Lysosomal lipid accumulation is a ubiquitous but poorly understood aspect of atherosclerosis in man and susceptible animals. The importance of this accumulation is suggested by its spatial and temporal association with important events in development of lesions from fatty streaks to plaques. Moreover, lysosomally sequestered cholesterol in lesion cells appears trapped and less available for efflux than that in cytoplasmic stores. Prolonged incubation of pigeon and THP1 macrophages (a human cell line) with oxidized (ox-) LDL produces accumulation of both free (FC) and esterified cholesterol (CE) in lysosomes similar to that in lesions. Morphological and biochemical studies show that an initial build up of lysosomal FC is followed by lysosomal CE accumulation. This suggests that initially, lipoprotein CE is hydrolyzed to FC but that subsequent to this, hydrolysis is impaired. We hypothesize that lysosomal FC accumulation, produced by inhibition of FC movement out of lysosomes, is a mediator of the impairment of hydrolysis. This proposal will test this hypothesis in order to define mechanism(s) of lysosomal accumulation. Using a combination of biochemical and structural tools the effects of lysosomal FC accumulation on lysosomal CE hydrolysis and exit of FC from lysosomes will be explored. Treatment of human cells with acetylated (ac-)LDL does not produce lysosomal accumulation, even in cells with equivalent cholesterol levels. Differences in responses of cells to ox- or ac-LDL loading will be used to identify potentially important factors for lysosomal engorgement. Mouse macrophages do not exhibit lysosomal loading with either ox- or ac-LDL. Based on this observation, suspected factors, principally FC, will be tested for the ability to induce lysosomal loading in mouse cells. Finally, specific lysosomal factors, such as sphingomyelin content or enzyme trafficking will be examined for the ability to trap FC and/or inhibit hydrolysis. This will determine the potential of these suspected factors under culture conditions to mediate lysosomal lipid accumulation. The analytical approach of integrating quantitative microscopic and biochemical data is designed to provide a broader analysis of the cellular consequences of specific manipulations. Completion of the aims will significantly expand understanding of lysosomal FC and CE accumulation and provide information to begin determining the role of this accumulation in atherogenesis.