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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

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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

	Introduction

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Accumulation of UC in the atherosclerotic lesion occurs in both rabbits and humans.^{1 2 3 4} We have shown⁵ that the CE moiety of Ox-LDL, as opposed to its apo B-100, is hydrolyzed normally in the macrophage lysosomes. The resulting UC, however, is trapped in the lysosomes, secondary to the effect of oxidized sterols that are present in Ox-LDL.⁵ UC is associated with phospholipids, including SM, in cell membranes^{6 7 8} as well as on the surface of plasma lipoproteins.⁹ There is a close relation between the metabolism of SM and cholesterol. Membrane SM regulates cholesterol absorption in intestinal cells¹⁰ and cholesterol biosynthesis in cell cultures.¹¹ SM is also involved in cellular cholesterol trafficking¹² as well as cellular binding and uptake of LDL.¹³ Moreover, when cells are deprived of or enriched with SM, macrophage acyl coenzyme A:cholesterol acyltransferase is stimulated or inhibited, respectively.¹⁴ NP-C, a genetic cholesterol lipidosis characterized by increased levels of SM and UC in lysosomes, provides an in vivo model with which to study the interaction between lysosomal SM and UC and reduced SMase activity after cell incubation with LDL.¹⁵ The attenuated lysosomal SMase activity in NP-C fibroblasts is probably mediated by some lipoprotein constituent, as this inhibition can be corrected by removal of LDL from the culture medium.^{16 17}

The mechanism responsible for the accumulation of excess UC and its possible interaction with SM within the lysosomes, however, has not been elucidated. Since Ox-LDL–derived UC is trapped in the lysosomes⁵ and SM binds UC with high affinity,^{18 19 20 21 22} we inquired whether macrophage lysosomes accumulate Ox-LDL–derived SM (in addition to UC) and if such a phenomenon could be responsible for lysosomal trapping of the lipoprotein-derived UC. Our results showed that inhibition of lysosomal SMase by 7-KC (the major oxysterol in Ox-LDL) leads to lysosomal accumulation of Ox-LDL–derived SM. This SM can bind the lipoprotein-derived UC to form SM-UC particles that accumulate in the macrophage lysosomes.

	Methods

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Materials
DMEM, fetal calf serum, penicillin, streptomycin, and L-glutamine were obtained from Beth-Haemek Biological Industries. [³H]Cholesteryl linoleate was purchased from Du Pont New England Nuclear. SMase was obtained from Boehringer Mannheim. SM, egg PC, and chlorpromazine were from Sigma. [methyl-³H]Choline chloride (83 Ci/mmol) was obtained from Amersham International, and Sephadex G-25 minicolumns were purchased from Pharmacia LKB Biotechnology Inc.

Cells
J-774 A.1 murine macrophage-like cell line was purchased from American Type Culture Collection. The cells were plated at 1x10⁶ cells/16-mm dish in DMEM supplemented with 10% fetal calf serum. The cells were fed every 3 days and were used for experiments within 7 days of plating.²³ Human monocytes were isolated by density-gradient centrifugation²⁴ from blood that was derived from two fasting NP-C patients and three normolipidemic subjects. Twenty milliliters of blood anticoagulated with 10 U/mL heparin was layered over 15 mL Ficoll-Paque and centrifuged at 500g for 30 minutes at 23°C. The mixed mononuclear cell band was removed by aspiration, and the cells were washed twice in RPMI 1640 culture medium containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. The cells were plated at 1x10⁶ monocytes/16-mm dish (Primaria, Falcon Labware, Becton Dickinson and Co) in the same medium. After 2 hours of incubation at 37°C in 5% CO₂ and 95% air, nonadherent cells were removed by three washes with serum-free medium. The cells were then placed in fresh medium containing 20% autologous serum and were fed twice weekly with the same medium. MDMs were used within 7 to 10 days of plating.

Patients
We studied UC accumulation in MDMs from two NP-C patients and three normolipidemic control subjects. The clinical criteria for a diagnosis of NP-C were splenomegaly and a progressive neurodegenerative course, including supranuclear disturbance of vertical gaze and ataxia. The diagnosis was supported by a finding of partial reduction in fibroblast SMase activity and a marked deficiency of esterification of LDL-derived cholesterol in the patients' cultured fibroblasts.^{25 26}

Lipoproteins
LDL was prepared by discontinuous density-gradient ultracentrifugation²⁷ from human plasma that was obtained from fasted normolipidemic volunteers. The LDL was washed at d=1.063 g/mL, dialyzed against 150 mmol/L NaCl and 1 mmol/L Na₂-EDTA (pH 7.4) under nitrogen in the dark at 4°C, sterilized by filtration (0.22 nm), and used within 2 weeks. LDL was radiolabeled in its cholesterol moiety with 1 mCi/mL [³H]cholesteryl linoleate.²⁸ Specifically, 2x10⁸ cpm [³H]cholesteryl linoleate and 100 µL dimethyl sulfoxide were warmed to 40°C for 5 minutes, after which 5 mg LDL and 0.9 mL of 20 mmol/L Tris-HCl containing 150 mmol/L NaCl and 1 mmol/L Na₂-EDTA, pH 7.4, were added. The mixture was incubated for 3 hours at 37°C. The final LDL preparation contained 98% of the radioactivity in CE (specific radioactivity, 271 to 359 cpm/µg CE). LDL was also radiolabeled with 1 mCi/mL (83 Ci/mmol) [methyl-³H]choline chloride based on the method for the incorporation of radiolabeled choline into phospholipids.²⁹ Specifically, 1 mCi [methyl-³H]choline chloride was incubated with 2 mg LDL in the presence of 0.5 mL lipoprotein-deficient serum and 0.9 mL of 20 mmol/L Tris base containing 130 mmol/L NaCl, 1 mmol/L MgCl₂, 1.5 mmol/L CaCl₂, and 1 mmol/L choline chloride, pH 7.4. The mixture was incubated for 5 hours at 37°C, and the labeled LDL was reseparated by ultracentrifugation at d=1.063 g/mL and dialyzed against 150 mmol/L NaCl and 1 mmol/L Na₂-EDTA, pH 7.4. By using this procedure we found that approximately 50% of the labeled choline was associated with LDL. About 90% of the LDL-associated [³H]choline was in the lipoprotein phospholipids (75% in PC and 25% in SM). Only about 10% of the labeled choline was associated with LDL as a free choline. Both [³H]CE- and [³H]choline-labeled LDL were also acetylated or oxidized. LDL was acetylated by repeated additions of acetic anhydride³⁰ to 4 mg LDL protein/mL and diluted 1:1 (vol/vol) with saturated ammonium acetate at 4°C. Acetic anhydride was added at a 40-fold molar excess with regard to total lysine residues in LDL, and the modification was confirmed by electrophoresis on cellulose acetate at pH 8.6 in barbital buffer.³¹ Ox-LDL was prepared by incubating dialyzed LDL (1 mg protein/mL in EDTA-free phosphate-buffered saline) with copper sulfate (10 µmol/L) for 24 hours at 37°C. Lipoprotein oxidation was confirmed by analysis of thiobarbituric acid–reactive substances, which measures malondialdehyde equivalents,³² as well as by determination of LDL lipid peroxide levels³³ and analysis of the lipoprotein-conjugated dienes content.³⁴

Metabolism of Lipoprotein-Derived Cholesterol by the Cells
J-774 A.1 macrophages were incubated with [³H]CE-labeled lipoprotein, after which changes in cellular radioactive [³H]UC and [³H]CE were analyzed. The cells were washed twice with phosphate-buffered saline at 4°C and incubated for 30 minutes with 1 mL hexane–isopropyl alcohol (3:2, vol/vol) in a 16-mm dish at room temperature to extract the cellular lipids. After two more similar extractions, the pooled lipid extract was dried under nitrogen and dissolved in chloroform. The labeled UC and CE were then isolated by TLC on silica-gel plates by using hexane–diethyl ether–acetic acid (130:40:1.5, vol/vol/vol). The appropriate spots of UC and CE were visualized by using iodine vapors, scraped into vials containing 4 mL scintillation fluid, and counted in a ß-scintillation counter. For measurement of cell protein, the cell layer was incubated for 2 hours at room temperature with 1 mL of 0.1N NaOH prior to protein determination.³⁵ The cellular contents of unlabeled UC and CE mass were determined after lipid extraction of washed cells by using the ferric chloride assay.³⁶ By using appropriate standards in this sensitive assay, we demonstrated 95% to 98% recovery of UC and CE.

LDL SM Hydrolysis by J-774 A.1 Macrophages
To measure the hydrolysis of lipoprotein-derived SM in macrophages, we radiolabeled the lipoproteins with 1 mCi/mL [³H]choline, which incorporated into the LDL phospholipids (SM and PC). J-774 A.1 macrophages were incubated with the [³H]choline-labeled lipoproteins for 3 hours ("pulse incubation") followed by cell wash and further "chase incubation" (without lipoproteins) for 24 hours at 37°C. At the end of the incubations cells were collected, and the lysosome-rich fraction was prepared. Lysosomal phospholipids (extracted with hexane-isopropanol [3:2, vol/vol]) were separated by TLC in a solvent system of chloroform-methanol–acetic acid–water (50:25:8:4, vol/vol/vol/vol). Spots corresponding to SM were scraped, and their radioactivity was counted. Lysosomal content of SM was also measured in macrophages that were incubated with unlabeled lipoproteins. The phospholipids were extracted from cells, separated by TLC, and SM was analyzed by using the method of Bartlett.³⁷

Cell Subfractionation
J-774 A.1 macrophages were incubated with [³H]CE- or [³H]choline-labeled lipoproteins under the conditions specified for each experiment. At the end of the incubation, cells (1x10⁶/35-mm dish) were washed three times with cold phosphate-buffered saline, harvested, and suspended in 2 mL of 250 mmol/L sucrose containing 5 mmol/L Tris-HCl buffer (pH 7.4). The cells were then sonicated twice at 20 W for 20 seconds, homogenized in a polytetrafluoroethylene/glass homogenizer (15 strokes), and centrifuged at 500g for 10 minutes to remove cell debris.⁵ The supernatant was centrifuged at 10 000g for 45 minutes to precipitate the lysosome-rich fraction. The lysosome-rich fraction was not contaminated by cytosolic or microsomal fractions, as markers of the lysosome (such as ß-glucoronidase, ß-galactosidase, and acid phosphatase) were shown in this fraction, whereas cytosolic marker (lactic dehydrogenase), endoplasmic reticulum marker (nicotinamide adenine dinucleotide phosphate–cytochrome C reductase), and plasma membrane marker (5'-nucleotidase) were not found in the lysosome-rich fraction.

The lysosome-rich fraction was resuspended in water and then frozen and thawed four times to disrupt the organelles. The SMase activity of the lysosomal extract was measured by using citrate-buffered DMEM at pH 4.5 and then compared with the activity at pH 9.0, at which lysosomal activity is completely blocked.

Liposome Preparation
[³H]-Labeled cholesterol, 7-KC, and SM (in chloroform) were evaporated under nitrogen, and DMEM containing 0.2% bovine serum albumin was added (final concentrations, 500, 250, and 500 µmol/L, respectively). The solution was then warmed to 45°C and sonicated continuously for 1 minute at 4°C. This process was repeated six times to ensure complete dispersion of the lipids. The suspension obtained was then centrifuged at 10 000g for 30 minutes prior to its use.³⁸

Characterization of Lipoprotein-Like Particles in the Lysosomal Extract
For gel-filtration chromatography, lysosomal extract was prepared from J-774 A.1 macrophages that had been incubated for 24 hours with [³H]CE- or [³H]choline-labeled Ox-LDL. The lysosomal extract (2 mL) was subjected to gel filtration on a Sephadex G-25 minicolumn (10x2 cm). The column was eluted with 0.05N NaCl solution, and fractions (1 mL) were collected. An aliquot of each fraction was removed for TLC analysis of the radioactivity in UC and phospholipids. The protein concentration in each fraction was estimated by monitoring the absorbance at 280 nm.

For density-gradient ultracentrifugation, lysosomal extract was also prepared from macrophages that had been incubated for 24 hours with Ox-LDL doubly labeled in its protein (with ¹²⁵I) and CE (with [³H]CE) moieties. The density of the lysosomal extract was adjusted to 1.250 g/mL by the addition of KBr, and a 6-mL sample was placed in a tube and overlaid with 6 mL saline-EDTA buffer (d=1.006 g/mL). After 48 hours of ultracentrifugation at 90 000g at 4°C with an SW41 rotor, one band was visualized. An aliquot (1 mL) of each fraction was removed by pipetting for analysis of the [³H]CE and ¹²⁵I-protein radioactivities.

Statistical Analysis
Results are given as mean±SD; significance was assayed by Student's t test.

	Results

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Accumulation of Ox-LDL–Derived SM in Macrophage Lysosomes
UC accumulates in macrophage lysosomes after cell incubation with Ox-LDL.⁵ To study the possible involvement of Ox-LDL–derived SM in the lysosomal accumulation of the lipoprotein-derived UC, J-774 A.1 macrophages (1x10⁶ cells/16-mm dish) were incubated with 25 µg protein/mL Ox-LDL or Ac-LDL for 24 hours at 37°C. Cells were then fractionated, and their lysosomal SM content was determined. Fig 1 shows that upon incubation of the cells with Ox-LDL the lysosomal content of SM increased by 63±8%, whereas in cells that were incubated with Ac-LDL, SM content increased by only 19±2% compared with control cells that were incubated without lipoprotein. In parallel, in cells that were incubated with 25 µg protein/mL [³H]CE-labeled Ox-LDL or [³H]CE-labeled Ac-LDL, lysosomal [³H]UC accounted for 75±7% and 18±4%, respectively, of the total lysosomal labeled cholesterol (n=3; data not shown).⁵

View larger version (17K): [in this window] [in a new window]   Figure 1. Bar graph showing lysosomal SM content in J-774 A.1 macrophages (1x106/16-mm dish) after they were incubated with 25 µg protein/mL Ox-LDL or Ac-LDL. After 24 hours of incubation at 37°C, the contents of 10 dishes were combined, and the lysosome-rich fraction was prepared and analyzed by TLC for its SM content. Results are mean±SD (n=3).

Under these experimental conditions, cellular cholesterol content increased by 37 or 48 µg/10⁷ cells upon incubation with Ox-LDL or Ac-LDL, respectively. Thus, normalization of SM lysosomal accumulation to the actual cellular cholesterol uptake resulted in 2.7-fold higher SM content in the cells after their incubation with Ox-LDL compared with Ac-LDL (2.3 µg SM · 37 µg cholesterol^-1 · 10⁷ cells^-1 versus 1.1 µg SM · 48µg cholesterol^-1 · 10⁷ cells^-1, respectively). To directly measure the accumulation of Ox-LDL–derived SM in macrophage lysosomes, the lipoproteins were prelabeled (in the presence of 10% lipoprotein-deficient serum) with 1 mCi/mL [³H]choline (which incorporated into the lipoprotein SM and PC) prior to their incubation with macrophages. Cells were then incubated with 25 µg protein/mL [³H]choline-labeled lipoproteins for 3 hours at 37°C (pulse incubation). The cells were then washed and further incubated in a lipoprotein-free medium for up to 24 hours at 37°C (chase incubation). The lysosomal [³H]SM content was expressed as the percentage of the lysosomal content of [³H]SM after the 3-hour pulse incubation and before the chase incubation. After 24 hours of chase incubation, 76±9% of the Ox-LDL–derived but only 40±5% of the Ac-LDL–derived [³H]SM remained in the lysosomes (n=3). Time-course study of the lysosomal accumulation of Ox-LDL–derived SM was performed by incubation of J-774 A.1 macrophages with 25 µg protein/mL [³H]choline-labeled Ox-LDL for up to 36 hours at 37°C. At the end of the incubation the lysosome-rich fraction was prepared and analyzed for its [³H]SM content. A time-dependent increment in macrophage lysosomal accumulation of labeled SM was found, and [³H]SM values of 22, 24, 32, 105, 557, 2237, and 5287 cpm/10⁷ cells were obtained in the lysosomes after 30 minutes and 1, 2, 4, 6, 24, and 36 hours of incubation, respectively (results are the mean of one experiment performed in triplicate). To further confirm this phenomenon, we studied the hydrolysis of the lipoprotein [³H]SM in a cell-free system by using lysosomal extract that was prepared from J-774 A.1 macrophages. [³H]Choline-labeled lipoproteins (10 µg protein/mL) were incubated with 10 µg lysosomal extract protein/mL for 3 hours at 37°C, pH 4.5; lysosomal activity of SMase is blocked at pH 9.0. Hydrolysis of the lipoprotein-labeled SM (measured as the percentage of radioactivity in the lipoprotein-labeled SM at pH 4.5 versus pH 9.0) was substantially higher when using labeled Ac-LDL (46%) compared with Ox-LDL (16%) (Table 1), suggesting again that the SM moiety in Ox-LDL was partially resistant to the macrophage lysosomal SMase. To find out whether acetylation of LDL affects the hydrolysis of lipoprotein-labeled SM compared with unmodified LDL, we measured the SM hydrolysis in native LDL by using the same lysosomal extract and found that 45±3% and 47±3% of native LDL and Ac-LDL–derived SM, respectively, were hydrolyzed under these experimental conditions (data not shown). This suggests that Ac-LDL does not increase the hydrolysis of SM compared with native LDL. Since PC, in addition to SM, is also labeled by [³H]choline, we measured the hydrolysis of Ac-LDL– and Ox-LDL–derived PC by using the same lysosomal extract. In contrast to [³H]SM, there was no significant difference in the hydrolysis of [³H]PC between the two modified lipoproteins: 52% of the PC in Ac-LDL and 49% of the PC in Ox-LDL were hydrolyzed. These results suggest that the PC moiety in Ox-LDL, unlike its SM constituent, is not resistant to hydrolysis by the macrophage lysosomes (Table 1).

View this table: [in this window] [in a new window]   Table 1. Lipoprotein SM and PC Hydrolysis by Lysosomal Extract From J-774 A.1 Macrophages

Macrophage SMase Activity and Cellular UC Accumulation
We hypothesized that the lysosomal accumulation of Ox-LDL–derived UC together with its SM is secondary to the attenuation of SMase in this organelle. To test this hypothesis, cells were incubated with [³H]CE-labeled Ac-LDL in the absence or presence of chlorpromazine at a concentration (20 µmol/L) at which chlorpromazine is a potent inhibitor of endogenous acid SMase but not toxic to the cells.³⁹ Chlorpromazine inhibited the hydrolysis of [³H]SM in choline-labeled, Ac-LDL that was incubated with lysosomal extract by 48±3% (n=3; data not shown). Macrophage incubation for 24 hours at 37°C with 25 µg protein/mL [³H]CE-labeled Ac-LDL or Ox-LDL was performed in the absence or presence of 20 µmol/L chlorpromazine (Fig 2). Up to 41±4% of the total cellular labeled UC was found in the lysosome-rich fraction from cells that were incubated with Ac-LDL and chlorpromazine; only 20±2% was found in lysosomes from cells after their incubation with Ac-LDL alone (Fig 2). In contrast, the accumulation of Ox-LDL–derived UC in the lysosomes was not further increased by the presence of chlorpromazine. Up to 71% and 69% of the total cellular labeled UC were found in the lysosome-rich fractions from cells that were incubated with Ox-LDL alone or with Ox-LDL and chlorpromazine, respectively (Fig 2). These results suggest that inhibition of the lysosomal SMase is responsible for the lysosomal accumulation of the lipoprotein-derived UC in the lipoprotein.

View larger version (15K): [in this window] [in a new window]   Figure 2. Bar graph showing effect of chlorpromazine (an SMase inhibitor) on lysosomal accumulation of UC after macrophage incubation with Ac-LDL or Ox-LDL. J-774 A.1 macrophages (1x106/16-mm dish) were incubated for 24 hours at 37°C with [3H]CE-labeled Ac-LDL or Ox-LDL (25 µg protein/mL) in the absence (control) or presence of 20 µmol/L chlorpromazine. Cells were fractionated after the incubation, and the [3H]UC radioactivity in the lysosome-rich fraction was determined. Results are given as the percentage of total cellular radioactive cholesterol and expressed as mean±SD (n=4). *P<.01 vs control.

Cellular UC Accumulation in MDMs From NP-C Patients
We next studied UC accumulation in Ox-LDL–incubated MDMs from two patients with NP-C, a genetic cholesterol lipidosis with impaired SMase activity.¹⁶ MDMs from these two patients and three control subjects (1x10⁶ cells/16-mm dish) were incubated with 25 µg protein/mL [³H]CE-labeled Ox-LDL or Ac-LDL for 24 hours at 37°C, after which the cellular content of [³H]UC was determined (Fig 3). NP-C MDMs accumulated 30% more UC than control MDMs after cell incubation with Ox-LDL (Fig 3A). A 130% increment in the cellular UC content was obtained in the NP-C patients' cells compared with cells from normal control subjects when cells were incubated with Ac-LDL, which does not cause macrophage UC accumulation in normal cells (Fig 3B). These results also support the role of an attenuated macrophage SMase activity in the cellular accumulation of UC.

View larger version (16K): [in this window] [in a new window]   Figure 3. Bar graphs showing cellular accumulation of lipoprotein-derived UC in MDMs obtained from two NP-C patients (P-1 and P-2) and three healthy subjects (control). Cells (1x106/16-mm dish) were incubated for 24 hours at 37°C with 25 µg protein/mL [3H]CE-labeled (A) Ox-LDL or (B) Ac-LDL. After the incubation the macrophage content of [3H]UC (expressed as percentage of total labeled cholesterol) was determined. Results are mean±SD of three experiments. *P<.01 vs control.

Association Between Cholesterol and SM in Macrophage Lysosomes
Since both Ox-LDL–derived SM and UC accumulated in the macrophage lysosomes after cell incubation with Ox-LDL, we questioned whether these lipids were associated together as a particle. For this purpose, J-774 A.1 macrophages were incubated with either [³H]CE- or [³H]choline-labeled Ox-LDL; incubation was followed by cell homogenization and fractionation. Lysosomal extract prepared as described above was then subjected to gel-filtration chromatography on a Sephadex G-25 minicolumn (10x2 cm), and fractions were collected and counted in a ß-scintillation counter. Fig 4 shows the elution pattern of the lysosomal extract as analyzed by determination of the [³H]cholesterol, [³H]phospholipid, and protein concentrations measured at 280 nm. The cholesterol-rich fraction was coeluted with phospholipids and protein and was recovered in the void volume.

View larger version (18K): [in this window] [in a new window]   Figure 4. Line graphs of gel-filtration chromatography of lysosomal extract. J-774 A.1 macrophages were incubated for 24 hours at 37°C with 25 µg protein/mL (A) [3H]CE-labeled Ox-LDL or (B) [3H]choline-labeled Ox-LDL. Lysosomal extracts (2 mL) were prepared after cell collection and subjected to a Sephadex G-25 minicolumn (2x10 cm). Fractions (1 mL) were collected by using 0.05N NaCl as eluant. Each fraction was analyzed for its content of (A) [3H]UC, (B) [3H]choline phospholipids, or (C) protein (absorbance at 280 nm). Elution patterns are representative of three different experiments.

Further analysis of this fraction revealed that 82±5% (n=3) of the [³H]cholesterol radioactivity was in the form of [³H]UC (Fig 5A). The [³H]CE accounted for 5±1% of the total cholesterol radioactivity, whereas 13±2% was found in 7-KC, the major oxysterol found in Ox-LDL.⁵ Analysis of the various phospholipids in this fraction (Fig 5B) showed that SM represented 65±4% of the total [³H]choline-labeled phospholipids, whereas PC and lyso-PC represented only 22±3% and 12±2%, respectively, of the total [³H]choline radioactivity in this fraction. To further analyze the protein component of this void-volume fraction, J-774 A.1 macrophages were incubated for 24 hours at 37°C with Ox-LDL (25 µg protein/mL) that was prelabeled in its protein moiety with Na¹²⁵I. Gel-filtration chromatography of the lysosomal extract that was prepared from these macrophages revealed that the iodinated apoB-100 eluted together with UC, 7-KC, and SM (data not shown), suggesting the combined presence of these constituents as a lipoprotein-like particle in the lysosomes.

View larger version (19K): [in this window] [in a new window]   Figure 5. Bar graphs. Lysosomal extract was prepared from J-774 A.1 macrophages that were incubated for 24 hours at 37°C with 25 µg protein/mL (A) [3H]CE-labeled Ox-LDL or (B) [3H]choline-labeled Ox-LDL. The lysosomal extract was then subjected to gel-filtration chromatography on a Sephadex G-25 minicolumn (2x10 cm), and the fractions eluted in the void volume were analyzed by TLC for (A) [3H]UC, 7-KC, and CE and (B) [3H]choline content in the various phospholipid spots. Results are mean±SD of three experiments. LPC indicates lyso-PC.

To further confirm the lysosomal accumulation of lipoprotein-like particles, J-774 A.1 macrophages were incubated for 24 hours at 37°C with Ox-LDL that was doubly labeled in the protein moiety (with ¹²⁵I) and in its core CE constituent (with [³H]CE). Density-gradient ultracentrifugation of the lysosomal extract that was prepared from these macrophages revealed that the [³H]cholesterol floated together with the iodinated apoB-100 (Fig 6) at d=1.02 to 1.04 g/mL.

View larger version (18K): [in this window] [in a new window]   Figure 6. Line graphs showing density-gradient ultracentrifugation of lysosomal extract. J-774 A.1 macrophages were incubated for 24 hours at 37°C with 25 µg protein/mL Ox-LDL that was doubly labeled in its protein moiety (with 125I) and core CE moiety (with [3H]CE). At the end of the incubation, lysosomal extract (6 mL) was prepared and subjected to density-gradient ultracentrifugation (90 000g for 24 hours). Fractions (1 mL) were collected from the top to the bottom of the tube. Each fraction was analyzed for the radioactivity of (A) [3H]CE or (B) 125I. [3H]Cholesterol floated together with the iodinated apoB-100. The elution patterns are representative of three different experiments.

Effect of 7-KC on Lysosomal Accumulation of Lipoprotein-Derived SM and UC
Oxysterols, which are present in Ox-LDL, contribute to the accumulation of Ox-LDL–derived UC in lysosomes; 7-KC, the major oxidized cholesterol derivative in Ox-LDL, was also trapped in the macrophage lysosomes.⁵ Since 7-KC was associated with Ox-LDL–derived UC in the same particle, we studied the possible effect of 7-KC on the lysosomal accumulation of UC in J-774 A.1 macrophages. Incubation of the macrophages with [³H]CE-labeled Ac-LDL (25 µg protein/mL) in the presence of 10 µg/mL Ox-LDL–derived oxysterols or 10 µg/mL pure 7-KC caused cellular accumulations of 60±4% or 55±3%, respectively, [³H]UC out of the total cellular labeled cholesterol compared with only 36% [³H]UC that was found in cells incubated with Ac-LDL alone (ie, in the absence of the oxysterols) (Fig 7). To further analyze the effect of 7-KC on lysosomal accumulation of UC, we prepared liposomes that contained [³H]UC and SM (500 µmol/L each) in the absence or presence of 7-KC. Incubation of J-774 A.1 macrophages with these liposomes (0.5 mL of the liposome preparation was added to 1x10⁶ cells) in the presence of 10 µg/mL 7-KC led to the accumulation of [³H]UC in the macrophage lysosomes. On using liposomes containing 7-KC, 50±8% [³H]UC of total cellular cholesterol radioactivity was found in the lysosomes compared with only 30±6% [³H]UC that was accumulated in lysosomes following cell incubation with liposomes that did not contain 7-KC (n=3).

View larger version (19K): [in this window] [in a new window]   Figure 7. Bar graph showing effect of oxysterols on Ac-LDL–mediated accumulation of UC in macrophages. J-774 A.1 macrophages were incubated for 24 hours at 37°C with 25 µg protein/mL [3H]CE-labeled Ac-LDL in the absence or presence of 10 µg/mL oxysterols (separated from Ox-LDL by TLC and scraped from the area between the phospholipids and the UC spots) or 10 µg/mL pure 7-KC. Cellular UC accumulation was determined after the incubation and is expressed as the percentage of total cellular labeled cholesterol. Results are mean±SD (n=3). *P<.01 vs control.

The effect of 7-KC on macrophage lysosomal SMase was also studied (Table 2). Upon incubation of 7-KC (10 µg/mL) with lysosomal extract in the presence of 25 µg protein/mL [³H]choline-labeled Ac-LDL for 3 hours at 37°C, the [³H]SM hydrolysis was 26%; the [³H]SM hydrolysis obtained in the absence of 7-KC was 45%.

View this table: [in this window] [in a new window]   Table 2. Effect of 7-KC on Hydrolysis of Ac-LDL–Derived SM and PC by Lysosomal Extract from J-774 A.1 Macrophages

To find out whether 7-KC specifically affected lysosomal SMase, we also measured the hydrolysis of Ac-LDL–labeled PC. In contrast to the impaired hydrolysis of the Ac-LDL–derived [³H]SM, we found that the lipoprotein [³H]PC hydrolysis was not influenced by 7-KC, as 47% of the Ac-LDL–derived PC was hydrolyzed by the lysosomal extract both in the absence and presence of 7-KC (Table 2). To further confirm this phenomenon, we studied the effect of 7-KC on the hydrolysis of nonlipoprotein SM (100 µmol/L) by lysosomal extract that was prepared from J-774 A.1 macrophages. The addition of 7-KC (10 µg/mL) to the lysosomal extract caused a 45±6% inhibition in SM hydrolysis compared with SM hydrolysis in the absence of 7-KC (data not shown).

As the effect of 7-KC on the accumulation of Ox-LDL–derived UC in the macrophage lysosomes can be secondary to its inhibitory effect on lysosomal hydrolysis of SM, the effect of 7-KC on lysosomal accumulation of Ac-LDL–derived [³H]SM was studied by using whole cells rather than the lysosomal extract system (Fig 8). Incubation of macrophages with [³H]choline-labeled Ac-LDL in the presence of 7-KC enhanced the lysosomal accumulation of Ac-LDL–derived SM compared with the effect of Ac-LDL alone (Fig 8A). Under similar conditions, upon incubation of the cells with [³H]CE-labeled Ac-LDL, 7-KC also caused lysosomal accumulation of Ac-LDL–derived [³H]UC (Fig 8B). Of the total cellular labeled cholesterol, 40±6% was found in the lysosomes as [³H]UC following macrophage incubation with [³H]CE-labeled Ac-LDL in the presence of 7-KC compared with only 20±2% that was found in the lysosomes after cell incubation with Ac-LDL alone (Fig 8B).

View larger version (16K): [in this window] [in a new window]   Figure 8. Bar graphs showing effect of 7-KC on Ac-LDL–mediated lysosomal accumulation of (A) SM and (B) UC. J-774 A.1 macrophages were incubated at 37°C for 3 hours ("pulse incubation") with (A) [3H]choline- or (B) [3H]CE-labeled Ac-LDL (25 µg protein/mL) in the absence or presence of 10 µg/mL 7-KC. This incubation was followed by a cell wash and a "chase incubation" for 24 hours at 37°C. After the cells were collected and fractionated, their lysosomes were separated. [3H]Choline-labeled SM and [3H]UC accumulations in the lysosome-rich fraction were determined by TLC analysis and are expressed as the percentage of control value, ie, the [3H]SM or [3H]UC content after the 3-hour pulse labeling and before the chase incubation. Results are mean±SD (n=3). *P<.01 vs Ac-LDL alone.

Effect of SM Content in the Plasma Membrane Macrophage on Ox-LDL–Mediated Accumulation of UC
Cellular SM content significantly influences the trafficking of cholesterol from the plasma membrane to the intracellular pool.¹² In this study we examined the relation between plasma membrane SM content and the trapping of Ox-LDL–derived UC in lysosomes. Enrichment of the plasma membrane of J-774 A.1 macrophages with SM was achieved by incubating the cells for 3 hours at 37°C with SM (250 µmol/L). This incubation resulted in the elevation of the macrophage SM content from 17.5±1.9 to 23.6±3.1 nmol SM/mg cell protein. Following macrophage incubation with 25 µg protein/mL [³H]CE-labeled Ox-LDL for 24 hours at 37°C, no significant changes in lysosomal accumulation of [³H]UC were found compared with macrophages that were incubated without SM (67±6% versus 63±7% of total cellular labeled cholesterol in SM-treated versus untreated cells, n=3). Depletion of plasma membrane SM (obtained by cell incubation for 3 hours at 37°C with 50 mU/mL SMase) did not significantly affect lysosomal [³H]UC accumulation. Following incubation of these SM-depleted macrophages with [³H]CE-labeled Ox-LDL, 66±7% [³H]UC out of the total cellular labeled cholesterol was found in the lysosomes compared with 63±7% [³H]UC obtained from macrophages that were not treated with SMase (n=3).

These results suggest that the accumulation of UC in the macrophage lysosomes following cell incubation with Ox-LDL (which was shown in the present study to be associated with the lysosomal SM content) is not affected by the SM content in the plasma membrane.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The CE moiety in Ox-LDL is hydrolyzed normally in lysosomes, but the resulting UC is trapped in the lysosomes secondary to the effect of oxysterols that are present in Ox-LDL.⁵ However, the mechanism responsible for trapping UC in the lysosomes has not been explored. Since SM binds UC with high affinity,^{18 19 20 21 22} the goal of this study was to examine whether SM accumulates in the macrophage lysosomes following cell incubation with Ox-LDL and whether SM accumulation can be responsible for trapping of the lipoprotein UC in the macrophage lysosomes. The present study indeed showed that Ox-LDL–derived SM accumulates in lysosomes as a result of an impaired SM hydrolysis by the lysosomal SMase. We also demonstrated that 7-KC, the major oxidized cholesterol derivative in Ox-LDL,^{5 40 41} is a potent inhibitor of macrophage lysosomal SMase. This oxysterol can thus lead to the lysosomal accumulation of Ox-LDL–derived UC secondary to SM accumulation in the macrophage lysosomes.

The mechanisms whereby 7-KC inhibits lysosomal SMase have not been elucidated. It might be that this oxysterol in Ox-LDL could induce allosteric changes in the lipoprotein SM that could then decrease its recognition of and/or affinity for SMase. Alternatively, 7-KC may bind directly to SMase and thus prevent its action on Ox-LDL–derived SM. 7-Dehydrocholesterol also inhibits lysosomal SMase activity,⁴² and cellular accumulation of 7-dehydrocholesterol may cause the accumulation of UC in lysosomes.¹⁶

A close association exists between SM and UC in cell membrane,^{6 7 8} and the SM content of cells influences intracellular trafficking of endogenous cholesterol.^{12 14} Depletion of SM from plasma membrane causes a loss of cholesterol from the membrane and its transfer into the cell cytosol.¹² This cholesterol inhibits cellular cholesterol synthesis and increases the cholesterol esterification rate.¹¹ Conversely, when fibroblasts are enriched with SM, cellular cholesterol synthesis increases.¹¹ Thus, SM, which has molecular attraction for cholesterol,^{18 21 22} may "trap" UC within a particular membrane and hence prevent its cellular trafficking, processing, and metabolism.³⁸ The SM content of macrophages can strongly influence the threshold level at which atherogenic lipoproteins stimulate macrophage acyl coenzyme A:cholesterol acyltransferase activity, most likely by affecting intracellular cholesterol trafficking.¹⁴ Our results indeed show that both Ox-LDL–derived SM and Ox-LDL–derived UC accumulated in the macrophage lysosomes and thus attenuated the release of Ox-LDL–derived UC from the lysosomes to the cytosolic compartment for the esterification reaction. In the present study we suggest that SM accumulates in the lysosomes as a result of impaired SMase activity. Chlorpromazine, an inhibitor of endogenous SMase,³⁹ induces the accumulation of Ac-LDL–derived SM in lysosomes and traps the UC in the same compartment. AY-9944, a cationic amphiphilic drug that causes a reduction in acid SMase activity in fibroblasts and various tissues in the rat,¹⁶ causes a marked reduction in cholesterol esterification and an excessive intracellular accumulation of UC.⁴³ Inhibition of lysosomal SMase in the present study by 7-KC also led to the accumulation of Ac-LDL–derived UC in the macrophage lysosomal compartment. The finding in J-774 A.1 macrophages that the attenuated lysosomal SMase activity induced by Ox-LDL is followed by lysosomal accumulation of SM and UC resembles the unique features described in fibroblasts from NP-C patients.⁴⁴ Lysosomal SMase activity in NP-C fibroblasts decreases following their incubation with LDL.^{15 16 17} Moreover, under these conditions, NP-C cells accumulate SM and UC, and the cellular cholesterol esterification rate is reduced.^{16 17} Unlike fibroblasts from NP-C patients, which demonstrate impaired SMase activity only after cell incubation with LDL, in cell lines from patients with either NP-A or NP-B, which lack SMase activity almost completely, LDL cholesterol transport and esterification rate are normal^{45 46 47} or reduced.^{48 49} A substantial increase in the amount of UC in the spleen and liver of patients with NP-A is reported.⁴⁸ Similarly, in some mouse strains that have been used as a model of NP-A, the fibroblast cholesterol esterification rate is significantly impaired.⁴⁹ Since cells with NP-A and NP-B completely lack SMase activity, it is possible that these cells can use an alternative pathway for cholesterol transport out of the lysosomes.

In the present study we have demonstrated for the first time that MDMs from NP-C patients accumulated UC following incubation with either Ac-LDL or Ox-LDL, suggesting that the attenuation of lysosomal SMase activity is probably the primary event leading to lysosomal accumulation of UC. In normal MDMs, Ox-LDL but not Ac-LDL caused lysosomal UC accumulation; this may have resulted from impaired lysosomal SMase activity (probably caused by 7-KC, which is present in Ox-LDL but not Ac-LDL). The increase in cellular UC content in the patients' MDMs compared with normal subjects' MDMs was by 30% or 130% following cell incubation with Ox-LDL or Ac-LDL, respectively. The lower stimulatory effect of Ox-LDL compared with Ac-LDL may be related to a limited maximal capacity of the lysosomes to accumulate UC. Since Ox-LDL, unlike Ac-LDL, stimulates lysosomal accumulation of UC by its inhibitory effect on lysosomal SMase (mediated by its 7-KC), the additional contributing effect of an impaired SMase activity in the patients' MDMs to cellular UC accumulation is limited. In contrast, upon incubation of the patients' macrophages with Ac-LDL, only the lipoprotein-induced inhibition of lysosomal SMase activity (but not an effect of 7-KC) in the patients' cells could contribute to the cellular UC accumulation.

According to another model, rapid lipoprotein uptake, which leads to the generation of a large amount of UC within a limited population of lysosomes, is the critical factor leading to cellular UC accumulation.⁵⁰ This is in contrast to a regulated LDL receptor–mediated uptake of lipoprotein, which is a relatively slow process that delivers the internalized lipoproteins into a large number of lysosomes and therefore does not result in a significant UC accumulation in the lysosomes. Phagocytic uptake of CE by macrophages^{50 51} results in lysosomal accumulation of UC, similar to the results obtained in the present study when macrophages were incubated with Ox-LDL. However, the accumulation of UC by phagocytic uptake of CE droplets leads to the formation of cholesterol monohydrate crystals,⁵¹ whereas Ox-LDL uptake by J-774 A.1 macrophages led to the accumulation of UC as a lipoprotein-like particle.

Cholesterol-enriched macrophages secrete cholesterol-containing multilamellar structures into their culture medium, and these structures (originated from macrophage lysosomes) are rich in UC (68%) and phospholipids (21%), mainly PC and SM.⁵² The availability of SM at the site of the lysosome-released cholesterol may thus determine the secretion rate of the lipoprotein-derived UC from the lysosomes.⁵² UC-enriched particles that accumulate in the extracellular space of the atherosclerotic lesions of human and cholesterol-fed rabbits have been isolated and characterized.^{1 2 3 4 53} Lysosomes from arterial cells in the atheroma may be the source of these cholesterol-phospholipid particles, as the lysosomal fraction from atheromatous aorta of cholesterol-fed rabbits has a UC/phospholipid ratio of 2.4:1.0, and SM represents 49% of the phospholipid fraction.³ Similarly, in the present study, the lysosomal extract from macrophages incubated with Ox-LDL had a UC/phospholipid mass ratio of 2.1:1.0, and SM represented about 65% of the total phospholipids. Thus, it may be that the Ox-LDL–derived UC that accumulates in the macrophage lysosomes in association with SM indeed represents the source of the UC-SM particles that have been demonstrated in atherosclerotic lesions.^{1 2 3 4 47 53}

In conclusion, the present study demonstrated that lipoprotein-like particles are formed after macrophage incubation with Ox-LDL. These particles accumulate in the macrophage lysosomes, and the Ox-LDL–derived UC cannot be further processed intracellularly. 7-KC was shown to cause lysosomal trapping of the lipoprotein UC together with its SM, and this effect was probably secondary to its inhibitory effect on lysosomal SMase. As 7-KC is a major oxidized sterol in Ox-LDL^{5 40 41} and its presence in the atherosclerotic lesion has been documented,⁵⁴ this oxysterol may have a major role in the formation of cholesterol-loaded macrophages that are enriched with lysosomal UC during early atherogenesis.⁵⁵

	Selected Abbreviations and Acronyms

 

Ac-LDL = acetylated LDL CE = cholesteryl ester DMEM = Dulbecco's modified Eagle's medium 7-KC = 7-ketocholesterol MDM = monocyte-derived macrophage NP-C = Niemann-Pick type C disease Ox-LDL = oxidatively modified LDL PC = phosphatidylcholine SM = sphingomyelin SMase = sphingomyelinase TLC = thin-layer chromatography UC = unesterified cholesterol

Received February 6, 1995; accepted May 26, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Chao FF, Amende LM, Blanchette-Mackie EJ, Skarlatos SI, Gamble W, Resau JH, Mergner WT, Kruth HS. Unesterified cholesterol-rich lipid particles in atherosclerosis lesions of human and rabbit aortas. Am J Pathol. 1988;131:73-83. [Abstract]

2. Chao FF, Blanchette-Mackie EJ, Chen YJ, Dickens BF, Berlin E, Amende LM, Skarlatos SI, Gamble W, Resau JH, Mergner WT, Kruth HS. Characterization of two unique cholesterol-rich lipid particles isolated from human atherosclerotic lesions. Am J Pathol. 1990;136:169-179. [Abstract]

3. Amanuma-Muto K, Kanaseki T, Imanaka T, Ohkuma S, Takano T. Lipid composition of low-density lysosomal membrane fraction prepared from atheromatous aorta of cholesterol-fed rabbits. Biochem Int. 1983;7:107-114. [Medline] [Order article via Infotrieve]

4. Chao FF, Blanchette-Mackie EJ, Dickens BF, Gamble W, Kruth HS. Development of unesterified cholesterol-rich lipid particles in atherosclerotic lesions of WHHL and cholesterol fed NZW rabbits. J Lipid Res. 1994;35:71-83. [Abstract]

5. Maor I, Aviram M. Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesteryl ester. J Lipid Res. 1994;35:803-819. [Abstract]

6. Lange Y, Swaisgood MH, Ramos BV, Steck TL. Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J Biol Chem. 1989;264:3786-3793. [Abstract/Free Full Text]

7. Ashworth LAE, Green C. Plasma membranes: phospholipid and sterol content. Science. 1970;151:210-211.

8. Lund-Katz S, Laboda HM, McLean LR, Phillips MC. Influence of molecular packing and phospholipid type on rates of cholesterol exchange. Biochemistry. 1988;27:3416-3423. [Medline] [Order article via Infotrieve]

9. Chao FF, Blanchette-Mackie EJ, Tertov VV, Skarlatos SI, Chen YJ, Kruth HS. Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome. J Biol Chem. 1992;267:4992-4998. [Abstract/Free Full Text]

10. Chen H, Born E, Mathur SN, Johlin FC Jr, Field FJ. Sphingomyelin content of intestinal cell membranes regulates cholesterol absorption. Biochem J. 1992;286:771-777.

11. Gupta AK, Rudney H. Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures. J Lipid Res. 1991;32:125-136. [Abstract]

12. Slotte P, Bierman EL. Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts. Biochem J. 1988;250:653-658. [Medline] [Order article via Infotrieve]

13. Gatt S, Bierman EL. Sphingomyelin suppresses the binding and utilization of low density lipoproteins by skin fibroblasts. J Biol Chem. 1979;255:3371-3376. [Abstract/Free Full Text]

14. Okwu AK, Xu X-X, Shiratori Y, Tabas I. Regulation of the threshold for lipoprotein induced acyl-CoA: cholesterol O-acyltransferase stimulation in macrophages by cellular sphingomyelin content. J Lipid Res. 1994;35:644-655. [Abstract]

15. Pentchev PG, Kruth HS, Comly ME, Butler JD, Vanier MT, Wenger DA, Patel S. Type C Niemann-Pick disease: a parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts. J Biol Chem. 1986;261:16775-16780. [Abstract/Free Full Text]

16. Yoshikawa H, Sakuragawa N. Removal of lipoprotein fraction from culture media corrects the reduction of acid sphingomyelinase activity induced by AY9944. J Inherit Metab Dis. 1992;15:155-156. [Medline] [Order article via Infotrieve]

17. Liscum L, Faust JR. Low density lipoprotein (LDL) mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts. J Biol Chem. 1987;262:17002-17008. [Abstract/Free Full Text]

18. Demel RA, Jansen JWCM, Van Dijck PWM, Van Deenen LLM. The preferential interaction of cholesterol with different classes of phospholipids. Biochim Biophys Acta. 1977;465:1-10. [Medline] [Order article via Infotrieve]

19. Nakagawa Y, Inoue K, Nojima S. Transfer of cholesterol between liposomal membranes. Biochim Biophys Acta. 1979;553:307-319. [Medline] [Order article via Infotrieve]

20. Fugler L, Clejan S, Bittman R. Movement of cholesterol between vesicles prepared with different phospholipids or sizes. J Biol Chem. 1985;260:4098-4102. [Abstract/Free Full Text]

21. Lange Y, D'Alessandro JS, Small DM. The affinity of cholesterol for phosphatidylcholine and sphingomyelin. Biochim Biophys Acta. 1979;556:388-398. [Medline] [Order article via Infotrieve]

22. Kudchodkar BJ, Albers JJ, Bierman EL. Effect of positively charged sphingomyelin liposomes on cholesterol metabolism of cells in culture. Atherosclerosis. 1983;46:353-367. [Medline] [Order article via Infotrieve]

23. Aviram M. Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells. J Biol Chem. 1992;267:218-225. [Abstract/Free Full Text]

24. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest Suppl. 1968;21:77-99.

25. Wenger DA. Niemann Pick disease. In: Glow RH, Peters SP, eds. Practical Enzymology of the Sphingolipidosis. New York, NY: AR Liss; 1977;1:39-70.

26. Vanier MT, Wenger DA, Comly ME, Rousson R, Brady RO, Pentchev PG. Niemann-Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterification. Clin Genet. 1988;33:331-348. [Medline] [Order article via Infotrieve]

27. Aviram M. Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients. Biochem Med. 1983;30:111-118. [Medline] [Order article via Infotrieve]

28. Brown MS, Dana SE, Goldstein JL. Receptor-dependent hydrolysis of cholesteryl ester contained in low density lipoprotein. Proc Natl Acad Sci U S A. 1975;72:2925-2929. [Abstract/Free Full Text]

29. Lasseque B, Alexander RW, Clark M, Greindling KK. Angiotensin II-induced phosphatidylcholine hydrolysis in cultured vascular smooth muscle cells. Biochem J. 1991;276:19-25.

30. Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3178-3182. [Abstract/Free Full Text]

31. Colf B, Verheyden Y. Electrophoresis and Sudan black staining of lipoprotein on gelatinized cellulose acetate. Clin Chim Acta. 1967;18:325-329.

32. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-310. [Medline] [Order article via Infotrieve]

33. El-Saadani M, Esterbauer H, El-Sayed M, Goher M, Nassar AY, Jurgens G. A spectrophotometric assay of lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627-631. [Abstract]

34. Esterbauer H, Rotheneder M, Striegel G, Waeg G, Ashy A, Sattler W, Jurgens G. Vitamin E and other lipophilic antioxidants protect LDL against oxidation. Fat Sci Technol. 1989;8:316-324.

35. Lowry OH, Rosebrough NJ, Farr AL, Randall RY. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;195:267-275.

36. Chiamori N, Henry RJ. Study of the ferric chloride method for determination of total cholesterol and cholesterol esters. Am J Clin Pathol. 1959;31:305-309. [Medline] [Order article via Infotrieve]

37. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959;234:466-472. [Free Full Text]

38. Chen H, Born E, Mathur SN, Field FJ. Cholesterol and sphingomyelin syntheses are regulated independently in cultured human intestinal cells, CaCO-2: role of membrane cholesterol and sphingomyelin content. J Lipid Res. 1993;34:2159-2167. [Abstract]

39. Albuz S, LeSaux F, Wenger DA, Hauw JJ, Baumann N. Modification of sphingomyelin and phosphorylcholine metabolism by tricyclic antidepressants and phenothiazines. Life Sci. 1986;38:357-363. [Medline] [Order article via Infotrieve]

40. Zhang H, Basra HJK, Steinbrecher UP. Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages. J Lipid Res. 1990;31:1361-1369. [Abstract]

41. Bhadra S, Arshad MAQ, Rymaszewski Z, Norman E, Wherley R, Subbiah MTR. Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu²⁺ ions: identification of major products and their effects. Biochem Biophys Res Commun. 1991;176:431-440. [Medline] [Order article via Infotrieve]

42. Maziere JC, Wolf C, Maziere C. Inhibition of human fibroblasts sphingomyelinase by cholesterol and 7-dehydrocholesterol. Biochem Biophys Res Commun. 1981;100:1299-1304. [Medline] [Order article via Infotrieve]

43. Yoshikawa H. Effects of drugs on cholesterol esterification in normal and Niemann-Pick type C fibroblasts: AY-9944, other cationic amphiphilic drugs and DMSO. Brain Dev. 1991;13:115-120. [Medline] [Order article via Infotrieve]

44. Vanier MT, Pentchev PG, Rodriguez-Lafrass C, Rousson R. Niemann-Pick disease type C: an update. J Inherit Metab Dis. 1991;14:580-595. [Medline] [Order article via Infotrieve]

45. Liscum L, Reggiero RM, Faust JR. The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. J Cell Biol. 1989;108:1625-1636. [Abstract/Free Full Text]

46. Pentchev PG, Comly ME, Kruth HS, Patel S, Proestel M, Weintroub H. The cholesterol storage disorder of the mutant BALB/c mouse. J Biol Chem. 1985;261:2722-2777.

47. Pentchev PG, Comly ME, Kruth HS, Vanier MT, Wenger DA, Patel S, Brady RO. A defect in cholesterol esterification in Niemann-Pick disease (type C) patients. Proc Natl Acad Sci U S A. 1985;82:8247-8251. [Abstract/Free Full Text]

48. Fredrickson DS, Solan HR. Sphingomyelin lipidosis: Niemann-Pick disease. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, eds. The Metabolic Basis of Inherited Disease. 3rd ed. New York, NY: McGraw-Hill; 1972:783-790.

49. Yamamoto T, Iwasawa K, Tokoro T, Eto Y, Maekawa K. A possible same genetic defect in two Niemann-Pick disease model mice [in Japanese]. No To Hattatsu. 1994;26:318-322. [Medline] [Order article via Infotrieve]

50. Tangirala RK, Mahlberg FH, Glick JM, Jerome WG, Rothblat GH. Lysosomal accumulation of unesterified cholesterol in model macrophage foam cells. J Biol Chem. 1993;268:9653-9660. [Abstract/Free Full Text]

51. Tangirala RK, Jerome WG, Jones NL, Small DM, Johnson WJ, Glick JM, Mahlberg FH, Rothblat GH. Formation of cholesterol monohydrate crystals in macrophage-derived foam cells. J Lipid Res. 1994;35:93-104. [Abstract]

52. Schmitz G, Beuck M, Fischer H, Nowicka G, Robenek H. Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoprotein mediated cholesterol efflux in macrophages. J Lipid Res. 1990;31:1741-1752. [Abstract]

53. Panganamala RV, Geer JC, Cornwell DG. Long-chain bases in the sphingolipids of atherosclerotic human aorta. J Lipid Res. 1969;10:445-455. [Abstract]

54. Hodis HN, Crawford DW, Sevanian A. Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis. Atherosclerosis. 1991;89:117-126.[Medline] [Order article via Infotrieve]

55. Aviram M, Bar M, Maor I. Macrophage localization of unesterified cholesterol affects cellular cholesterol regulation and its cytotoxicity: atherogenicity of excess plasma membrane cholesterol. Bull Mol Biol Med. 1995;20:49-54.

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