Evaluation of Antiatherogenic Properties of Ezetimibe Using³H-Labeled Low-Density-Lipoprotein Cholesterol and^99mTc-cAbVCAM1–5 SPECT in ApoE^−/−Mice Fed the Paigen Diet

Abstract

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E^-/- mice. <b>Methods:</b> Apolipoprotein E^-/- mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (<i>n</i> = 15), we intravenously injected ³H-cholesteryl oleate-labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (<i>n</i> = 20), we used the imaging agent ^99mTc-cAbVCAM1-5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (<i>n</i> = 21), we compared VCAM-1 expression with ^99mTc-cAbVCAM1-5 uptake in various tissues. <b>Results:</b> Mice treated with ezetimibe showed a 173% higher LDL-cholesteryl ester plasma disappearance rate (<i>P</i> < 0.001 vs. control) after ³H-cholesteryl oleate-labeled LDL injection. At 96 h after injection, the hepatic fraction of ³H-tracer was 61% lower in mice treated with ezetimibe (<i>P</i> < 0.001). Meanwhile, LDL-derived ³H-cholesterol excretion in the feces was 107% higher (<i>P</i> < 0.001). The antiatherogenic effect of ezetimibe monitored by ^99mTc-cAbVCAM1-5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; <i>P</i> < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with ^99mTc-cAbVCAM1-5 uptake (<i>r</i> = 0.75; <i>P</i> < 0.05). <b>Conclusion:</b> Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.