Apolipoprotein E (apoE) is a ligand for receptors that clear remnants of chylomicrons and very low density lipoproteins. Lack of apoE is, therefore, expected to cause accumulation in plasma of cholesterol-rich remnants whose prolonged circulation should be atherogenic. ApoE-deficient mice generated by gene targeting were used to test this hypothesis and to make a mouse model for spontaneous atherosclerosis. The mutant mice had five times normal plasma cholesterol, and developed foam cell-rich depositions in their proximal aortas by age 3 months. These spontaneous lesions progressed and caused severe occlusion of the coronary artery ostium by 8 months. The severe yet viable phenotype of the mutants should make them valuable for investigating genetic and environmental factors that modify the atherogenic process.

We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, pJPB63 and pNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.

Apolipoprotein E-deficient mice have spontaneous elevations of total plasma cholesterol and triglycerides and reduced high-density lipoprotein. The mice develop arterial lesions in a time-dependent manner. Lesional distribution was centered in the aortic sinus in young mice, and the lesions were widely distributed throughout the arterial tree in mice at 8 to 9 months of age. In young mice, subendothelial foam cell deposits were present in the aortic sinus adjacent to valveattachment sites. By 5 months of age, foam cell deposits, free cholesterol, and admixed smooth muscle cells composed the developing atherosclerotic lesions. After 8 to 9 months of age, the arterial lesions showed increased complexity, and fibrous cap lesions were present. Transmission electron microscopy showed foam cells, smooth muscle cells (both contractile and synthetic varieties), cellular debris, and acicular cholesterol deposits within the plaques. By scanning electron microscopy, subendothelial collections of foam cells were present within the aortic sinus and ascending aorta. The results show that the complexity of the atherosclerotic lesions that develop in these apo E deficient-mice are similar to those described in other species and therefore represent an important model for studies of genetic and environmental influences on the atherosclerotic process. {Arteriosder Thromb. 1994;14:141-147.) Key Words • atherosclerotic lesions • mice • apo E • gene targeting • animal models • electron microscopy • lipid deposition • fibrous plaques • microthrombiA therosclerotic vascular disease is associated with a /\ variety of etiologic factors. Studies have shown A. \. that both genetic and environmental factors, either singly or in combination, have an important role in the etiology of atherosclerosis. Of the numerous risk factors identified, diet-induced hypercholesterolemia and inherited defects in lipid metabolism have received the most study. In experimental studies, animals fed high-fat diets 13 and those with a genetic predisposition to atherosclerosis 4 -3 show an accelerated rate of diet-induced vascular lesions. Lesional development was shown to proceed through a series of arterial changes that initially consisted of intimal foam cell deposits. In advanced lesions, mixed plaques were described that contained smooth muscle and lipidfilled cells, necrotic debris, extracellular cholesterol deposits, and calcification.Mice with genetic abnormalities of lipid metabolism created by gene targeting may develop atherosclerotic lesions when fed regular mouse chow. While some inbred strains have been shown to be susceptible to diet-induced atherosclerosis, mice are normally resistant to the development of atherosclerosis.6 Long periods of feeding are necessary to produce arterial lesions, and the diets used are extremely rich in cholesterol content. We 7 and others 8 have previously described mice lacking apolipoprotein E (apo E) that are generated by gene targeting, have spontaneous hypercholesterolemia, and develop atherosclerotic lesi...

With the aim of establishing whether a genetically reduced capability of producing apolipoprotein E (apo E) can affect atherogenesis, we have compared the consequences of dietary stress on normal mice and on mice heterozygous or homozygous for a disrupted apo E gene. A dramatically accelerated development of lesions occurred in the vasculature of the homozygous mutants as a result of feeding an atherogenic diet for 12 wk, and extensive deposition of lipidfilled macrophages was found outside the cardiovascular system. In nine heterozygotes fed the atherogenic diet for 12 wk, the amount of apo E in their total plasma lipoproteins increased to a level comparable to normal, but all nine developed much larger foam cell lesions in their proximal aorta than those found in 3 of 9 normal mice fed the same diet. The other six normals had no lesions. Our study demonstrates that heterozygous mice with only one functional apo E gene are more susceptible to diet-induced atherosclerosis than are normal, two-copy mice. Genetically determined quantitative limitations of apo E could, therefore, have similar effects in humans when they are stressed by an atherogenic diet. (J. Clin. Invest. 1994. 94:937-945.)

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