CHARACTERIZATION OF LIPID-LADEN AORTIC-CELLS FROM CHOLESTEROL-FED RABBITS .3. INTRACELLULAR-LOCALIZATION OF CHOLESTEROL AND CHOLESTERYL ESTER

Abstract

The subcellular sites of accumulation of cholesterol and cholesteryl esters in rabbit atheromatous cells were investigated by morphologic and biochemical techniques. EM of lipid-filled cells in situ in atheromatous aortas of cholesterol-fed rabbits revealed lipid accumulation in the cytoplasm as lipid droplets and within lysosomes in the form of lipid globules, membranous whorls and crystals. When such cells were isolated from the rabbit aortas by enzymic digestion and treated with Flickinger''s aldehyde fixative containing 0.2% digitonin, characteristic digitonide-lipid complexes (spicules) were observed in discrete sites of the cytoplasm distinct from the cytoplasmic droplets. If these cells were first stained cytochemically for acid phosphatase and then treated with digitonin-aldehyde fixative, enzyme reaction product was found associated with the spicules, indicating that the lysosomes of the atheromatous cells possess digitonin-reactive lipid. Subcellular fractionation of isolated rabbit aortic foam cells by sucrose density gradient centrifugation demonstrated the coequilibration of most of the intracellular unesterified cholesterol with low density lysosomes. Some cholesteryl ester was also associated with low density lysosomes, although most was found in a lipid droplet fraction of very low density. In rabbit atheromatous cells, lysosomes are apparently the site of accumulation of intracellular cholesterol in excess of that structurally associated with membranes and cytoplasmic droplets. Lysosomes are apparently depot sites for cholesteryl esters.