Lysosomes of the arterial wall: II. Subcellular fractionation of aortic cells from rabbits with Experimental Atheroma

Abstract

Cells were isolated from the aortas of control rabbits and of rabbits with experimental atheroma induced by cholesterol feeding. They were homogenized and fractionated by three different techniques relying mainly on differences in equilibrium density of cell particles in sucrose gradients. After cholesterol feeding, the aortic smooth muscle cells transformed progressively into foamy cells. Their protein to DNA ratio was unchanged. The mitochondrial marker cytochrome oxidase and the presumed plasma membrane markers 5′-nucleotidase and leucyl-β-naphthylamidase showed no significant change in specific activity, nor in density distribution, even in severely atheromatous lesions. Only minimal changes were seen for α-glucosidase, an enzyme probably associated largely with microsomes. Catalase was increased up to 7-fold; one-third of this activity was in particles of about 1.18 density, the rest was soluble. The specific activity of all lysosomal acid hydrolases increased with increasing severity of atheroma, up to a little more than 2-fold for N-acetyl-β-galactosaminidase and α-mannosidase, 3- to 4-fold for N-acetyl-β-glucosaminidase, acid phosphatase, and cathepsin C, more than 10-fold for β-galactosidase and β-glucuronidase. At the same time the lysosome became less fragile and their density distribution underwent a progressive broadening in the direction of decreasing density, extending to a value as low as 1.03. Incipient changes in lysosome density could be detected already in the early stages of the cholesterol-induced atheromatous transformation and in animals with moderate atheroma induced by a diet rich in saturated fatty acids. The cholesterol content of the cells increased up to 16-fold and the distribution of this substance was altered from a plasma membrane type in normal smooth muscle cells to one resembling that of lysosomal enzymes in foamy cells. Most likely, cholesterol storage is responsible for the decrease in lysosome density, and cholesterol-containing vacuoles in foamy cells are of lysosomal nature. Much of this cholesterol was in esterified form. In other experiments, no age- or sex-related changes in the aortic cell organelles could be demonstrated. After repeated intravenous injection of Triton WR-1339 there was a significant decrease in equilibrium density of the lysosomes indicating uptake of the detergent by this organelle. No change was observed after repeated injection of dextrans of various molecular weight, injection of neutral red, or starvation.