Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2.

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 (normolipidemic) mice that are expressing human apoE3 (3/3) made in an identical manner. The 2/2 mice are markedly defective in clearing beta-migrating VLDL particles, and spontaneously develop atherosclerotic plaques, even on a regular diet. An atherogenic diet, high in fat and cholesterol, exacerbates development of atherosclerosis and xanthomas in the 2/2 mice. Thus, comparisons between the 2/2 and 3/3 mice unequivocally demonstrate that a single amino acid difference (Arg158 Cys) in the apoE protein is sufficient to cause type III HLP and spontaneous atherosclerosis in mice

Targeted Replacement of the Mouse Apolipoprotein E Gene with the Common Human APOE3 Allele Enhances Diet-induced Hypercholesterolemia and Atherosclerosis

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: beta-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

Role of Hepatic Lipase and Endothelial Lipase in High-Density Lipoprotein—Mediated Reverse Cholesterol Transport

Reverse cholesterol transport (RCT) constitutes a key part of the atheroprotective properties of high-density lipoproteins (HDL). Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol levels. Although overexpression of EL decreases overall macrophage-to-feces RCT, knockout of both HL and EL leaves RCT essentially unaffected. With respect to important individual steps of RCT, current data on the role of EL and HL in cholesterol efflux are not conclusive. Both enzymes increase hepatic selective cholesterol uptake; however, this does not translate into altered biliary cholesterol secretion, which is regarded the final step of RCT. Also, the impact of HL and EL on atherosclerosis is not clear cut; rather it depends on respective experimental conditions and chosen models. More mechanistic insights into the diverse biological properties of these enzymes are therefore required to firmly establish EL and HL as targets for the treatment of atherosclerotic cardiovascular disease

Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2.

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 (normolipidemic) mice that are expressing human apoE3 (3/3) made in an identical manner. The 2/2 mice are markedly defective in clearing beta-migrating VLDL particles, and spontaneously develop atherosclerotic plaques, even on a regular diet. An atherogenic diet, high in fat and cholesterol, exacerbates development of atherosclerosis and xanthomas in the 2/2 mice. Thus, comparisons between the 2/2 and 3/3 mice unequivocally demonstrate that a single amino acid difference (Arg158 Cys) in the apoE protein is sufficient to cause type III HLP and spontaneous atherosclerosis in mice

Apo E structure determines VLDL clearance and atherosclerosis risk in mice

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 cholesterol in their diet. However, on all diets tested, the 4/4 mice had approximately twice the amount of cholesterol, apo E, and apo B-48 in their VLDL as did 3/3 mice. The 4/4 VLDL competed with human LDL for binding to the human LDL receptor slightly better than 3/3 VLDL, but the VLDL clearance rate in 4/4 mice was half that in 3/3 mice. On an atherogenic diet, there was a trend toward greater atherosclerotic plaque size in 4/4 mice compared with 3/3 mice. These data, together with our earlier observations in wild-type and human APOE*2-replacement mice, demonstrate a direct and highly significant correlation between VLDL clearance rate and mean atherosclerotic plaque size. Therefore, differences solely in apo E protein structure are sufficient to cause alterations in VLDL residence time and atherosclerosis risk in mice