Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100

Abstract

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.

Figure 1

Lipid and lipoprotein levels in Apoe^–/–Apob^100/100 (n = 44), Apoe^–/–Apob^+/+ (n = 40), Ldlr^–/–Apob^100/100 (n = 42), and Ldlr^–/–Apob^+/+ (n = 40) mice. The top panel shows the mean plasma cholesterol levels at 8, 16, 24, 32, and 40 weeks of age; error bars represent SEM. When all five cholesterol values from the different time points were averaged, the total cholesterol level was somewhat higher in Ldlr^–/–Apob^100/100 than in Apoe^–/–Apob^100/100 mice (298 ± 5 versus 280 ± 7 mg/dl; P < 0.05). The cholesterol levels in Ldlr^–/–Apob^100/100 and Apoe^–/–Apob^100/100 mice were significantly different from those in the two control groups (P < 0.0001). The middle panel shows the distribution of cholesterol within the lipoprotein fractions. The bottom panel shows the plasma triglyceride levels. Mean triglyceride levels (from all measurements) were significantly higher in the Apoe^–/–Apob^100/100 mice (150 ± 5 mg/dl) than in the other groups (88 ± 4 mg/dl for Apoe^–/–Apob^+/+ mice, 89 ± 2 mg/dl for Ldlr^–/–Apob^100/100 mice, and 91 ± 3 mg/dl for Ldlr^–/–Apob^+/+ mice; P < 0.0001 for each comparison).

Figure 2

Lipoprotein sizes in the four different groups of mice. The median diameter of lipoproteins in Apoe^–/–Apob^100/100 mice (n = 15) was 140% larger than in Ldlr^–/–Apob^100/100 (n = 21) mice, 90% larger than in Ldlr^–/–Apob^+/+ mice (n = 17), and 50% larger than in Apoe^–/–Apob^+/+ mice (n = 16). The size of VLDL (d < 1.006 g/ml) particles ranged from an average of 33.4 nm in Ldlr^–/–Apob^100/100 mice to 61 nm in Apoe^–/–Apob^100/100 mice; the size of IDL (d = 1.006–1.020 g/ml) particles ranged from an average of 27 nm in Ldlr^–/–Apob^100/100 mice to 38 nm in Apoe^–/–Apob^100/100 mice; the size of LDL (d = 1.020–1.052 g/ml) particles was 23 nm in Apoe^–/–Apob^100/100 plasma, 20 nm in Apoe^–/–Apob^+/+ plasma, 22 nm in Ldlr^–/–Apob^100/100 plasma, and 19 nm in Ldlr^–/–Apob^+/+ plasma. The difference in size between the bottom and top deciles of particles was 64.0 nm for Apoe^–/–Apob^100/100 mice, 27.2 nm for Apoe^–/–Apob^+/+ mice, 17.8 nm for Ldlr^–/–Apob^+/+ mice, and 17.0 nm for Ldlr^–/–Apob^100/100 mice.

Figure 3

Apo-B100 levels in Apoe^–/–Apob^100/100 (n = 36) and Ldlr^–/–Apob^100/100 mice (n = 34) at 16 and 32 weeks of age. Apo-B100 levels were not measured in the two control groups (Apoe^–/–Apob^+/+ and Ldlr^–/–Apob^+/+) because the RIA is specific for apo-B100, and the plasma of those mice contains both apo-B100 and apo-B48. Previously, however, we showed that Apoe^–/–Apob^+/+ mice have lower apo-B100 levels in their plasma than Apoe^–/–Apob^100/100 mice, and that Ldlr^–/–Apob^+/+ mice have lower apo-B100 levels than Ldlr^–/–Apob^100/100 mice (50).

Figure 4

Morphometric assessment of atherosclerotic lesions in Apoe^–/–Apob^100/100 (n = 44), Apoe^–/–Apob^+/+ (n = 40), Ldlr^–/–Apob^100/100 (n = 42), and Ldlr^–/–Apob^+/+ (n = 40) mice. The top panel shows the extent of atherosclerotic lesions in the entire aorta. Differences between all groups were significant at the P < 0.0001 level with one exception: the lesions in Apoe^–/–Apob^+/+ mice were different from those in Apoe^–/–Apob^100/100 mice at P = 0.0004. The bottom panel shows the extent of lesions in the top and middle third of the aorta. The top and middle thirds of the aortas contained greater than 90% of the total aortic lesions.

Figure 5

Representative Sudan IV–stained thoracic aortas. The amount of atherosclerosis in these four aortas matched the mean level for each group.

Figure 6

Oil Red O–stained sections of proximal aortic roots.

Figure 7

Relationship between atherosclerotic lesions and the total plasma cholesterol levels and plasma apo-B100 levels in Apoe^–/–Apob^100/100 (n = 41) and Ldlr^–/–Apob^100/100 (n = 40) mice. The top shows a plot lot of lesions, as assessed by morphometric techniques, versus total plasma cholesterol levels (mean of the five measurements). The bottom shows a plot of lesions versus the plasma apo-B100 levels (measured at 32 weeks).

Figure 8

Scoring of aortic pathology in the four groups of mice according to the aortic content of free and esterified cholesterol and aortic DNA synthesis rate. (a) Cholesterol ester content of aortas in Apoe^–/–Apob^100/100 (n = 43), Apoe^–/–Apob^+/+ (n = 38), Ldlr^–/–Apob^100/100 (n = 39), and Ldlr^–/–Apob^+/+ (n = 34) mice. (b) Free cholesterol content of aortas in the four different groups of mice (numbers of mice are identical to those for a). (c) Aortic DNA synthesis rates in Apoe^–/–Apob^100/100 (n = 18), Apoe^–/–Apob^+/+ (n = 17), Ldlr^–/–Apob^100/100 (n = 25), and Ldlr^–/–Apob^+/+ (n = 14) mice.

Figure 9

Mean extent of atherosclerotic lesions plotted against the mean total plasma cholesterol level. The atherosclerosis data (assessed by morphometric techniques) and the cholesterol data represent means calculated from all of the mice in each group. The steep increase in atherosclerosis in the LDL receptor–deficient mice between total cholesterol concentrations of ∼200 and 300 mg/dl may be a consequence of the small apo-B–containing lipoproteins in those mice. However, this finding may in part reflect the lower HDL cholesterol levels in Ldlr^–/–Apob^100/100 mice than in Ldlr^–/–Apob^+/+ mice (see Figure 1).