Inhibition of intestinal absorption of cholesterol by ezetimibe or bile acids by SC-435 alters lipoprotein metabolism and extends the lifespan of SR-BI/apoE double knockout mice

Abstract

SR-BI/apoE double knockout (dKO) mice exhibit many features of human coronary heart disease (CHD), including hypercholesterolemia, occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, cardiac dysfunction and premature death. Ezetimibe is a FDA-approved, intestinal cholesterol absorption inhibitor that lowers plasma LDL cholesterol in humans and animals and inhibits aortic root atherosclerosis in apoE KO mice, but has not been proven to reduce CHD. Three-week-ezetimibe treatment of dKO mice (0.005% (w/w) in standard chow administered from weaning) resulted in a 35% decrease in cholesterol in IDL/LDL-size lipoproteins, but not in VLDL- and HDL-size lipoproteins. Ezetimibe treatment significantly reduced aortic root (57%) and coronary arterial (68%) atherosclerosis, cardiomegaly (24%) and cardiac fibrosis (57%), and prolonged the lives of the mice (27%). This represents the first demonstration of beneficial effects of ezetimibe treatment on CHD. The dKO mice were similarly treated with SC-435 (0.01% (w/w)), an apical sodium codependent bile acid transporter (ASBT) inhibitor, that blocks intestinal absorption of bile acids, lowers plasma cholesterol in animals, and reduces aortic root atherosclerosis in apoE KO mice. The effects of SC-435 treatment were similar to those of ezetimibe: 37% decrease in ILD/LDL-size lipoprotein cholesterol and 57% prolongation in median lifespan. Thus, inhibition of intestinal absorption of either cholesterol (ezetimibe) or bile acids (SC-435) significantly reduced plasma IDL/LDL-size lipoprotein cholesterol levels and improved survival of SR-BI/apoE dKO mice. The SR-BI/apoE dKO murine model of atherosclerotic occlusive, arterial CHD appears to provide a useful system to evaluate compounds that modulate cholesterol homeostasis and atherosclerosis.

Figure 1. Effects of ezetimibe on plasma lipids and lipoproteins

dKO mice (animal number in parentheses) were fed standard chow without (‘no drug’, black bars or symbols) or with ezetimibe (white bars or symbols) from weaning. Plasma was harvested at 39–44 days of age. A. Average plasma lipid concentrations for total cholesterol (TC), phospholipids (PL), and triglycerides (TG). B. Plasma lipoprotein cholesterol profiles determined by FPLC size fractionation (TC, mg/dL plasma, average of profiles from six untreated and seven ezetimibe-treated mice). Brackets indicate sizes of standard human lipoproteins. C. For each mouse, total cholesterol levels in the indicated pooled fractions corresponding to VLDL-, IDL/LDL- or HDL-size particles were summed and averages for six untreated and seven ezetimibe-treated mice were calculated. * P=0.043 for no drug vs. ezetimibe-treated values.

Figure 2. Effects of ezetimibe on atherosclerosis and heart disease

dKO mice (animal number in parentheses) were fed standard chow without (‘no drug’, black bars or symbols) or with ezetimibe (white bars or symbols) from weaning. Hearts were harvested at 39–44 days of age. A. Oil red O-stained lesions in the aortic root. Average lesion areas (mm², horizontal lines) were: no drug, 0.109±0.032 (n=4); ezetimibe 0.047±0.037 (n=6, P=0.024). B. Coronary arterial atherosclerosis. Left: Representative oil red O stained sections of coronary arteries (neutral lipid stains red) from untreated (no drug, top) and ezetimibe-treated (bottom) mice. Bar: 50 μm. Right: Extent of coronary arterial occlusions (average percent of coronary vessels with 0–10%, 10–50% or 50–100% occlusions). *P ≤ 0.01 for no drug vs. ezetimibe-treated. C. Cardiac fibrosis. Left: Representative trichrome-stained (healthy myocardium, red; fibrotic tissue, blue) transverse cryosections of hearts from untreated (no drug; top) or ezetimibe-treated (bottom). RV: right ventricle; LV: left ventricle; arrowhead: myocardial infarct; * extensive fibrosis in the outflow tract area. Bar: 500 μm. Right: Quantitative analysis of cardiac fibrosis. *P = 0.015 for no drug vs. ezetimibe-treated values. D. Heart-to-Body Weight Ratios. * P<0.001 for comparison to no drug control.

Figure 3. Effect of ezetimibe on survival

Survival curves for dKO mice fed standard chow without (‘no drug’, black line) or with ezetimibe (grey line) from weaning. Inset indicates median survival time (animal number in parentheses); P < 0.0001.

Figure 4. Effects of the ASBT inhibitor SC-435 on plasma lipids, lipoprotein profiles and survival

dKO mice were fed standard chow without (‘no drug’; black line, bars or symbols) or with SC-435 (‘ASBT inh.’; grey line or white bars or symbols) from weaning. A and B. Lipoprotein cholesterol profiles, plasma total cholesterol (TC) levels and lipoprotein cholesterol determined at 40–41 days of age. A. Lipoprotein cholesterol profiles (averages from four untreated and seven SC-435-treated mice). Inset shows the average plasma levels of TC. B. For each mouse, TC levels in the indicated pooled fractions corresponding to VLDL-, IDL/LDL- or HDL-size particles were summed and averages of four untreated and seven ASBT inhibitor-treated mice were calculated. * P=0.009 for no drug vs. SC-435-treated. C. Survival curves. Inset indicates median survival time (animal number in parentheses); P < 0.0001. Mice housed at the Massachusetts (panels A and B) or Missouri (panel C) facilities. SC-435 treatment also prolonged the lives of dKO mice in a much smaller scale survival study in the Massachusetts facility (n=3/group), although to a lesser extent (2 week extension; P=0.04).