Neointimal formation in two apolipoprotein E-deficient mouse strains with different atherosclerosis susceptibility

C57BL/6 (B6) and C3H/HeJ (C3H) are two commonly used mouse strains that differ markedly in atherosclerosis susceptibility. In this study, we determined plaque formation after removal of the endothelium in the two strains carrying the mutant apolipoprotein E gene (apoE(-/-)). At 10 weeks of age, male B6.apoE(-/-) and C3H.apoE(-/-) mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, B6.apoE(-/-) mice developed significantly larger neointimal lesions in the vessel than their C3H.apoE(-/-) counterparts, although they had comparable plasma cholesterol levels on a chow diet. Feeding of a Western diet aggravated lesion formation in both strains, but the increase was more dramatic in B6.apoE(-/-) mice than in C3H.apoE(-/-) mice. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells in neointimal lesions. We then compared neointimal growth in F1 mice reconstituted with bone marrow from B6.apoE(-/-) and C3H.apoE(-/-) mice. No significant lesions were observed 2 weeks after endothelial denudation in the mice reconstituted with bone marrow from either donor. Thus, these data indicate that foam cell formation contributes to neointimal growth in the hyperlipidemic apoE(-/-) model and that neither endothelial cells nor blood cells alone explain the dramatic difference between B6 and C3H mice in plaque formation.     

Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice fed a Western diet exhibit severe hyperlipidemia and develop significant atherosclerosis. Apolipoprotein E (apoE) is a multifunctional protein synthesized by hepatocytes and macrophages. We sought to determine effect of macrophage apoE deficiency on severe hyperlipidemia and atherosclerosis. Female LDLR(-/-) mice were lethally irradiated and reconstituted with bone marrow from either apoE(-/-) or apoE(+/+) mice. Four weeks after transplantation, recipient mice were fed a Western diet for 8 weeks. Reconstitution of LDLR(-/-) mice with apoE(-/-) bone marrow resulted in a slight reduction in plasma apoE levels and a dramatic reduction in accumulation of apoE and apoB in the aortic wall. Plasma lipid levels were unaffected when mice had mild hyperlipidemia on a chow diet, whereas IDL/LDL cholesterol levels were significantly reduced when mice developed severe hyperlipidemia on the Western diet. The hepatic VLDL production rate of mice on the Western diet was decreased by 46% as determined by injection of Triton WR1339 to block VLDL clearance. Atherosclerotic lesions in the proximal aorta were significantly reduced, partially due to reduction in plasma total cholesterol levels (r=0.56; P<0.0001). Thus, macrophage apoE-deficiency alleviates severe hyperlipidemia by slowing hepatic VLDL production and consequently reduces atherosclerosis in LDLR(-/-) mice.     

Effects of atherogenic diet consumption on lipoproteins in mouse strains C57BL/6 and C3H

Inbred mouse strains C57BL/6J (B6) (susceptible) and C3H/HeJ (C3H) (resistant) differ in atherosclerosis susceptibility due to a single gene, Ath-1. Plasma lipoproteins from female mice fed chow or an atherogenic diet displayed strain differences in lipoprotein particle sizes and apolipoprotein (apo) composition. High density lipoprotein (HDL) particle sizes were 9.5 +/- 0.1 nm for B6 and 10.2 +/- 0.1 nm for C3H. No major HDL particle size subclasses were observed. Plasma HDL level in the B6 strain was reduced by the atherogenic diet consumption while the HDL level in the resistant C3H mice was unaffected. The reduction in HDL in the B6 strain was associated with decreases in HDL apolipoproteins A-I(-34%) and A-II(-60%). The HDL apoC content in mice fed chow was two-fold higher in C3H than B6. Lipoproteins containing apolipoprotein B (VLDL, IDL, LDL) shifted from a preponderance of the B-100 (chow diet) to a preponderance of the B-48 (atherogenic diet). The LDL-particle size distribution was strain-specific with the chow diet but not genetically associated with the Ath-1 gene. In both strains on each diet, apolipoprotein E was largely distributed in the VLDL, LDL, and HDL fractions. The B6 strain became sixfold elevated in total lipoprotein E content which in the C3H strain was not significantly affected by diet. However, the C3H LDL apoE content was reduced. On both diets, the C3H strain exhibited apolipoprotein E levels comparable to the atherogenic diet-induced levels of the B6 mice.     

Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice

Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.     

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.     

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.     

Myriocin slows the progression of established atherosclerotic lesions in apolipoprotein E gene knockout mice

The serine palmitoyl transferase inhibitor myriocin potently suppresses the development of atherosclerosis in apolipoprotein E (apoE) gene knockout (apoE(-/-)) mice fed a high-fat diet. This is associated with reduced plasma sphingomyelin (SM) and glycosphingolipid levels. Furthermore, oral administration of myriocin decreases plasma cholesterol and triglyceride (TG) levels. Here, we aimed to determine whether myriocin could inhibit the progression (or stimulate the regression) of established atherosclerotic lesions and to examine potential changes in hepatic and plasma lipid concentrations. Adult apoE(-/-) mice were fed a high-fat diet for 30 days, and lesion formation was histologically confirmed. Replicate groups of mice were then transferred to either regular chow or chow containing myriocin (0.3 mg/kg/day) and maintained for a further 60 days. Myriocin significantly inhibited the progression of established atherosclerosis when combined lesion areas (aortic sinus, arch, and celiac branch point) were measured. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. The inhibition of lesion progression was associated with reductions in hepatic and plasma SM, cholesterol, and TG levels and increased hepatic and plasma apoA-I levels, indicating that the modulation of pathways associated with several classes of atherogenic lipids may be involved.     

Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice.

Lipoprotein lipase (LPL) is known to play a crucial role in lipoprotein metabolism by hydrolyzing triglycerides; however its role in atherogenesis has yet to be determined. We have previously shown that low density lipoprotein receptor knockout mice overexpressing LPL are resistant to diet-induced atherosclerosis due to the suppression of remnant lipoproteins. Plasma lipoproteins and atherosclerosis of apolipoprotein (apo) E knockout mice which overexpress the human LPL transgene (LPL/APOEKO) were compared with those of control apoE knockout mice (APOEKO). On a normal chow diet, LPL/APOEKO mice showed marked suppression of the plasma triglyceride levels compared with APOEKO mice (54 vs. 182 mg/dl), but no significant changes in plasma cholesterol and apoB levels. Non-high density lipoproteins (HDL) from LPL/APOEKO mice had lower triglyceride content, a smaller size, and a more positive charge compared with those from APOEKO mice. Cholesterol, apoA-I, and apoA-IV were increased in HDL. Although both groups developed hypercholesterolemia to a comparable degree in response to an atherogenic diet, the LPL/APOEKO mice developed 2-fold smaller fatty streak lesions in the aortic sinus compared to the APOEKO mice. In conclusion, overproduction of LPL is protective against atherosclerosis even in the absence of apoE.     

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.     

Mouse models of experimental atherosclerosis.

Since 1992 the mouse has become an excellent model for experimental atherosclerosis research. Until 1992, the diet -- induced atherosclerosis mouse model has been used effectively, but the lesions tended to be small and were limited to early fatty-streak stage. This model was also criticized because of the toxicity and inflammatory responses due to the diet. In 1992 the first line of gene targeted animal models, namely apolipoprotein E -- knockout mice was developed. Of the genetically engineered models, the apoE -- deficient model is the only one that develops extensive atherosclerotic lesions on a chow diet. It is also the model in which the lesions have been characterized most thoroughly. The lesions develop into fibrous plaques; however, there is no evidence that plaque rupture occurs in this model. The LDL receptor - deficient model has elevated LDL levels, but no lesions, or only very small lesions, form on the chow diet, however, robust lesions do form on the western-type diet. The creation of apoE -- knockout mice has changed the face of atherosclerosis research.     

ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia

To establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE(-/-)) and Fas(lpr/lpr) (Fas(-/-)) C57BL/6 mice. On a normal chow diet, 5 month old apoE(-/-)Fas(-/-) mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE(-/-)Fas(+/+) littermates. Moreover, female apoE(-/-)Fas(-/-) mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE(-/-)Fas(+/+) mice. Compared with apoE(-/-)Fas(+/+) and apoE(+/+)Fas(-/-) mice, apoE(-/-)Fas(-/-) mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = -0.57), and BV/TV (r = -0.72). These results suggest that the apoE(-/-)Fas(-/-) mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes.     

Folic acid supplementation delays atherosclerotic lesion development in apoE-deficient mice

Folic acid is a vitamin that when used as a dietary supplementation can improve endothelial function. To assess the effect of folic acid on the development of atherosclerosis, male apolipoprotein E-deficient mice fed a standard chow diet received either water (control group) or an aqueous solution of folic acid that provided a dose of 75 microg/kg/day, for ten weeks. At the time of sacrifice, blood was drawn and the heart removed. The study measured plasma homocysteine, lipids, lipoproteins, low-density lipoprotein (LDL) oxidation, isoprostane, paraoxonase, and apolipoproteins, and aortic atherosclerotic areas. In folic acid-treated animals, total cholesterol, mainly carried in very low-density and low-density lipoproteins, increased significantly, and homocysteine, HDL cholesterol, paraoxonase, and triglyceride levels did not change significantly. Plasma isoprostane and apolipoprotein (apo) B levels decreased. The resistance of LDL to oxidization and plasma apoA-I and apoA-IV levels increased with a concomitant decrease in the area of atherosclerotic lesions. The administration of folic acid decreased atherosclerotic lesions independently of plasma homocysteine and cholesterol levels, but was associated with plasma levels of apolipoproteins A-I, A-IV and B, and decreased oxidative stress.     

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.     

Hypomorphic apolipoprotein E mice: a new model of conditional gene repair to examine apolipoprotein E-mediated metabolism.

In creating an allelic variant of mouse Apoe designed to resemble human apolipoprotein E4 (apoE4), we generated hypomorphic apoE (hypoE) mice that express only approximately 5% of normal apoE mRNA levels in all tissues. Insertion of a neo cassette flanked by loxP sites in the third intron of Apoe reduced expression of the Arg-61 allelic variant in hypoE mice and resulted in plasma apoE levels that were approximately 2-5% of normal. Unlike other mouse models with low levels of circulating apoE, hypoE mice had a nearly normal lipoprotein cholesterol profile when fed a chow diet. Further reduction of apoE expression in hypoE/Apoe(-/-) heterozygous mice led to an increase in remnant lipoprotein-associated cholesterol levels, demonstrating that hypoE mice express close to the threshold level of Arg-61 apoE required for a normal lipoprotein profile. Unlike wild type mice, hypoE mice were susceptible to diet-induced hypercholesterolemia, which was fully reversed within 3 weeks after resumption of a chow diet. In Mx1-Cre transgenic hypoE mice, plasma apoE levels returned to normal within 10 days after gene repair and removal of the neo cassette following induction of Cre recombinase. HypoE mice provide the opportunity for conditional gene repair by crossing with inducible or lineage/cell type-specific Cre transgenic mice, generating new models to dissect the roles of apoE in atherosclerosis regression, immunoregulation, and neurodegeneration.     

Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P < 0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.     

Evidence for differential effects of apoE3 and apoE4 on HDL metabolism

We present a murine model that examines the effects of macrophage-produced apolipoprotein E3 (apoE3) and apoE4 on VLDL and high density lipoprotein (HDL) metabolism. Mice expressing apoE3 on the Apoe(-/-) background had substantially lower VLDL levels than mice expressing apoE4. In addition, there were differences between the HDL of apoE3- and apoE4-expressing mice. Apoe(-/-) mice have low levels of HDL. Low level expression of either apoE3 or apoE4 was able to restore near-normal HDL levels, which increased dramatically when the mice were challenged with a high-fat diet. ApoE4-expressing mice had smaller HDL than apoE3-expressing mice on both chow and high-fat diets. In addition, plasma from apoE4-expressing mice was less efficient at transferring apoA-I from VLDL to HDL and at generating HDL in vitro than that from apoE3-expressing mice. Thus, we present experimental evidence for differential effects of apoE3 and apoE4 on HDL metabolism that supports epidemiological observations made in humans, which suggested that individual homozygous for the epsilon 4 allele had lower HDL than others.     

Phenotypic characterization of the Ath-1 gene controlling high density lipoprotein levels and susceptibility to atherosclerosis

The Ath-1 gene determines the levels of high density lipoprotein (HDL) lipid in response to a high fat diet challenge as well as susceptibility to diet-induced atherosclerosis in mice (Paigen et al. 1987. Proc. Natl. Acad. Sci. USA. 84: 3763-3767). As yet, the identity of the Ath-1 gene and how it acts to affect HDL levels are completely unknown. In an effort to clarify the nature of the gene, we have examined HDL phenotypes in strains carrying either the susceptible or resistant alleles. When challenged with a high fat diet, the susceptible strain C57BL/6 exhibited a marked decrease in the levels of HDL cholesterol and apolipoprotein A-I (apoA-I), the major protein of HDL, whereas the resistant strains C3H and BALB/c maintained high levels of both. Separation of HDL subfractions by polyacrylamide gradient gel electrophoresis revealed that the decrease was particularly striking among the larger HDL species. The rates of synthesis of apoA-I in liver and intestine were similar in the strains and were unaffected by the high fat diet. Although the rates of synthesis of apoA-II and the levels of apoA-II mRNA were decreased in response to the high fat diet, similar decreases were observed in both the susceptible and resistant strains. We conclude that the Ath-1 gene results in a rapid decrease in both HDL lipid and HDL apolipoprotein levels in the susceptible strain in response to the high fat diet and that this is mediated primarily at the level of HDL catabolism.     

Altered activities of anti-atherogenic enzymes LCAT, paraoxonase, and platelet-activating factor acetylhydrolase in atherosclerosis-susceptible mice

We examined whether the putative anti-atherogenic enzymes LCAT, paraoxonase (PON), and platelet-activating factor acetylhydrolase (PAF-AH) are impaired in 8 week old atherosclerosis susceptible apolipoprotein E (apoE)(-/-) and LDL receptor (LDLr)(-/-) mice and whether plasma concentrations of bioactive oxidized phospholipids accumulate in plasma. ApoE(-/-) mice had reduced (28%) LCAT activity and elevated lysophosphatidylcholine and bioactive oxidized phospholipids (1-palmitoyl-2-oxovaleryl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine) compared with controls on the chow diet. Elevated oxidized phospholipids and reduced LCAT activity may, in part, contribute to spontaneous lesions in these mice on a chow diet. A Western diet decreased LCAT activity further (50% of controls) and PON activity was decreased 38%. The LDLr(-/-) mice showed normal LCAT activity on chow diet and little accumulation of oxidized phospholipids. On a Western diet, LDLr(-/-) mice had reduced LCAT activity (21%), but no change in PON activity. All genotypes had reduced PAF-AH activity on the Western diet. ApoE(-/-) and LDLr(-/-) mice, but not controls, had elevated plasma bioactive oxidized phospholipids on the Western diet.We conclude that impairment of LCAT activity and accumulation of oxidized phospholipids are part of an early atherogenic phenotype in these models.     

Quantitative trait locus analysis of atherosclerosis in an intercross between C57BL/6 and C3H mice carrying the mutant apolipoprotein E gene

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) differ significantly in atherosclerosis susceptibility and plasma lipid levels on the apolipoprotein E-deficient (apoE-/-) background when fed a Western diet. To determine genetic factors contributing to the variations in these phenotypes, we performed quantitative trait locus (QTL) analysis using an intercross between the two strains carrying the apoE-/- gene. Atherosclerotic lesions at the aortic root and plasma lipid levels of 234 female F2 mice were analyzed after being fed a Western diet for 12 weeks. QTL analysis revealed one significant QTL, named Ath22 (42 cM, LOD 4.1), on chromosome 9 and a suggestive QTL near D11mit236 (20 cM, LOD 2.4) on chromosome 11 that influenced atherosclerotic lesion size. One significant QTL on distal chromosome 1, which accounted for major variations in plasma LDL/VLDL cholesterol and triglyceride levels, coincided with a QTL having strong effects on body weight. Plasma LDL/VLDL cholesterol or triglyceride levels of F2 mice were significantly correlated with body weight, but they were not correlated with atherosclerotic lesion sizes. These data indicate that atherosclerosis susceptibility and plasma cholesterol levels are controlled by separate genetic factors in the B6 and C3H mouse model and that genetic linkages exist between body weight and lipoprotein metabolism.     

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.