Purification and partial characterization of extracellular liposomes isolated from the hyperlipidemic rabbit aorta

Abstract

Extracellular liposomes (EL) that accumulated in the aortic intima of rabbits on 2 weeks (prelesional stage) and 16 weeks (lesional stage) of diet-induced hyperlipidemia were isolated and purified by gel filtration, ultracentrifugation, and affinity chromatography on anti-apoB and anti-albumin Sepharose. The material obtained after each step was examined by negative staining electron microscopy, by protein analysis (SDS-PAGE, immunoblotting, autoradiography, uronic acid), and by lipid analysis for unesterified cholesterol (UC), cholesteryl esters (CE), phospholipids (PL), triglycerides (TG), thiobarbituric acid reactants (TBAR). EL represented the major constituent of intimal lipid deposits; their predominance on particulate beta-lipoproteins (LP) increased with the duration of hyperlipoproteinemia. As compared with serum low density lipoproteins (LDL) and beta-very low density lipoproteins (beta-VLDL), the crude EL fraction obtained after gel filtration and ultracentrifugation had a decrease in CE and TG, with augmentation of UC, PL, and apoB. After removal of apoB and some albumin by immunoadsorption, the purified EL fraction consisted only of UC, PL, and albumin. The albumin was resistant to proteolytic digestion with pronase, and reacted with anti-albumin antibody only after delipidation of EL. This indicated that albumin was trapped in the aqueous core of vesicles, presumably acting as a scavenger of oxygen-free radicals. TBAR was highly associated with intact or degraded beta-LP. The EL that accumulate in the aortic intima of hyperlipidemic rabbits represent the predominant form of lipid deposits, resulting from the transcytosed excess beta-LP, which is degraded and reassembled upon interaction with the extracellular matrix components.