Moderately decreased cholesterol absorption rates are associated with a large atheroprotective effect

Abstract

Objective: Human treatment with ezetimibe results in a moderate 50% to 54% decrease in cholesterol absorption and a 15% to 20% decrease in plasma LDL-cholesterol levels; nevertheless, the efficacy of ezetimibe therapy has been recently challenged by the ENHANCE trial. We examined the efficacy of a moderate decrease in cholesterol absorption in preventing atherosclerosis formation in the mouse.

Methods and results: Congenic 14DKK animals, consisting of a castaneus (CASA/Rk) chromosome 14 interval introgressed onto the C57BL/6J background, displayed a moderate decrease in cholesterol absorption rates. The effect of moderately decreased absorption on atherosclerosis formation was determined in 14DKK apolipoprotein E knockouts (14DKK-apoEKO). When compared to chow diet-fed control apoEKO mice, congenic 14DKK-apoEKO displayed a moderate 41% decrease in cholesterol absorption rates, 30% to 37% decrease in plasma cholesterol levels, and a 70% decrease in atherosclerosis formation. Studies on cholesterol efflux and reverse cholesterol transport (RCT) from 14DKK bone marrow-derived macrophages rejected a 14DKK interval-dependent atheroprotective effects that operate in macrophages. In contrast, 14DKK-apoEKO congenics were characterized by a 60% increase in RCT from peripheral tissue macrophages.

Conclusions: These studies strongly suggest that moderately decreased cholesterol absorption rates result in a large atheroprotective effect attributable to a decrease in plasma cholesterol levels and an increase in RCT from peripheral tissue macrophages.

Figure 1

Effect of the 14DKK interval on cholesterol absorption from the intestine. Control apoEKO and 14DKK-apoEKO males were placed in metabolic cages, received a gastric bolus of olive oil supplemented with radiolabeled cholesterol and β-sitostanol, and cholesterol absorption rates determined as described in the Methods section (N=6 per group; values shown are mean ± SD).

Figure 2

Effect of the 14DKK interval on plasma cholesterol levels. Plasma cholesterol levels were determined in fasted animals as described in the Methods section. (N=14–19 animals per group; values shown are mean ± SD).

Figure 3

Plasma lipoprotein cholesterol profiles in apoEKO and 14DKK-apoEKO animals: ApoEKo and 14DKK-apoEKO animals were fasted, plasma samples isolated, plasma of animals in each group pooled, and plasma lipoprotein cholesterol profiles were analyzed as described in the Methods section (N=5 males in each group).

Figure 4

Effect of the 14DKK interval on aortic root atherosclerotic lesions. Chow diet fed control apoEKO and 14DKK-apoEKO congenics males (A) and females (B) were fed with rodent chow diet, sacrificed at 16 weeks of age, and atherosclerosis lesion area at the aortic root was determined as described in the Methods section (N=14–19 animals per group).

Figure 5

Effect of the 14DKK interval on in-vitro cholesterol efflux and in-vivo RCT from peripheral tissue macrophages. (A) Bone marrow macrophages (BMM) from control C57BL/6J and 14DKK congenic animals were loaded with cholesterol and labeled with ³[H]cholesterol. Cholesterol efflux was determined in macrophages incubated in absence or presence of the indicated concentrations of human HDL protein. Percent cholesterol efflux was determined as described in the Methods section. Data is a representative one experiment out of a total of three experiments. (B) Donor bone marrow macrophages of control C57BL/6J and 14DKK congenics were loaded with cholesterol and labeled with radiolabeled cholesterol and β-sitostanol. Labeled donor macrophages were injected into recipient C57BL/6J control animals and RCT determined as described in the Methods section (N=5 per group; values shown are mean ± SD).

Figure 6

Effect of the 14DKK interval on RCT from apoEKO bone marrow macrophages: Donor bone marrow macrophages of control apoEKO were loaded with cholesterol, injected subcutaneously into recipient apoEKO and 14DKK-apoEKO congenic animals and RCT was determined as described in the Methods section (N=6 nimals per group; values shown are mean ± SD).