Apo E structure determines VLDL clearance and atherosclerosis risk in mice

Abstract

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.

Figure 1

(a) Mice with the 3 human apo E isoforms. Delipidated plasma samples from 2/2 (lane 2), 3/3 (lane 3), and 4/4 (lane 4) mice were separated by isoelectric focusing, and the apo E proteins were viewed by silver staining after immunofixation with an anti-human apo E antibody. The positions of the human apo E isoforms — E2, E3, and E4 — are indicated. Lane 1 shows apo E2, apo E3, and apo E4 from mixed human plasma as controls. (b) Competition assay of human LDL receptor binding and internalization of VLDL from 2/2, 3/3, and 4/4 mice. VLDL was isolated from 2/2 (filled squares), 3/3 (filled circles), and 4/4 (open triangles) mice maintained on an HFC diet by ultracentrifugation of plasma pooled from at least 6 animals, and it was used to compete with DiIC₁₈-labeled human LDL for binding to human fibroblasts. Values are the mean ± SD of 4 wells from a representative experiment carried out at 37°C. VLDL amounts are expressed as micrograms of total protein. The apo E content of the isolated VLDL was approximately 17 ± 3%, 7 ± 3%, and 4 ± 1% of total protein for 2/2, 3/3, and 4/4 VLDL, respectively (P < 0.001 by one-way ANOVA).

Figure 2

Distribution of plasma lipoproteins, apo E, and apo B-100 in mice fed normal chow (a–e), HFW (f), and HFC (g). Pooled plasma (100 μL) from 6 age-matched female mice was fractionated by gel filtration chromatography on a Superose 6B column, and 0.5-mL fractions were collected. Fractions containing VLDL, IDL/LDL, and HDL are shown. (a) Total cholesterol was measured in micrograms per fraction. (b) Triglycerides were measured in micrograms per fraction. (c) Apo E (micrograms per fraction) was measured using an ELISA with antibodies specific for human apo E. (d) Apo B-100 was measured using an ELISA with antibodies specific for mouse apo B-100 and is expressed as arbitrary units per fraction. (e) Apo B-100 and apo B-48 of isolated VLDL (80 μL plasma equivalent) or total lipoproteins (25 μL plasma equivalent) from 3/3 and 4/4 mice were viewed by Coomassie blue staining after SDS-PAGE on 3–20% gradient gel. (f) Total cholesterol was measured in micrograms per fraction in at least 5 female mice maintained on an HFW diet for 2 months. (g) Total cholesterol was measured in micrograms per fraction in at least 5 female mice maintained on an HFC diet for 2 months.

Figure 3

VLDL cholesterol in 3/3 and 4/4 mice on 3 different diets. VLDL from plasma of 6 individual female mice maintained on normal chow (a), HFW diet (b), or atherogenic diet (HFC) (c) was isolated by ultracentrifugation. Amounts are expressed in milligrams of cholesterol per deciliter of plasma ± SD (n = 6 for all groups).

Figure 4

The plasma clearance of VLDL in 3/3 and 4/4 mice. Three male mice maintained on normal chow of each group (filled circles, 3/3; open triangles, 4/4) were injected with radiolabeled remnant particles from apo E–deficient donor mice (a) or from donor mice of the same genotype (b), and the fraction of radioactivity remaining in plasma was measured for 2 hours. Each data point shows mean ± SD. The SD of some data is smaller than the data points.

Figure 5

Correlation between atherosclerotic lesion size and circulation time of VLDL particles. Atherosclerotic lesion area from at least 6 female mice maintained on an HFC diet for 3 months was determined. The log of the mean lesion size was plotted against β-VLDL retention time (inverse of the fractional catabolic rate; Figure 4 and refs. 15, 16), as determined from at least 3 mice injected with apo E–deficient labeled VLDL. The symbols are as follows: wild-type (open diamond), 2/2 (filled square), 3/3 (filled circle), and 4/4 (open triangle).