Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse

Abstract

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5-6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between approximately 3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease.

Fig. 1.

Effects of probucol on survival. (A) dKO mice (red line, n = 13, chow diet, ref. 1), dKO-P mice (blue line, n = 10, 0.5% probucol diet from conception), and dKO-Pww mice (black line, n = 9, probucol diet from conception until weaning). (B) dKO-P<5 mice (probucol diet administered only after weaning but before 5 weeks of age, green line; n = 9) and dKO-P>5 (probucol diet administered immediately after 5 weeks of age, purple line; n = 7).

Fig. 2.

Effects of probucol on heart function and size. Parental and offspring mice were fed either a low-fat chow or probucol (0.5% wt-wt)-supplemented diet. (A) Representative short-axis MRI images of hearts from mice (Upper, untreated dKO, n = 4; Lower, probucol-treated dKO-P, n = 5) at end diastole (Left) and end systole (Right). Blood-filled heart chambers are white, and arrows indicate left ventricles (LV). (B) Representative ECGs from 6-week-old unanesthetized, untreated (Upper, dKO; ref. 1) or probucol-treated (Lower, dKO-P, n = 11) mice. (C) Mean values (±standard deviations) of heart weights normalized to body weights for 6-week-old untreated (Left) or probucol-treated (Right) apoE KO (open bars) and dKO (filled bars) mice. Untreated dKO hearts were statistically different from the others (*, P < 0.0001, ANOVA test). Probucol treatment significantly altered the weights of dKO (P < 0.0001, t test) but not apoE KO (P = 0.09) hearts. Numbers of mice studied were: dKO, 18; dKO-P, 11; apoE KO, 8; and apoE KO-P, 4.

Fig. 3.

Histologic analysis of hearts. (A–F) Representative longitudinal sections of hearts from 6-week-old untreated dKO (A and D), probucol-treated dKO-P (B and E), and untreated apoE single KO (C and F) mice stained with Masson's trichrome (healthy myocardium, red; fibrotic infarcted tissue, blue). (G and H) Oil red O-stained sections through the aortic sinuses of probucol-treated dKO (G) and apoE KO (H) mice. (Scale bars: A–C, 1 mm; D–F,10 μm; G and H, 100 μm.)

Fig. 4.

Lipoprotein cholesterol profiles and morphology. Parental and offspring mice were fed a low-fat chow diet without or with probucol [0.5% (wt/wt) supplementation]. (A) Representative chromatograms of the TC (mg/dl of plasma) from size-fractionated (Superose 6-FPLC) plasma lipoproteins from 6-week-old untreated dKO (•) or probucol-treated dKO (○) mice are shown with the approximate elution positions of VLDL, intermediate-density lipoproteins (IDL)/LDL, and HDL. (Inset) Expanded HDL region. (B) Electron micrographs of negatively stained lipoproteins from pooled FPLC fractions. Abnormal morphologies are indicated by arrows (lamellar/vesicular), arrowheads (stacked discoidal), and asterisks (aggregated). (Scale bar = 60 nm.)

Fig. 5.

Effects of probucol on RBCs. RBCs from 5-week-old untreated (-) or probucol-treated (+) apoE KO and dKO mice were examined by either transmission electron microscopy (Upper) or confocal fluorescence microscopy after staining with the cholesterol-binding, blue fluorescing dye filipin (Lower). [Scale bar (Upper) = 2 μm; magnification (Lower) = ×100.)