Apolipoprotein (apo) E, a constituent of several lipoproteins, is a ligand for the low density lipoprotein receptor, and this interaction is important for maintaining cholesterol and triglyceride homeostasis. We have used a gene replacement strategy to generate mice that express the human apoE3 isoform in place of the mouse protein. The levels of apoE mRNA in various tissues are virtually the same in the human apoE3 homozygous (3/3) mice and their littermates having the wild type mouse allele (؉/؉). Total cholesterol and triglyceride levels in fasted plasma from the 3/3 mice were not different from those in the ؉/؉ mice, when maintained on a normal (low fat) chow diet. We found, however, notable differences in the distribution of plasma lipoproteins and apolipoprotein E between the two groups: ␤-migrating lipoproteins and plasma apoB100 levels are decreased in the 3/3 mice, and the apoE distribution is shifted from high density lipoproteins to larger lipoprotein particles. In addition, the fractional catabolic rate of exogenously administered remnant particles without apoE was 6-fold slower in the 3/3 mice compared with the ؉/؉ mice. When the 3/3 and ؉/؉ animals were fed a high fat/high cholesterol diet, the 3/3 animals responded with a dramatic increase (5-fold) in total cholesterol compared with the ؉/؉ mice (1.5-fold), and after 12 weeks on this same diet the 3/3 animals developed significantly (at least 13-fold) larger atherosclerotic plaques in the aortic sinus area than the ؉/؉ animals. Thus the structural differences between human APOE3 and mouse ApoE proteins are sufficient to cause an increased susceptibility to dietary-induced hypercholesterolemia and atherosclerosis in the 3/3 mice.

Apo E is a 34-kDa plasma protein important for the metabolism of plasma lipoproteins (1). Like other apolipoproteins, apo E contains multiple 22-amino acid repeats that form amphipathic helices, enabling it to associate with the surface of plasma lipoproteins. Apo E also contains a stretch of basic residues (136-150) that is important for high-affinity binding to the LDL receptor and subsequent endocytosis of the associated lipoprotein particle (2). In addition, apo E mediates lipoprotein interactions with LDL receptor-related protein (LRP) (3), the VLDL receptor (4), other lipoprotein receptors (5), endothelial heparin sulfate (6), and plasma lipases (7,8). The phenotype of severe hyperlipidemia and spontaneous development of atherosclerosis in mice lacking apo E clearly demonstrates the central role of apo E in mammalian lipid metabolism (9, 10).In humans, the APOE gene is polymorphic and has 3 alleles: APOE*2, APOE*3, and APOE*4. These alleles have frequencies of 7%, 77%, and 15%, respectively, in the general population (11). The APOE*3 allele codes for cysteine at position 112 and for arginine at 158. The APOE*2 allele codes cysteines at both positions, whereas the APOE*4 allele codes for arginines at both positions. Various population-based studies have suggested that the different APOE alleles have distinct influences on lipid metabolism in humans. Possession of at least 1 copy of the APOE*2 allele has been associated with higher plasma apo E (12) and lower plasma cholesterol, LDL cholesterol, and apo B levels (11) when compared with APOE*3 homozygotes. The APOE*2 allele is also associated with lower risk of coronary artery disease (13), except in 5-10% of APOE*2 homozygotes who develop type III hyperlipoproteinemia and premature atherosclerosis (14). On the other hand, the presence of at least 1 APOE*4 allele is associated with lower plasma apo E (12) and increased plasma cholesterol, LDL cholesterol, and apo B levels (11), and a greater risk of coronary artery disease (13), when compared with APOE*3 homozygotes. Davignon et al. (11) estimate that the apo E polymorphism accounts for 2.8% of the variation of risk for atherosclerosis, which is a large contribution for a single locus in this complex, polygenic disease.The availability of a well-defined model system should benefit studies of the role of the human apo E polymorphism in lipid metabolism and atherosclerosis. To develop such a model, we have used gene targeting to replace the murine Apoe gene with the 3 human APOE alleles. These mice retain the murine Apoe regulatory sequences and solely produce human apo E proteins with different We have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform. Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and choles...

To study isoform-specific effects of apolipoprotein E (apoE) in vivo, we generated mice with a human APOE*2 allele in place of the mouse Apoe gene via targeted gene replacement in embryonic stem cells. Mice expressing human apoE2 (2/2) have virtually all the characteristics of type III hyperlipoproteinemia. Their plasma cholesterol and triglyceride levels are both twice to three times those in (

A B S T R A C T The addition of apoprotein E isolated from human very low density lipoproteins to both rat lymph chylomicrons and a triglyceride emulsion significantly increased the hepatic uptake of these particles in a nonrecycling isolated rat liver perftision system. The cleared triglyceride was removed without apparent hydrolysis by the hepatocyte. When lymph chylomicrons were loaded with both Apo E and Apo C proteins by exposture to rat plasma, no increment in hepatic clearance was observed. Sequential evaluations of the influence of the C apoproteins on the hepatic clearance of both emulsions an(l chylomicrons revealed that the CIII (CIII-1) protein had a pronounced inhibitory effect on hepatic removal. The inhibition was observed for both Apo E-enriched chylomicrons and those containing little of this apoprotein.

Dietary administration of probucol (0.5%, wt/wt) efficiently reduced total plasma cholesterol levels in apolipoprotein E-deficient mice (apoE Ϫ / Ϫ ) by 40%, with decreases in high density lipoprotein (HDL) and apoAI by 70 and 50%, respectively. Paradoxically, however, aortic atherosclerotic plaques in the probucol-treated apoE Ϫ / Ϫ mice formed more rapidly than in the untreated apoE Ϫ / Ϫ mice, and the lesions were two to four times larger and more mature regardless of sex, age, and genetic background ( P Ͻ 10 Ϫ 6 ). Histologically, lesions in probucol-treated mice contained increased fibrous materials and cells other than foam cells, and were commonly associated with focal inflammation and aneurysmal dilatation. Probucol treatment also accelerated lesion development in apoE ϩ / Ϫ mice fed an atherogenic diet, indicating that the adverse effect is not dependent on the complete absence of apoE. Furthermore, mice lacking apoE and apoAI have plasma lipoprotein profiles very similar to the probucol-treated apoE Ϫ / Ϫ mice, but do not have accelerated plaque development. Thus, the enhanced atherosclerosis in the probucol-treated animals is unlikely to be caused by the reduction of HDL and apoAI levels. Our data indicate that a reduction in plasma cholesterol caused by probucol does not necessarily lead to an antiatherogenic effect. ( J. Clin. Invest. 1997. 99:2858-2866.)