Dietary Correction of Hypercholesterolemia in the Rabbit Normalizes Endothelial Superoxide Anion Production

Abstract

Background We have shown that hypercholesterolemia increases vascular superoxide anion (O₂⁻) production, which could be responsible for augmented inactivation of endothelium-derived vascular relaxing factor. We sought to determine whether this increased vascular O₂⁻ production is due to infiltration of macrophages into the intima and whether dietary treatment of hypercholesterolemia normalizes O₂⁻ production. Methods and Results A specific and sensitive assay for O₂⁻ based on chemiluminescence of lucigenin was used; the amount of O₂⁻ produced by vascular ring segments was quantified based on known quantities of O₂⁻ produced by xanthine–xanthine oxidase standards. O₂⁻ production of aortic segments from normal rabbits (n=9), cholesterol-fed rabbits (1% cholesterol diet for 1 month, n=7), and rabbits fed a 1% cholesterol diet for 1 month followed by a normal diet for 1 month (regression rabbits, n=5) was measured. At the end of these diets, serum cholesterol levels were 1.5±0.2, 26.0±3.9, and 1.8±0.5 mmol/L (58±6, 1000±150, and 71±19 mg/dL) in the normal, cholesterol-fed, and regression animals, respectively. Vessels from normal rabbits with endothelium produced 0.32±0.06 nmol O₂⁻/mg dry wt per minute, whereas those without endothelium produced approximately twice as much O₂⁻ (0.66±0.12 nmol O₂⁻ mg dry wt per minute. Vessels with endothelium from cholesterol-fed rabbits produced 4.5-fold more O₂⁻ than vessels from normal animals. This increased production of O₂⁻ was normalized by endothelial removal. This increased production of O₂⁻ was not due to infiltration of macrophages in the intima, because there was no correlation between vascular O₂⁻ production and macrophage infiltration assessed by immunohistochemistry with use of a specific antibody against rabbit macrophage. O₂⁻ production by vessels from regression rabbits was similar to that observed in normal animals, and as in the normal rabbits, endothelial removal increased O₂⁻ production. Aortic rings from these animals also were studied in organ chambers. Dietary lowering of cholesterol dramatically improved vasodilator responses to acetylcholine and A23187 (P<.05 versus cholesterol-fed rabbits). Conclusions Dietary lowering of cholesterol not only improves endothelium-dependent vascular relaxation but also normalizes endothelial O₂⁻ production. Decreases of O₂⁻ production by dietary lowering of cholesterol not only may improve vasomotor control but also may improve other aspects of vascular integrity in atherosclerosis.