Localization of unesterified cholesterol in human atherosclerotic lesions. Demonstration of filipin-positive, oil-red-O-negative particles.

Both unesterified and esterified cholesterol accumulate in human atherosclerotic lesions. Whereas previous studies have established that esterified cholesterol deposits intra- and extracellularly, less is known concerning the distribution of lesion unesterified cholesterol. The objective of this study was to establish the location and in what structures unesterified cholesterol accumulates in lesions. The fluorescent probe filipin has been used to detect unesterified cholesterol. In addition, the lipid-soluble dye oil red O (which does not stain unesterified cholesterol) was used to stain hydrophobic lipids, including esterified cholesterol. Filipin staining occurred in association with three extracellular structures: spherical particles, elongated crystals, and granular or amorphous calcium deposits. These structures were not stained by oil red O. Filipin-stained particles sometimes accumulated within cells, which did not contain any oil-red-O-stained lipid. Interestingly, extracellular filipin-stained particles occurred in loci separate from extracellular oil-red-O-stained particles. The results of this study suggest that accumulation of unesterified and esterified cholesterol occurs within many diverse structures in atherosclerotic lesions. Extracellular filipin-stained particles constituted a significant component of accumulated cholesterol. These cholesterol-rich particles have not been previously observed because they are not stained by lipid-soluble dyes such as oil red O.