Cellular and Molecular Mechanisms of Atherosclerosis with Mouse Models

Cellular and Molecular Mechanisms of Atherosclerosis with Mouse Models Ryuji Ohashi, Hong Mu, Qizhi Yao, and Changyi Chen*

for 10 weeks. Although C57BL/6 mice were most susceptible to the development of diet-induced atherosclerosis, the lesions were restricted to the aortic root. Therefore, the use of this model gradually diminished as genetically engineered mice exhibiting larger lesions were induced (discussed below). 

Recently, there has been an explosion in the number of in vivo studies using genetically engineered mouse models. Atherosclerosis research using mice began with the invention of traditional atherosclerotic mice including low-density lipoprotein receptor knockout (LDLR / ) and apolipoprotein E knockout (apoE / ) mice, which provided tremendous progress in atherosclerosis research. Since then, a number of modified atherosclerotic mouse models have been reported to generate lesions that more closely characterize human atherosclerotic lesions. Those modifications include inflammation, hypertension, proteinases and extracellular matrix, glucose metabolism, and immune systems. This article focuses on various kinds of mouse models with atherosclerosis and their contributions to the current advances of research. (Trends Cardiovasc Med 2004;14:187–190) n 2004, Elsevier Inc.

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the intimal layer of arteries. Following endothelial injury, the lesion develops to a fatty streak consisting mainly of lipid-laden macrophages and progresses to atherosclerosis silently over many years before the disease is manifested by rupture or erosion of the lesion. In the last few decades, a substantial number of atherosclerosis studies have been carried out in different animal models. Recently, there

Ryuji Ohashi, Hong Mu, Qizhi Yao, and Changyi Chen are at the Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine and The Methodist Hospital, Houston, Texas, USA. * Address correspondence to: Changyi (Johnny) Chen, MD, PhD, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, NAB 2010, Surgery, Houston, TX 77030, USA. Tel.: (+1) 713798-4401; fax: (+1) 713-798-6633; e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter

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has been an explosion in the number of in vivo studies using genetically engineered mouse models. This may have been due to the advantages offered by the availability of numerous inbred strains, new genetic information, and new biologic tools and resources, as well as to the small size of mice, which facilitates the use of a large number of animals at a time. This review summarizes the use of mouse models in atherosclerosis research and further focuses on the updates of the most commonly used mouse models and related molecular and cellular mechanisms. 

Diet-Induced Atherosclerosis in Mice

Atherosclerotic mice were first reported by Thompson (1969). A lesion was induced in C57BL/6J inbred mice by feeding them a 50% fat-containing diet for 5 weeks instead of a regular diet containing 5%. However, this diet had a disadvantage of high mortality rates. Later on, Paigen et al. (1985) invented a 15% fat-containing diet, which gave notable atherosclerosis and lower mortality rate after feeding C57BL/6J mice

Gene-Targeted Models in Mice

In gene-targeted mice, a specific allele is deleted that permits definition of a protein with very high specificity. The most widely used atherosclerotic mice were generated by modulating apolipoprotein E (apoE) and low-density lipoprotein receptor (LDLR) genes, both of which are pivotal for lipid metabolism. ApoE is an important ligand for the uptake of lipoproteins by many receptors in the LDLR gene family, and deficiency of apoE leads to the accumulation of cholesterol ester-enriched particles. Mice deficient in apoE (apoE / ) were first produced in 1992 (Plump et al. 1992). These mice, which develop severe atherosclerosis on a 4.5% fat-containing diet, shortly became a powerful tool in atherosclerosis research. The arterial cell types seen in these mice are similar to those seen in human atherosclerotic lesions: macrophages, T cells, and smooth muscle cells (Nakashima et al. 1994). Unlike diet-induced atherosclerotic mice, the lesions of apoE / mice form throughout the arterial tree, with sites of predilection at the aortic root. When apoE / mice are fed a ‘‘Western diet,’’ which approximates the human fatty fast food diet, they develop fatty streaks and fibroproliferative lesions sooner than on regular chow diet (Nakashima et al. 1994). A major shortcoming of apoE / mice is that their lipoprotein profiles are dissimilar from those of most human subjects with atherosclerosis. In these mice, most plasma cholesterol is carried in very low-density lipoprotein (VLDL), rather than in LDL as in humans. Deficiency of LDLRs causes pronounced hypercholesterolemia with cardiovascular complications in humans. In 1993, LDLR / mice were created by gene targeting of embryonic stem cells (Ishibashi et al. 1993). These mice showed a 2-fold increase in total cholesterol levels due to high LDL and VLDL levels when fed with a 10% fat diet (Ishibashi et al. 1993). Although LDLR /

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mice on a regular diet do not succumb to atherosclerosis, this model should be distinguished from the diet-induced atherosclerotic model, because the lesion in LDLR / mice is more extensive than is that in C57BL/6 mice on a Western diet (Masucci-Magoulas et al. 1997). Furthermore, plasma lipid profiles of LDLR / mice are more similar to those in human hyperlipidemia than those of apoE / mice. Following invention of apoE / and LDLR / mice, various genetic modifications have been performed to obtain phenotypes that resemble human atherosclerotic lesions. Although a number of atherosclerotic models have been generated, many attempts have also been made to reverse the lesions in order to explore potential therapies for humans. 

Inflammation

In the early stage of atherosclerosis, macrophages infiltrate vessel walls due to endothelial dysfunction. To elucidate the role of macrophages, their infiltration was blocked. Osteopetrotic (op) mice have a mutation in the gene for macrophage colony stimulating factor, resulting in a reduction of the number of macrophages. ApoE / mice crossbred with op mice develop much smaller lesions than do control apoE / mice (Smith et al. 1995). Monocyte chemoattractant protein-1 (MCP-1) plays a major role in recruitment of macrophages, and the absence of MCP-1 can decrease the lesions in atherosclerotic mice (Dawson et al. 1999). On the other hand, overexpression of MCP-1 generates more extensive atherosclerotic lesions (Aiello et al. 1999). Leukotriene B4 (LTB4) is also a potent chemotactic agent that activates monocytes through the LTB4 receptor (BLTR). BLTR blockade can slow atherosclerotic progression by inhibiting monocyte recruitment in both LDLR / and apoE / mice (Aiello et al. 2002). Interferon g (IFN-g) is known to stimulate the expression of proatherogenic molecules such as intracellular adhesion molecule 1, and decreases antiatherogenic effects by upregulating the expression of lipoprotein receptors on macrophages. Mice with a combined deficiency of IFN-g and apoE exhibit a substantial reduction in lipid accumulation in arteries, presumably resulting

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from an increase of atheroprotective phospholipid/apoA-IV-rich particles (Gupta et al. 1997). Although the mechanism of IFN-g action remains to be elucidated, it is evident that IFN-g promotes atherosclerosis. Tumor necrosis factor a (TNF-a) can alter lipid metabolisms by decreasing the activity of lipoprotein lipase (Feingold and Grunfeld 1992). However, C57BL/6 mice lacking the TNF receptor p55 developed aortic sinus fatty streak lesions larger than those in wild-type mice (Schreyer et al. 1996), indicating that TNF p55 receptors protect against atherosclerotic lesions. This report is contrary to a proatherogenic role of TNF-a. 

Proteinases and Extracellular Matrix

When atherosclerotic plaques become unstable, they may rupture and trigger the onset of systemic cardiovascular complications. The unstable plaques are characterized by substantial loss in vascular smooth muscle cells and collagen accumulation, leading to fibrous cap disruption. Transforming growth factor h (TGF-h) is an anti-inflammatory cytokine that also has a protective role against the development of atherosclerotic plaques (Mallat et al. 2001). A recent study (Gojova et al. 2003) revealed that mice expressing dominant-negative TGF-h showed increased vascular inflammation along with a decrease in smooth muscle cells and collagen content, a plaque phenotype that is potentially vulnerable to rupture. This finding indicates a key role of TGF-h in stabilizing atherosclerotic plaque. Mice lacking the matrix GLA protein (Mgp / ) show spontaneous calcification of all elastic and muscular arteries (Luo et al. 1997). The mice usually die within 2 months as a result of rupture of the aorta without atherosclerotic plaque formation. Plasminogen (Plg) is a proenzyme that is converted to its active form, plasmin, by physiologic activators, and is involved in the degradation of extracellular matrix. Therefore, it seems reasonable that the absence of Plg greatly increases the size of atherosclerotic lesions and infiltration of foamy cells in apoE / and Plg / mice (Xiao et al. 1997). In contrast, Plg activator inhibitor 1 (PAI-1) is another member of the fibrin/fibrinolytic pathway and functions

to convert Plg to plasmin. Interestingly, neither the absence of PAI-1 nor its overexpression by the transgene affects lesion development of atherosclerosis, possibly because alternative inhibitors of Plg activators may exist in mice (Sjoland et al. 2000). Matrix metalloproteinases (MMPs) degrade extracellular matrix (ECM) and play a pivotal role in atherosclerosis by balancing between proteolytic and antiproteolytic activities. MMP-3 null mice have more extensive atherosclerosis and collagen accumulation in the lesions, but less frequent aneurysms (Silence et al. 2001). Conversely, transgenic mice overexpressing human MMP-1 have decreased atherosclerotic lesions due to accelerated digestion of febrile collagens of neointimal ECM (Lemaitre et al. 2001). 

Immune Modulation

In atherosclerotic lesions, infiltrates mostly consist of macrophages and T cells, suggesting that the immune system may be involved in the lesion formation. Thus, a large number of mouse models have been generated by modulating the immune system. The importance of T cells in atherogenesis has been emphasized recently. One study (Emeson et al. 1996) demonstrated that CD4+ and CD8+ cell depletion reduced fatty streak formation in C57BL/6 mice, indicating that T cells aggravate fatty streak formation. If CD4+ cells are transferred from immunocompetent mice to immunodeficient apoE / mice, the atherosclerotic lesions increase drastically, suggesting that CD4+ cells may be responsible for development of atherosclerosis (Zhou et al. 2001). Cytotoxic CD8+ T cells can induce apoptosis in vascular smooth muscle cells in apoE / mice, indicating a contribution of CD8+ T cells to atherogenesis (Ludewig et al. 2000). Furthermore, MHC-I- or MHC-II-deficient mice show exacerbated lesions compared with normal mice (Fyfe et al. 1994). Recombinase activator gene-2 deficient (RAG2 / ) mice have a total deficiency of B and T lymphocytes (Daugherty et al. 1997). Despite the defect, atherosclerotic plaque size of RAG-2 / mice is comparable with that in an immunocompetent group, suggesting that the absence of autoantibodies and T cells does not influence the atherosclerotic lesions. TCM Vol. 14, No. 5, 2004



Glucose Metabolism

Diabetes is a risk factor for cardiovascular disease, but the mechanisms involved are poorly understood. Work using steptozotocin-treated mice, which spontaneously become diabetic, has shed light on a mechanism of hyperglycemic vascular injury (Vischer 1999). Advanced glycation end products (AGEs)—by-products of hyperglycemia—interact with the receptor for AGEs (RAGE), and induce several inflammatory markers that could increase atherosclerosis (Schmidt et al. 1996, Vischer 1999). Atherosclerotic lesions are seen in streptozotocin-treated diabetic apoE / mice, but these lesions are suppressed by daily injection of soluble forms of RAGEs, which inhibit actions of AGEs (Park et al. 1998, Tse et al. 1999). These findings indicate a strong link between AGEs and diabetes-related atherosclerosis (Kunjathoor et al. 1996). Controversially, there is a report that diabetes can be an antiatherogenic factor. Lyngdorf et al. (2003) induced obesity and diabetes in apoE / mice by injecting goldthioglucose (GTG), which destroys the hypothalamic satiety center. Unexpectedly, GTG-injected apoE / mice developed less atherosclerosis than did lean, nondiabetic, control, apoE / mice. This finding questions the theory that diabetes is atherogenic, and further studies should be done to solve this paradox. Clinically, patients with type I and type II diabetes have different pathophysiologies and cardiovascular complications (Fisher 2004). However, the underlying mechanisms of these differences are not understood. There is limited information of a differential impact of type I and type II diabetes on atherosclerosis using mouse models, (Hattori et al. 1986, Mathews et al. 2004). Thus, this is an important area warranting future investigation in order to elucidate human disease.



Hypertension

Hypertension is believed to be a cause of atherosclerosis, and endothelial nitric oxide synthase (eNOS) plays a major role in controlling blood pressure-relaxing vasculature by producing NO. Indeed, mice that lack eNOS gene are known TCM Vol. 14, No. 5, 2004

to exhibit hypertension (Shesely et al. 1996). When eNOS / mice are crossbred with apoE / mice, the offspring become hypertensive and also show atherosclerotic lesions (Knowles et al. 2000). However, some studies have shown that eNOS or NO may have proatherosclerotic properties by generating superoxide anions, which could oxidize LDL and promote atherosclerosis (O’Donnell and Freeman 2001). A recent article (Shi et al. 2002) demonstrated that the offspring of eNOS / mice cross-bred with apoE / mice developed much smaller aortic lesions than did apoE / control mice. These studies indicate that the precise roles of eNOS and NO in atherosclerosis still remain uncertain, and additional work is needed in this area. Angiotensin-converting enzyme (ACE) plays a central role in the renin & angiotensin system through generation of the peptide angiotensin II (Ang II) from its inactive precursor Ang I. Ang II accelerates atherosclerosis by stimulating smooth muscle cell proliferation and cholesterol accumulation in arterial macrophages (Daugherty et al. 2000). When ACE knockout mice are cross-bred with apoE / mice, the offspring demonstrates a marked reduction of atherosclerotic lesions and oxidative stress as compared with apoE / and ACE+/+mice, indicating the involvement of ACE in atherogenesis (Hayek et al. 2003). 

Other Important Factors

Hyperhomocysteinemia has been reported to be a risk factor for atherosclerosis (Thambyrajah and Townend 2000). ApoE / mice with diet-induced hyperhomocysteinemia have a greater atherosclerotic lesion size, exacerbated vascular inflammation, and multiple complications as compared with controls (Hofmann et al. 2001). Ascorbic acid (vitamin C) is essential for stabilization of collagen by the addition of polar hydroxyl groups to amino acids such as proline and lysine. In a recent report (Nakata and Maeda 2002), Gulo / mice, which lack ascorbic acid synthesis, were generated and cross-bred with apoE / mice. Although no difference in atherosclerotic lesions was seen, the offspring demonstrated less collagen accumulation in the lesions.



Conclusion

This article reviews the major mouse models of atherosclerosis and their underlying mechanisms reported to date. Development and application of apoE / and LDLR / mice, as well as other genetic modifications in mice, have made significance advances in cardiovascular research. However, there are conflicts among reports on the role of some molecules such as TNF-a and eNOS. In acuality, these types of inconsistencies are often encountered in studies of knockout models. To avoid this, it is necessary to utilize models in which the genes are specifically impaired in target organs. The application of the Cre-lox technique, which generates organ-specific knockout mice, makes it possible to evaluate specific roles of specific genes in the vascular system. Although there appear to be accumulated issues that need to be solved, it is likely that mouse models will still be increasingly used to define mechanisms of atherosclerosis. On the other hand, small rodents may not accurately reflect human cardiovascular physiology in certain aspects, although the study of the cardiovascular system has benefited significantly from the use of gene-targeted and transgenic mouse models. 

Acknowledgments

This work was partially supported by research grants from the National Institutes of Health (AI 49116 and DE15543 to Q.Y. and HL61943, HL60135, HL65916, HL72716, and EB-002436 to C.C.).

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