Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions: A cytochemical and ultrastructural study

Abstract

In atherosclerotic lesions of rabbits fed a cholesterol-rich diet, the lipid deposits of foam cells derived from monocytes, smooth muscle and endothelial cells were studied by physical, cytochemical and ultrastructural methods. Beginning with the third week of diet, the lipid material that could be visualized at the light microscope level by Oil red O and Nile red staining was progressively accumulated in the intimal cells of the atherosclerotic lesions. In the early stages of foam cell formation, the deposits occurred especially as intracytoplasmic non-membrane bound lipid inclusions (lipid droplets). In polarizing microscopy these appeared as a mixture of iso-, and anisotropic material. The latter were birefringent and showed an axial symmetry with a black cross image, suggesting that the lipids were in a liquid crystalline state. In chemically-fixed specimens, the content of lipid inclusions was preserved in various degrees. In freeze-fractured preparations they displayed a layered onion-like arrangement with smooth cleavage faces surrounding an amorphous core. Upon incubation with filipin, that specifically binds to 3β-hydroxysterols, the peripheral layers of the inclusions were labeled, revealing the existence of unesterified cholesterol. In the advanced stages of foam cell formation, lipids were additionally accumulated in the lysosomal compartment as polymorphic multilamellar structures concentrically arranged, with cleavage faces devoid of intralamellar particles. The presence of acid phosphatase showed that these features were modified lysosomes and were tentatively named lysosomal lipid bodies. In the latest stages examined cholesterol crystals developed within lysosomal lipid bodies usually enclosed in multilamellar structures. This lipid coat may represent the place of crystal formation and presumably acts as barrier for the turnover of the crystalline cholesterol, thus impeding plaque regression.