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Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermediate voltage electron microscopy was used to examine monocyte-derived macrophages (monocyte/macrophages) during early in vitro uptake of beta migrating very-low-density lipoproteins (beta VLDL). Lysosomes were identified using acid phosphatase cytochemistry, and in control cells these organelles constituted 3.5% of the total cytoplasmic volume. Both primary and secondary lysosomes were observed. Upon beta VLDL uptake, the total volume of acid-phosphatase-positive organelles increased threefold over 30 minutes, and the reaction product was found in three additional morphologically distinct structures: tubular lysosomes, membrane stacks, and endoplasmic reticulum with widened cisternae. The proportion of the cell occupied by each of the five acid-phosphatase-positive organelles was quantitated at 10 minutes, 30 minutes, 1 hour, and 4 hours of beta VLDL incubation, and their relative abundance was compared with controls that were processed either with no lipoprotein challenge or albumin incubation for 1 hour. Secondary lysosomes compartment volume peaked at 30 minutes; over the ensuing 3.5 hours, however, the reaction progressively shifted to three new membrane-limited locations. Our observations document the complex 3D organization and spacial relationships among the acid-phosphatase-positive structures induced by lipoprotein uptake. The 3D organization patterns for acid-phosphatase-positive lysosomes in lipoprotein-stimulated pigeon monocyte/macrophages were similar in several aspects to the complex lysosomes previously observed in the macrophages of pigeon arterial lesions.

The addition of cholesterol (0.5%) to the diet of White Carneau pigeons induces site specific, temporally predictable, atherosclerotic lesions. The earliest lesions, which occurred after 3 weeks, were small (less than 2500 sq mu in surface area) and were composed primarily of macrophage foam cells (94% of lesion volume). With a prolonged time on the diet the lesions expanded due to increases in the number and size of foam cells, increases in the amount of extracellular space, and influx of smooth muscle cells. Macrophage foam cells in advanced lesions composed 61% of the lesion volume, smooth muscle cells 25%, and extracellular space 14%. Concurrent with the alteration in the constituency of the lesion, redistribution of lipid within foam cells was noted. Lipid in small lesions was primarily cytoplasmic (88%), with the remaining 12% in acid-phosphatase-positive secondary lysosomes. In more advanced lesions, 34% of the lipid was cytoplastic and 66% was lysosomal. The changes in large lesions appeared to be a function of lesion age, because at the growing edge of large lesions both composition and lipid distribution resembled those of small early lesions.

The addition of 0.2% or more cholesterol to the diet of young White Carneau pigeons produced atherosclerotic lesions within 10 weeks in a 2-sq mm area of the lower thoracic aorta. Concurrent with lesion development, a shift in the shape of endothelial cells from fusiform to polygonal was noted. This shift changed the ratio of endothelial cell width to length from 0.34 in pigeons receiving a control diet to 0.50 in pigeons receiving cholesterol. In contrast, endothelial cells in an atherosclerosis-resistant region 6 cm superior to the test region retained their fusiform shape despite the addition of cholesterol to the pigeon's diet. Cholesterol diets also increased adherence of leukocytes to the luminal surface of the aorta. This was most prevalent at the edge of large lesions (2430 +/- 180 cells/sq mm) and over small lesions (2240 +/- 150 cells/sq mm). Leukocyte adherence was also increased in the central region of large lesions (960 +/- 140 cells/sq mm). In addition, leukocyte activation, as evidence by crawling or spreading cells, was increased almost twofold over small lesions and the edge of large lesions when compared with adherent cells over nonlesion areas.

The role of lysosomes in aortic atherogenesis in White Carneau pigeons was examined by means of acid phosphatase cytochemistry. Foam cells were the major constituent of nascent atherosclerotic lesions in pigeons fed a 0.5% cholesterol diet for either 5 or 10 weeks. Seventy-four percent of foam cell lipid from animals at 5 weeks was in cytoplasmic droplets. The remaining lipid appeared in secondary lysosomes. After 10 weeks of cholesterol feeding, lysosomal lipid accounted for 73% of the lipid volume. The lipid accumulation correlated with increases in both size and number of lysosomes. An average of 2.4 lysosomes per 10(4) cu mu of cytoplasm was observed at 5 weeks. This value doubled by 10 weeks. The average lysosome diameter also increased between 5 and 10 weeks from 2.2 mu to 5.75 mu. Concomitantly, the complexity of lysosomes increased from simple, spherical organelles at 5 weeks to complex, multichambered organelles at 10 weeks. In contrast, lipid storage within cytoplasmic lipid droplets did not change either in size or in number. These observations suggest that by 5 weeks lipid storage within cytoplasmic droplets was maximized, and continued increases in lipid stores occurred predominantly through lysosomal loading.