A common phenotype associated with atherogenesis in diverse mouse models of vascular lipid lesions.

The introduction of a range of different genetic modifications in mice results in altered lipoprotein metabolism and the development of vascular lipid lesions. At present, however, it is unclear to what extent the molecular events underlying lipid lesion formation are similar in these different mouse models of atherosclerosis. The aim of this study was to compare the protein expression pattern of lipid lesions from seven different mouse lines with varying susceptibility to vascular lipid lesion development, to determine to what extent lesions induced by different genetic interventions have a similar composition. The proteins we have measured, using quantitative immunofluorescence, are proteins whose expression is known to be modulated during atherogenesis in humans, including plasminogen activator inhibitor (PAI)-1, transforming growth factor (TGF)-beta 1, osteopontin and the macrophage marker CD11b. In all the mice lines we have investigated, PAI-1 was elevated wherever lesions developed. Active TGF-beta was depressed in the vessel wall of mice which developed lipid lesions, particularly in the intima. In contrast, TGF-beta 1 antigen (active plus latent TGF-beta 1) was increased at lesion sites. Accumulation of osteopontin and, with the marked exception of apolipoprotein(a) transgenic mice, tissue macrophages occurred at sites of lipid deposition in the vessel wall. Each lesion, irrespective of its size and the mouse strain in which it developed, had similar amounts of PAI-1, active TGF-beta and osteopontin per unit area of lesion. These data are consistent with a common phenotype accompanying atherogenesis, irrespective of the genetic basis of susceptibility.

Monocyte chemoattractant protein-1 but not tumor necrosis factor-alpha is correlated with monocyte infiltration in mouse lipid lesions.

BACKGROUND: Apolipoprotein (apo)(a) transgenic mice and C57BL/6 mice fed a high fat diet develop similar-sized lipid lesions, but lesions in apo(a) mice are devoid of macrophages. We used this observation to identify which proinflammatory proteins might be involved in mediating monocyte recruitment during atherogenesis. METHODS AND RESULTS: Macrophage-deficient apo(a) transgenic mouse lesions contained similar levels of several different proinflammatory proteins, both adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and cytokines (tumor necrosis factor-alpha [TNF-alpha] and macrophage inflammatory protein-1alpha [MIP-1alpha]), similar to the macrophage-rich lesions of C57BL/6 mice. CONCLUSIONS: From this we conclude that ICAM-1, VCAM-1, TNF-alpha, and MIP-1alpha may all be necessary for vascular monocyte recruitment in vivo, but they cannot be sufficient. Monocyte chemoattractant protein-1 (MCP-1) protein was undetectable in the vessel wall taken from apo(a) transgenic mice fed a high fat diet compared with high expression in mice with lipid lesions (C57BL/6 and apoE knockout mice). Therefore elevated expression of MCP-1 but not TNF-alpha, MIP-1alpha, ICAM-1, or VCAM-1 is correlated with vascular macrophage accumulation. To test the hypothesis that monocyte infiltration during atherogenesis is MCP-1 dependent, it will be necessary to develop specific pharmacological inhibitors of MCP-1 activity.

HDL deficiency in genetically engineered mice requires elevated LDL to accelerate atherogenesis.

In humans, a low HDL concentration is one of the strongest indicators of increased risk for coronary heart disease. Apolipoprotein A-I (apo A-I) synthetic defects results in extremely low HDL levels and are frequently although not invariably associated with premature atherosclerosis. To investigate atherosclerosis susceptibility associated with HDL deficiency alone and in combination with other risk factors, such as high levels of LDL, we have quantified diet-induced atherogenesis in a series of genetically engineered mice, including mice with low HDL levels due to targeted disruption of both apo A-I alleles (AI KO mice), mice with high LDL levels due to expression of a human apolipoprotein B transgene (Btg mice), and mice with combined high LDL and low HDL levels due to the presence of the human apo B transgene and apo A-I knockout alleles, respectively (AI KO/Btg mice). After exposure to an atherogenic diet, AI KO and control mice had negligible lesions. All mice expressing the apo B transgene developed extensive lesions, but AI KO/Btg mice developed significantly larger lesions than Btg mice: 56, 260 +/- 4630 micron 2 for AI KO/Btg (n = 27) versus 38, 120 +/- 3350 micron 2 for Btg mice (n = 19) (P < .02). Results of this study, consistent with several human epidemiological studies, indicate that HDL deficiency in the mouse does not by itself lead to the development of atherosclerosis but does increase atherosclerosis susceptibility when accompanied by other risk factors, in this case elevated LDL.

Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase.

Studies in vitro have shown that copper-zinc superoxide dismutase (CuZn-SOD) inhibits a number of events putatively involved in atherogenesis, including cell-mediated oxidation of LDL. To investigate whether increased activity of CuZn-SOD reduces atherogenesis in vivo, we examined diet-induced fatty streak formation in CuZn-SOD transgenic mice (n = 24) as compared with their nontransgenic littermates (n = 28). Transgenic animals were originally created by introduction of an EcoRI-BamHI human genomic DNA fragment containing the CuZn-SOD gene and its regulatory elements into B6SJL zygotes. For the current studies, the transgene was bred for 12 generations into the atherosclerosis-susceptible C57BL/6 background. Animals were fed atherogenic diets (15% fat, 1.25% cholesterol, 0.5% Na cholate) starting at 100 weeks of age and extending for 18 weeks. At the end of the diet period, aortic SOD activity was two-fold higher in transgenics than nontransgenics (mean +/- SE: 46.7 +/- 5.8 versus 20.1 +/- 2.4 units/mg of protein, P < .001). Levels of protein-bound amino acid oxidation products (meta-, ortho-, and dityrosine) were either similar or lower in aorta and heart from transgenics as compared with nontransgenics, suggesting that amplification of CuZn-SOD activity above the normal complement had modest inhibitory effects on basal oxidative stress in these tissues. CuZn-SOD overexpression did not reduce the extent of lesion development as analyzed by quantitative lipid staining of serial sections of the proximal aorta; mean lesion areas (+/- SE) were 997 +/- 478 and 943 +/- 221 mu 2 in transgenics and nontransgenics, respectively. Notably, the range of values for lesion area was 2.2-fold greater in transgenics (0-8403 versus 0-3868 mu 2 in nontransgenics). Moreover, within this group, lesion area showed a significant positive correlation with SOD activity (r = .611, P < .03). These results do not support an antiatherogenic effect of Cu-Zn-SOD over expression and raise the possibility that high tissue SOD activity may potentiate atherogenesis in fat-fed atherosclerosis-susceptible mice [corrected].

Lipoprotein(a) vascular accumulation in mice. In vivo analysis of the role of lysine binding sites using recombinant adenovirus.

Although the mechanism by which lipoprotein(a) [Lp(a)] contributes to vascular disease remains unclear, consequences of its binding to the vessel surface are commonly cited in postulated atherogenic pathways. Because of the presence of plasminogen-like lysine binding sites (LBS) in apo(a), fibrin binding has been proposed to play an important role in Lp(a)'s vascular accumulation. Indeed, LBS are known to facilitate Lp(a) fibrin binding in vitro. To examine the importance of apo(a) LBS in Lp(a) vascular accumulation in vivo, we generated three different apo(a) cDNAs: (a) mini apo(a), based on wild-type human apo(a); (b) mini apo(a) containing a naturally occurring LBS defect associated with a point mutation in kringle 4-10; and (c) human- rhesus monkey chimeric mini apo(a), which contains the same LBS defect in the context of several additional changes. Recombinant adenovirus vectors were constructed with the various apo(a) cDNAs and injected into human apoB transgenic mice. At the viral dosage used in these experiments, all three forms of apo(a) were found exclusively within the lipoprotein fractions, and peak Lp(a) plasma levels were nearly identical (approximately 45 mg/dl). In vitro analysis of Lp(a) isolated from the various groups of mice confirmed that putative LBS defective apo(a) yielded Lp(a) unable to bind lysine-Sepharose. Quantitation of in vivo Lp(a) vascular accumulation in mice treated with the various adenovirus vectors revealed significantly less accumulation of both types of LBS defective Lp(a), relative to wild-type Lp(a). These results indicate a correlation between lysine binding properties of Lp(a) and vascular accumulation, supporting the postulated role of apo(a) LBS in this potentially atherogenic characteristic of Lp(a).

Atherogenesis in transgenic mice with human apolipoprotein B and lipoprotein (a).

The engineering of mice that express a human apoB transgene has resulted in animals with high levels of human-like LDL particles and through crosses with human apo(a) transgenics, high levels of human-like lipoprotein (a) (Lp[a]) particles. In this study, these animals have been used to compare the atherogenic properties of apo(a), LDL, and Lp(a). The presence of the high expressing apoB (apoBH) transgene was associated with a 2.5-fold increase in VLDL-LDL cholesterol (primarily in the LDL fraction) and a 15-fold increase in proximal lesions compared with non-transgenic mice (P < or = 0.0001), while the presence of the low expressing human apoB (apoBL) transgene was not associated with major changes in lipoprotein profiles or increases in aortic lesion size. Examination of aortas of apoBH mice demonstrated lesions along the entire length of the aorta and immunochemical analysis of the lesions revealed features characteristically seen in human lesions including the presence of oxidized lipoproteins, macrophages, and immunoglobulins. Unlike animals with the apoBL transgene, animals with the apo(a) transgene had significant increases in proximal aortic fatty streak lesions compared to nontransgenic control animals (threefold; P < 0.02), while animals with both transgenes, the apo(a)/apo BL double transgenics, had lesions 2.5 times greater than animals expressing the apo(a) transgene alone and eightfold (P < 0.0006) greater than nontransgenic animals. These murine studies demonstrate that marked increases in apoB and LDL resulted in atherosclerotic lesions extending down the aorta which resemble human lesions immunochemically and suggest that apo(a) associated with apoB and lipid may result in a more pro-atherogenic state than when apo(a) is free in plasma.

Human apolipoprotein A-I prevents atherosclerosis associated with apolipoprotein[a] in transgenic mice.

Elevated levels of apolipoprotein[a] (apo[a]) and apolipoprotein A-I (apoA-I) are associated, respectively, with increased and decreased atherosclerosis risk, in both humans and transgenic mice. To investigate the interactions of these two important lipid-associated proteins, we assessed the effect of expression of human apoA-I and apo[a] transgenes, both singularly and together, on murine atherogenesis. Mice expressing the apo[a] transgene have a lipoprotein profile similar to nontransgenic controls, yet have significantly increased susceptibility to diet-induced atherosclerosis. Compared to mice expressing only the apo[a] transgene, mice expressing both apo[a] and apoA-I transgenes have twofold greater high density lipoprotein (HDL) concentrations and approximately a 20-fold decrease in development of early atherosclerotic lesions. The finding of decreased atherosclerosis in the setting of elevated apo[a] and apoA-I suggests that elevations of apoA-I and HDL have a dominant effect in reducing atherosclerosis susceptibility in various settings, including those not associated with alterations of plasma lipids.

Effect of human apoE4 on the clearance of chylomicron-like lipid emulsions and atherogenesis in transgenic mice.

Apolipoprotein (apo) E is a ligand for lipoprotein receptors and mediates the cellular uptake of several different lipoproteins. Human apoE occurs in three allelic forms designated E2, E3, and E4. The E2 isoform is associated with changes in lipoprotein metabolism, and the E4 isoform is associated with Alzheimer's disease and an increased risk of coronary heart disease. In this study transgenic mice were generated to assess the effect of a sustained increase in plasma apoE4 concentration. The transgenic animals had three- to sixfold increases in total plasma apoE, associated primarily with the non-high-density lipoprotein (HDL) fractions of plasma lipoproteins. In response to an atherogenic diet the transgenic mice developed hypercholesterolemia similar to that in nontransgenic mice but did not experience the decrease in HDL cholesterol normally observed in this strain of C57BL/6 mice. The rate of plasma clearance of a lipid emulsion mimicking lymph chylomicrons was measured in transgenic mice expressing the human apoE4 gene and compared with the clearance rate in nontransgenic control animals. In animals fed a low-fat diet the emulsion lipids were cleared significantly more rapidly from the plasma of transgenic than control mice. In animals adapted to a high-fat diet, the clearance of chylomicron remnants was slowed markedly in both transgenic and control mice and was not significantly accelerated in transgenic compared with control animals. We also investigated the effect of increasing the plasma concentration of apoE4 on the progression of atherosclerotic heart disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice.

Apolipoprotein E (apo E)-deficient mice are severely hypercholesterolemic and develop advanced atheromas independent of diet. The C57BL/6 strain differs from most inbred strains by having lower HDL concentrations and a high risk of developing early atherosclerotic lesions when fed an atherogenic diet. The relative HDL deficiency and atherosclerosis susceptibility of the C57BL/6 strain are corrected with the expression of a human apolipoprotein AI (apo AI) transgene in this genetic background. To examine if increases in apo AI and HDL are also effective in minimizing apo E deficiency--induced atherosclerosis, we introduced the human apo AI transgene into the hypercholesterolemic apo E knockout background. Similar elevations of total plasma cholesterol occurred in both the apo E knockout and apo E knockout mice also expressing the human apo AI transgene. The latter animals, however, also showed a two- to threefold increase in HDL and a sixfold decrease in susceptibility to atherosclerosis. This study demonstrates that elevating the concentration of apo AI reduces atherosclerosis in apo E deficient-mice and suggests that elevation of apo AI and HDL may prove to be a useful approach for treating unrelated causes of heightened atherosclerosis susceptibility.

Atherosclerosis in genetically obese mice: the mutants obese, diabetes, fat, tubby, and lethal yellow.

Mice with five different mutations conferring an obese or diabetic phenotype were evaluated for fatty streak lesions after consuming an atherogenic diet containing 15% fat and 1.25% cholesterol (wt/wt) for 14 weeks. The five mutations, fat, obese, tubby, diabetes, and lethal yellow, are maintained as congenic strains with C57BL/6J (B6) or C57BL/KsJ (BKs) as genetic backgrounds. None of the mutants exhibited accelerated fatty streak lesion formation; the mutant fat had aortic lesions comparable in size to those of its control strain, and the mutants obese, diabetes, tubby, and lethal yellow had significantly reduced lesion area in comparison to controls. Although B6 and BKs are closely related strains, we observed that the BKs strain was more prone to early-stage atherogenesis. Fatty streak lesion area was twice as large in BKs mice than those found in B6 mice; likewise, in comparison, the mutants obese and diabetes had larger lesions if they were carried as congenic strains in the BKs rather than the B6 genetic background. Plasma triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and combined low-density and very-low-density lipoprotein cholesterol (LDL-C and VLDL) levels were also measured in the mice. Lipid profiles differed among the mutant mice, but in general, elevations in plasma total cholesterol, triglycerides, and HDL-C were observed. Whereas the hypertriglyceridemia and hypercholesterolemia are consistent with an atherogenic lipid profile, HDL-C levels, which are normally decreased in individuals with non-insulin-dependent diabetes mellitus, were increased in the mouse mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein composition determines the anti-atherogenic properties of HDL in transgenic mice.

High-density lipoprotein (HDL) contains two major proteins, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), comprising about 70% and 20% of the total HDL protein mass, respectively. HDL exists in human plasma in two main forms, one containing apoA-I with apoA-II (AI/AII-HDL) and another containing apoA-I without apoA-II (AI-HDL). A strong inverse relationship exists between total plasma HDL concentration and atherosclerosis, but the results of studies examining the relationship between AI-HDL and AI/AII-HDL and atherosclerosis have been conflicting. To determine whether these two HDL populations have different effects on atherogenesis, human apoA-I (AI) and human apoA-I and apoA-II (AI/AII) transgenic mice were produced in an atherosclerosis-susceptible strain. Following an atherogenic diet, despite similar total cholesterol and HDL cholesterol concentrations, the area of atherogenic lesions in the AI/AII mice was 15-fold greater than in the AI animals. These studies show that the protein composition of HDL significantly affects its role in atherogenesis and that AI-HDL is more antiatherogenic than AI/AII-HDL.

Effects of dietary fats from animal and plant sources on diet-induced fatty streak lesions in C57BL/6J mice.

This study was designed to determine the effects of a variety of naturally occurring saturated fats on aortic lesion formation in C57BL/6J mice that are susceptible to diet-induced fatty streak lesions. Groups of female mice were randomly assigned to one of seven treatment groups and were fed diets containing 15% (w/w) hydrogenated coconut oil, hydrogenated soy oil, hydrogenated palm oil, cocoa butter, lard, tallow, or dairy butter, 1% cholesterol, and 0.5% cholic acid. Plasma lipid levels were measured to determine whether lesion formation was related to specific changes in these parameters. Lesions, which were observed in all groups of mice, ranged from 420 to 3220 microns2/aortic cross section. Lesion area was positively correlated to the percentage of saturated fatty acids contained in the fat sources and the ratio of combined VLDL plus LDL-cholesterol to HDL-cholesterol and inversely correlated to monounsaturated fatty acids content and to HDL-cholesterol levels. Results from this study demonstrate that inbred mice may provide a good model for dissecting the genetic basis for the differential atherogenic responses to diet-induction and for studying the effects of dietary factors on aortic lesion development.

Atherogenesis in transgenic mice expressing human apolipoprotein(a)

Elevated plasma levels of the lipoprotein Lp(a) are associated with increased risk for atherosclerosis and its manifestations, myocardial infarction, stroke and restenosis (for reviews, see refs 1-3). Lp(a) differs from low-density lipoprotein by the addition of the glycoprotein apolipoprotein(a), a homologue of plasminogen that contains many tandemly repeated units which resemble the fourth kringle domain of plasminogen, and single homologues of its kringle-5 and protease domain. As plasma Lp(a) concentration is strongly influenced by heritable factors and is refractory to most drug and dietary manipulation, the effects of modulating it are difficult to mimic experimentally. In addition, the absence of apolipoprotein(a) from virtually all species other than primates precludes the use of convenient animal models. Here we show that transgenic mice expressing human apolipoprotein(a) are more susceptible than control mice to the development of lipid-staining lesions in the aorta, and that apolipoprotein(a) co-localizes with lipid deposition in the artery walls.

Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.

apoE-deficient mice have been created by homologous recombination in ES cells. On a low fat, low cholesterol chow diet these animals have plasma cholesterol levels of 494 mg/dl compared with 60 mg/dl in control animals, and when challenged with a high fat Western-type diet, these animals have plasma cholesterol levels of 1821 mg/dl compared with 132 mg/dl in controls. This marked hypercholesterolemia is primarily due to elevated levels of very low and intermediate density lipoproteins. At 10 weeks of age, apoE-deficient mice have already developed atherosclerotic lesions in the aorta and coronary and pulmonary arteries. apoE-deficient mice are a promising small animal model to help understand the role of apoE in vivo and the genetic and environmental determinants of atherosclerosis.

Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.

Epidemiological surveys have identified a strong inverse relationship between the amount in the plasma of high density lipoproteins (HDL), apolipoprotein AI (ApoA-I), the major protein component of HDL, and the risk for atherosclerosis in humans. It is not known if this relationship arises from a direct antiatherogenic effect of these plasma components or if it is the result of other factors also associated with increases in ApoA-I and HDL levels. Because some strains of mice are susceptible to diet-induced formation of preatherosclerotic fatty streak lesions, and because of available techniques for the genetic manipulation of this organism, the murine system offers a unique setting in which to investigate the process of early atherogenesis. To test the hypothesis that induction of a high plasma concentration of ApoA-I and HDL would inhibit this process, we studied the effects of atherogenic diets on transgenic mice expressing high amounts of human ApoA-I. We report that transgenic mice with high plasma ApoA-I and HDL levels were significantly protected from the development of fatty streak lesions.

Synthetic low and high fat diets for the study of atherosclerosis in the mouse.

Diets currently used to produce atherosclerotic lesions in mice are often undefined and cause accumulation of fat in the liver and gallstone formation. Therefore, synthetic low and high fat diets of known composition were formulated in this study. A synthetic diet containing 50% sucrose, 15% cocoa butter, 1% cholesterol, and 0.5% sodium cholate was found to produce a depression in high density lipoprotein cholesterol (HDL-C) and an elevation of very low density lipoprotein (VLDL) and low density lipoprotein cholesterol (LDL-C) in the atherosclerosis-susceptible strain, C57BL/6J. This diet was able to consistently produce aortic lesions and led to a decrease in liver damage and gallstone formation. The synthetic low fat diet did not produce HDL-C levels as high as those found in mice fed chow, but resulted in similar VLDL/LDL-C levels. Lipoprotein and apolipoprotein parameters were compared in C57BL/6J and the atherosclerosis-resistant strain, C3H/HeJ, consuming the synthetic low fat or high fat diets. As reported earlier, when consuming a high fat diet C57BL/6J mice have significantly lower HDL-C and apoA-I levels than C3H/HeJ mice. Further analysis shows that the molar ratio of plasma HDL-C to apoA-I is significantly lower in C57BL/6J mice, suggesting that HDL in the susceptible strain has a lower cholesterol-carrying capacity. This conclusion is consistent with the observation that the HDL particle size is smaller for C57BL/6J mice than for C3H/HeJ. Both strains increased their apoE levels when fed the synthetic high fat diet, but C3H/HeJ mice had higher levels of apoE on both diets. The major response to consumption of the high fat diet for both strains was an increase in apoB-48 from 5 micrograms/ml on a low fat diet to 54 and 109 micrograms/ml for C57BL/6J and C3H/HeJ, respectively. ApoB-100 showed minimal response to the high fat diet. The defined high fat diet can be used to study atherosclerosis in the mouse since it produces aortic lesions but reduces or eliminates other pathological changes such as gallstone formation and liver damage.

Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice.

Female mice of 16 inbred mouse strains were fed an atherogenic diet for 14 weeks and were then evaluated for atherosclerotic lesions in the aorta. Strains C57BL/6, C57BR/cd, C57L, and SM were very susceptible to atherosclerosis, with lesion area/aortic cross-sections in the range of 4500 to 8000 microns 2. Strains C58 and SWR were intermediate in susceptibility, with lesion area/sections in the range of 1670 to 1690 microns 2. Strains 129, AKR, DBA/2, and BALB/c had only small lesions in the range of 20 to 350 microns 2/section; strains C3H, NZB, CBA, HRS, SJL, and A had no lesions after 14 weeks. Lesion formation in five strains was compared at several time points. Strain C57BL/6 mice developed lesions by 7 weeks, and these continued to grow until all mice had large atheromatous plaques in the aorta and coronary arteries. Strains AKR and DBA/2 also had fatty streak lesions as early as 7 or 8 weeks, but these lesions had not progressed in size by 14 weeks. Strains BALB/c and C3H, which were both resistant to lesion formation at 14 weeks, diverged from each other as time progressed. By 1 year, BALB/c mice had large lesions, but C3H mice had none. Most of the inbred strains chosen for evaluation are the progenitors of recombinant inbred sets of strains, a genetic tool that greatly facilitates the analysis of strain differences. This survey indicates seven additional recombinant inbred sets of strains whose progenitors differ in atherosclerosis susceptibility: BXD, AKXL, SWXJ, NX8, 129XB, NXSM, and B6NXAKRN. An analysis of these recombinant inbred strains may reveal additional mouse genes affecting atherosclerosis susceptibility.