was converted to m 2 with a proper reference measurement (hemocytometer grid). Serial sections (#3, #13, and #23) were used for Masson's trichrome stain to identify collagen accumulation in the plaque. These procedures were as described in our previous report [15].

2.7.
Immunohistochemistry. All slides were deparaffinized and then blocked with avidin block, biotin block, and Peroxoblock (Zymed Laboratories, South San Francisco, CA), before incubating in specific antibodies. For fibrin(ogen) staining, the slides were blocked only with Peroxo-block. These following procedures were as described in our previous report [15].

Results
It is known that female mice are more vulnerable to atherosclerosis than male mice due to sex steroid hormone fluctuations [16,17]. Thus, the progression of spontaneous atherosclerosis in only male mice maintained on a low-fat diet has been assessed. The distribution of cholesterol in the various lipoprotein fractions, as determined from FPLC profiles of the type shown in Figure 1, for mice aged up to 72 weeks, is listed in Table 1 (Figure 3(a)) shows that lipid-containing plaque forms initially in the aortic sinuses and then spreads throughout the entire aortic trees. The percentage of the trees containing plaque progresses from approximately 2% in mice 12 weeks of age to approximately 60% in 72 weeks (Figure 3(b)), thus showing the extensive nature of plaque deposition in these mice. Aortic sinuses have been microsectioned from L −/− /A −/− mice. The sizes of the plaques in the aortic sinuses of L −/− /A −/− mice were measured on 3 equally spaced H&E stained sections from each mouse aortic sinus. The data show that plaque continues to increase throughout the lifespans of the mice (Figure 3(c)). No significant plaque was found in Wt mice fed the same diet as young mice (12 weeks of age).     (Figure 4(a)). At 18 weeks of age, H&E staining shows a diffuse thickening of the neointima that is primarily due to foam cells and focal acellular areas above the media (Figure 4(b)). At 24 weeks of age, H&E staining demonstrates a well-formed fibrous cap overlaying a foam cell laden core. Arterial wall thickening, with collagen development and stretching of lamella (Figure 4 Figure 7. At 24 weeks of age, anti-CD31 immunostaining (Figure 7(a)) indicates that an intact endothelium separates the lumen from the fibrous cap. At 36 weeks of age, anti-CD31 immunostains (Figure 7(b)) reveal an intact endothelium separating the lumen from the lesion. At 48 weeks of age, anti-CD31 immunostaining (Figure 7(c)) reveals multiple focal breakpoints within the endothelial layer. At 60 weeks of age, anti-CD31 immunostaining (Figure 7(d)) indicates the presence of focal rounded areas in the endothelium, with a small breakpoint. There are bulging endothelial regions with underlying foam cells.
Antifibrin(ogen) immunostaining of sectioned aortic sinuses from L −/− /A −/− mice at various ages are presented in Figure 8. At 24 weeks of age, antifibrinogen immunostains (Figure 8(a)) indicate that diffuse fibrin deposits are present in the underlying endothelial spaces and at the base of the lesion above the media. At 36 weeks of age, antifibrinogen immunostains (Figure 8(b)) demonstrate variable fibrin(ogen) in the underlying endothelial region, as well as some focal, but faint, positive areas associated with the endothelium, and in the lipid core above the media. At 48 weeks of age, antifibrin(ogen) immunostaining (Figure 8(c)) demonstrates that the underlying endothelial region is abundant in fibrin deposits. Patchy areas of fibrin are also associated with the endothelium and fibrin is also identified at breakpoints on the endothelium. Diffuse areas of fibrin are evident at the base of the lipid core above the media. At 60 weeks of age, antifibrin(ogen) immunostaining (Figure 8(d)) demonstrates fibrin deposition scattered throughout the underlying endothelium and core region. Antimacrophage immunostaining of sectioned aortic sinuses from L −/− /A −/− mice at various ages is presented in Figure 9. At 24 weeks of age, the antimacrophage immunostains (Figure 9(a)) demonstrate that the core consists predominantly of macrophages. At 36 weeks of age, antimacrophage immunostains (Figure 9(b)) show that the cap now consists of scattered macrophages. At the broadest area of the cap, the macrophages appear as foam cells adjacent to the core. At 48 weeks of age, antimacrophage immunostaining (Figure 9(c)) shows macrophages within the thinned cap in the subendothelium. Some are associated with breakpoints of the endothelium underlying some faintly positive foam cells. At 60 weeks of age, antimacrophage immunostaining (Figure 9(d)) reveals small clusters of macrophages in the endothelial and subendothelial areas.
Anti-SMA immunostaining of sectioned aortic sinuses from L −/− /A −/− mice at various ages are presented in Figure 10. At 24 weeks of age, anti-SMA immunostaining (Figure 10(a)) reveals a cellular multilayered SMC -actinpositive region associated with the fibrous cap, as well as with the positive medial compartment. However, at the base of the lipid core, within the media, an area devoid of positive cells is evident, with a faintly positive region immediately underlying the core. Furthermore, several SMemb-positive cells are identified in the core associated with an area devoid of SMA positive cells (Figure 10(b)). At 36 weeks of age, anti-SMA immunostains (Figure 10(c)) indicate numerous single layered positive cells at the endothelium. They consist of weak to highly positive SMA foci among other negative cells. The medial compartments of the arterial wall are populated with both SMA positive and negative cells. The inner medial compartment (below the lipid core) appears thickened and is mainly negative for SMA. Furthermore, several diffuse SMemb-positive foci are identified within the broad end of the foam cell-containing cap (Figure 10(d)). Additional foam cell clusters within the core are also positive. These areas within the core were negative for SMA. At 48 weeks of age, the anti-SMA immunostained pattern (Figure 10 Figure 11. H&E stains at two different depths (Figures 11(a) and 11(b)) show sections covered (Figure 11(a)) and noncovered (Figure 11(b)) by plaque. The plaque contains an irregular cap with many broken areas (Figure 11(c)). Diffuse fibrin is also present in the remaining part of the plaque core (Figures 11(e) and 11(f)) and dense fibrin deposits are observed on the cap, which colocalize with ruptured areas identified from endothelial cell staining (Figure 11(e)). A small number of SMA positive cells are observed in the cap area covered by plaque (Figure 11(g)), but the aortic wall is heavily stained with SMA positive SMC cells at a depth not covered by plaque (Figure 11(h)). SMemb-positive cells were detected in the cap area of the plaque and light staining is also in the aortic walls (Figure 11(i)). No expression of SMemb is observed in aortic wall areas in sections at a depth where plaque does not cover the wall (Figure 11(j)).

Discussion
Atherosclerosis in humans begins focally in lesion-prone vascular areas where blood flow is compromised [18]. This reduced flow then facilitates recruitment of monocytes to intimal locations, via their interaction with cell adhesion molecules (CAMs), followed by monocyte differentiation to macrophages, which subsequently become foam cells. This disease then progresses in a series of AHA-defined classifications, from simple Type I to advanced Type VI lesions [19]. Complications of atherosclerosis include thinning and rupture of unstable plaques and aneurysm. Rupture of the plaque leads to thrombotic disease and possibly sudden death. Certain characteristics of plaques, including the size and composition of the lipid core, the structure and composition of the fibrous cap, apoptosis and/or dedifferentiation of collagen-synthesizing SMCs, and/or the presence of a local inflammatory process, predispose the plaque to disruption [20,21]. Plaque rupture is frequently observed in calcified plaques [22]. The nature and level of plasma lipoproteins, especially of HDL-C, are of predictive significance in the risk of coronary artery [23] and plasma HDL-C has been found to be a potent inverse risk factor in a variety of clinical endpoints of this disease [23][24][25]. On the other hand, high LDL levels are associated with risk of cardiovascular disease [26], mainly through formation of its oxidized product(s) [27]. oxLDL in the vessel wall originates from mild oxidation of vascular LDL [28] and circulating oxLDL stimulates vascular monocyte/macrophage infiltration [29], as well as vascular EC [30] and SMC [31,32] migration and proliferation. Thus, it has become clear that a variety of genes, including those that influence lipid and carbohydrate metabolism, those that influence the hemodynamic state of the organism, those that mediate many phases of the inflammatory response, and those that affect hemostasis, can affect the development and progression of atherosclerosis [33], and murine models of atherosclerosis are extensively employed to investigate the effects of gene alterations on the characteristics of this disease.
In this investigation, we have employed a mouse strain that contains a total combined deficiency of the Apobec1 and Ldlr genes in order to both eliminate the liver production of apoB-48 and inhibit clearance of the produced apoB-100-containing particle. This has led to a lipid profile in these mice, which is very similar to that of atherosclerotic prone humans with familial hypercholesteremia, wherein the very high levels of cholesterol reside in LDL particles. These excessive LDL-C levels then predispose these mice, even on low fat, low cholesterol diets, to spontaneous development of atherosclerostic lesions, similar to the human condition. Whereas this mouse model has been introduced in a previous study [13], a systematic characterization of the lesions that develop has not been offered.
The lesions that spontaneously develop in −/− / −/− mice progressively worsen with time and begin as fatty streaks in the proximal aortic regions as early as 12 weeks of age. These lesions spread to distal regions and, by 72 weeks of age, occupy >60% of the entire arterial tree. Intimal thickening occurs with foam cells and collagen-producing SMCs, which then progresses to a fibrous cap containing atheroma, overlaying a well-established necrotic core. The fibrous cap then thins and the core becomes calcified. We have only sporadically observed mice that experience sudden death, despite clear evidence that the cap progressively thins, loses collagenproducing SMCs, perhaps through dedifferentiation, and gains macrophages, which may degrade the stabilizing collagen within the cap. This may indicate that cap rupture is not a feature of this model, a usual situation for murine models of atherosclerosis, but evidence for cap erosion is present in this model. WHHL (Watanabe Heritable Hyperlipidemic) and WHHLMI (WHHL Myocardial Infarction) rabbits are used as arteriosclerosis model animals. The function of LDLr is impaired in these rabbits due to spontaneously occurring mutations in Ldlr. Moreover, Apobec1 is not expressed in rabbit liver; thus, the majority of cholesterol is packaged in LDL fractions similar to humans [34]. However, a small number of cap ruptures are observed in these rabbit models. Cap rupture and/or cap erosion are asymptomatic without thrombotic complications even in humans. While these animals exhibit cap rupture and cap erosion, it is very hard technically to detect sudden death due to thrombotic complications. Previously, we reported that prothrombin times and activated partial thromboplastin times were shorter in −/− / −/− mice than Wt mice. Moreover, the platelets and von Willebrand factors in −/− / −/− mice were more activated [15]. However, considering the average lifespan of −/− / −/− mice is almost equal to that of Wt mice, these mice might not expire due to thrombotic complications following cap rupture and/or cap erosion, and the severity of thrombotic complications in humans might be much worse than that in other animals.

Conclusions
In conclusion, the L −/− /A −/− model is very suitable for the study of features of LDL-C driven spontaneous atherosclerosis in humans. It offers many advantages over the Apoe −/− and L −/− models in terms of generation of the initial stages of the disease and also appears to incorporate many of the progressive features of the human disease, beginning with fatty streaks, developing to a clear human stage IV atheroma and then to a thin cap fibrous atheroma. While this model is ideal for investigation of genetic influences of a variety of pathways on atherosclerosis development and progression, generation of mice with further deficiencies is a more complex and timeconsuming process. Despite this, we have successfully developed desirable strains containing additional gene deficiencies. In our previous report, some of the serum parameters other than factors related to coagulation and fibrinolysis in L −/− /A −/− mice were reported [15]. Global assessment of this model other than functions of any specific genes integrated also will be the subject of future communications.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
No conflicts of interest are declared in this report.