High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Abstract

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Fig. 1.

Foam cell formation with native LDL. BMMs were treated for 24 h with the indicated concentration of LDL in medium B. Cells were then washed and stained with Oil Red O (A), or cell lipids were extracted and used for determination of TC, FC, and CE content (B, shown as the mean ± SEM, n = 3). Results are representative of four independent experiments.

Fig. 2.

Macrophage selective LDL CE uptake. BMMs were treated in medium B with the indicated concentration of [³H]CEt/¹²⁵I-LDL for 4 h (A) or with 250 µg/ml [³H]CEt/¹²⁵I-LDL for the indicated time periods (B, C: panel C is a magnification of panel B for clarification). D: BMMs were pretreated for 24 h in medium A with or without 500 µg/ml LDL or 25 µg/ml acetylated LDL and were then washed and treated for 4 h in medium B with 250 µg/ml [³H]CEt/¹²⁵I-LDL. CE uptake was determined from [³H]CEt and ¹²⁵I-LDL uptake, and selective CE uptake [(³H)-(¹²⁵I)] was calculated as the difference between these two values, as described in Materials and Methods. Values in panels A–C are the mean ± SEM (n = 3), and values in panel D are the mean ± SEM (n = 4) expressed as the percent of the control value (not treated with LDL or acLDL). * P < 0.05 compared with ¹²⁵I or control; ** P < 0.01; *** P < 0.001. Results are representative of three independent experiments.

Fig. 3.

Comparison of CEt, CE, and FC uptake from LDL. BMMs were treated in medium B with 250 µg/ml of ¹²⁵I-LDL labeled with [³H]CEt, [³H]CE, or [³H]FC for the indicated time periods. A: LDL CE or FC uptake was determined from cell-associated ³H after treatment with the indicated ligand. a, P < 0.05, [³H]CEt versus [³H]FC; b, P < 0.05, [³H]CE versus [³H]FC; c, P < 0.001, [³H]CEt versus [³H]FC; d, P < 0.001, [³H]CE versus [³H]FC. B: FC uptake from [³H]FC/¹²⁵I-LDL was determined from [³H]FC and ¹²⁵I-LDL uptake, and selective FC uptake [(³H)-(¹²⁵I)] was calculated as the difference between these two values, as described in Materials and Methods. Values are the mean ± SEM (n = 3). Results are representative of three independent experiments.

Fig. 4.

Effect of LDL modification or scavenger receptor deficiency on selective CE uptake. A: BMMs were treated with 10 µg/ml [³H]CEt/¹²⁵I-labeled LDL, aggregated LDL (agLDL), or acetylated LDL (acLDL) for 4 h. a, P < 0.05 compared with the corresponding [³H]CEt from the same ligand; b, P < 0.05 compared with LDL [³H]CEt; c, P < 0.001 compared with LDL ¹²⁵I; d, P < 0.001 compared with LDL [³H]CEt. B: BMMs from wild type, SR-BI-null, CD36-null, or DKO mice were treated with 10 µg/ml [³H]CEt/¹²⁵I-LDL for 4 h. CE uptake was determined from [³H]CEt and ¹²⁵I-LDL uptake, and selective CE uptake [(³H)-(¹²⁵I)] was calculated as the difference between these two values, as described in Materials and Methods. Values are the mean ± SEM (n = 3).

Fig. 5.

Role of actin polymerization in selective LDL CE uptake. BMMs were pretreated for 30 min with the indicated concentration of cytochalasin D in medium A, then washed and treated for 4 h with the same addition of cytochalasin D and 500 µg/ml [³H]CEt/¹²⁵I-LDL or 100 µg/ml ¹²⁵I-BSA in medium B. ¹²⁵I-LDL uptake (A), ¹²⁵I-BSA uptake (B), and selective [³H]CEt uptake (C) were determined as described in Materials and Methods. Values are the mean of duplicate determinations, and results are representative of three independent experiments.

Fig. 6.

LDL CE uptake and hydrolysis. BMMs were treated with 1,000 µg/ml [³H]CE/¹²⁵I-LDL for the indicated time period, then lipid was extracted from cells (A) and media (B) and separated by TLC. [³H]CE and [³H]FC were quantified as described in Materials and Methods. C: In the same experiment, CE uptake was determined from [³H]CEt and ¹²⁵I-LDL uptake, and selective CE uptake [(³H)-(¹²⁵I)] was calculated as the difference between these two values, as described in Materials and Methods. ***, P < 0.001, [³H]CE compared to ¹²⁵I. Values are the mean ± SEM (n = 3). Results are representative of two independent experiments.

Fig. 7.

LDL-derived CE hydrolysis and efflux. BMMs were treated for 30 min in medium B with 250 µg/ml [³H]CE/¹²⁵I-LDL and then washed thoroughly and chased with 250 µg/ml unlabeled LDL in medium B for the indicated time period, after which lipid was extracted from cells (A) and media (B), and [³H]CE and [³H]FC were quantified as described in Materials and Methods. Values are the mean ± SEM (n = 3). Results are representative of two independent experiments.

Fig. 8.

Effect of chloroquine and UBP on CE uptake and hydrolysis. BMMs were pretreated for 2 h in medium A with or without 200 µM chloroquine (chlor) or 400 µM UBP, then washed and treated under the same conditions in medium B plus 250 µg/ml [³H]CE/¹²⁵I-LDL for 4 h. Cells were then washed thoroughly, and media and cell lysates were collected and used for determination of ¹²⁵I-LDL uptake, association, and degradation as described in Materials and Methods (A). Cell lipid was extracted and separated by TLC, and [³H]CE and [³H]FC were quantified as described in Materials and Methods (B). Values are the mean ± SEM (n = 4). Results are representative of three independent experiments. ** P < 0.01 compared with control; *** P < 0.001.

Fig. 9.

ACAT-dependent reesterification of LDL CE-derived cholesterol. A: BMMs were treated for 24 h in medium B with 250 µg/ml [³H]CE-LDL or [³H]FC-LDL with or without 10 µg/ml of the ACAT inhibitor CI-976. Lipid from cells was extracted and separated by TLC, and [³H]CE (left panel) and CE or FC uptake (right panel) were quantified as described in Materials and Methods. * P < 0.05 compared with DMSO control. B: BMMs were treated for the indicated time with 1,000 µg/ml [³H]CE-LDL in medium C (containing 0.2 mM [¹⁴C]oleate, left panel) or in medium C with the addition of 10 µg/ml CI-976 (right panel), then washed, and cellular [³H]CE and [¹⁴C]CE were extracted and quantified as described in Materials and Methods. C: BMMs were treated for 24 h in medium B with 500 µg/ml [³H]CE-LDL (left panel) or 25 µg/ml [³H]CE-acLDL (right panel). Cells were then washed, and [³H]CE and [³H]FC were quantified as described in Materials and Methods. Aliquots of lipid extracts from the same cells were also used to determine the CE and FC content, as described in Materials and Methods. Values are the mean ± SEM (n = 3). Results are representative of two independent experiments.