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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1378-1387.)
© 1995 American Heart Association, Inc.

Articles

Macrophage Uptake of Oxidized LDL Inhibits Lysosomal Sphingomyelinase, Thus Causing the Accumulation of Unesterified Cholesterol–Sphingomyelin–Rich Particles in the Lysosomes

A Possible Role for 7-Ketocholesterol

Irit Maor; Hanna Mandel; Michael Aviram

From the Lipid Research Laboratory and the Pediatric Department (H.M.), Rambam Medical Center, The Bruce Rappaport Faculty of Medicine, Technion, and the Rappaport Family Institute for Research in the Medical Sciences, Haifa, Israel.

Correspondence to M. Aviram, Lipid Research Laboratory, Rambam Medical Center, Haifa, Israel.

Abstract Macrophage uptake of oxidatively modified LDL (Ox-LDL), unlike the uptake of acetylated LDL (Ac-LDL), resulted in lysosomal accumulation of unesterified cholesterol (UC). As sphingomyelin (SM) binds UC with high affinity, we considered whether lysosomes also accumulate Ox-LDL–derived SM, and if such a phenomenon could be involved in the lysosomal trapping of Ox-LDL–derived UC. Incubation of J-774 A.1 macrophages with Ox-LDL increased the lysosomal accumulations of UC by 75% and SM by 63% compared with the effect of Ac-LDL. The addition of chlorpromazine, an inhibitor of lysosomal sphingomyelinase (SMase), to macrophages that were incubated with [³H]cholesteryl ester–labeled Ac-LDL also led to lysosomal accumulation of both SM and UC. 7-Ketocholesterol (7-KC), the major oxysterol in Ox-LDL, inhibited lysosomal SMase in a cell-free system. The addition of 7-KC to cells in the presence of [³H]choline- or [³H]cholesteryl ester–labeled Ac-LDL led to macrophage accumulation of SM or UC, respectively. Niemann-Pick type C disease (NP-C) is an inherited cholesterol-storage disease in which lysosomal SMase activity is attenuated after uptake of LDL. Incubation of monocyte-derived macrophages from two NP-C patients with Ac-LDL or Ox-LDL resulted in an accumulation of UC in the lysosomes, whereas normal monocyte-derived macrophages accumulate UC in their lysosomes after incubation with Ox-LDL but not Ac-LDL. These results suggest that inhibition of lysosomal SMase in NP-C cells or by 7-KC is required for lysosomal accumulation of UC. Analysis of the macrophage lysosomal extract (following cell incubation with Ox-LDL) by density-gradient ultracentrifugation and gel-filtration chromatography revealed the presence of a particle consisting of UC, SM, 7-KC, and apoB-100. We conclude that 7-KC in Ox-LDL can inhibit lysosomal SMase, thus leading to the accumulation of SM, which binds UC avidly and inhibits its further cellular processing out of the lysosome. As UC-SM particles of lysosomal origin exist in the atherosclerotic lesion, the formation of such particles may result from an impaired processing of Ox-LDL by arterial wall macrophages during early atherogenesis.

Key Words: unesterified cholesterol • oxidized LDL • sphingomyelinase • sphingomyelin • macrophages

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