Abstract
A critical event in atherogenesis is the interaction of arterial wall macrophages with subendothelial lipoproteins. Although most studies have investigated this interaction by incubating cultured macrophages with monomeric lipoproteins dissolved in media, arterial wall macrophages encounter lipoproteins that are mostly bound to subendothelial extracellular matrix, and these lipoproteins are often aggregated or fused. Herein, we utilize a specialized cell-culture system to study the initial interaction of macrophages with aggregated low density lipoprotein (LDL) bound to extracellular matrix. The aggregated LDL remains extracellular for a relatively prolonged period of time and becomes lodged in invaginations in the surface of the macrophages. As expected, the degradation of the protein moiety of the LDL was very slow. Remarkably, however, hydrolysis of the cholesteryl ester (CE) moiety of the LDL was 3-7-fold higher than that of the protein moiety, in stark contrast to the situation with receptor-mediated endocytosis of acetyl-LDL. Similar results were obtained using another experimental system in which the degradation of aggregated LDL protein was delayed by LDL methylation rather than by retention on matrix. Additional experiments indicated the following properties of this interaction: (a) LDL-CE hydrolysis is catalyzed by lysosomal acid lipase; (b) neither scavenger receptors nor the LDL receptor appear necessary for the excess LDL-CE hydrolysis; and (c) LDL-CE hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis. In summary, experimental systems specifically designed to mimic the in vivo interaction of arterial wall macrophages with subendothelial lipoproteins have demonstrated an initial period of prolonged cell-surface contact in which CE hydrolysis exceeds protein degradation.
Highlights
A critical event in atherogenesis is the interaction of arterial wall macrophages with subendothelial lipoproteins
Additional experiments indicated the following properties of this interaction: (a) low density lipoprotein (LDL)-cholesteryl ester (CE) hydrolysis is catalyzed by lysosomal acid lipase; (b) neither scavenger receptors nor the LDL receptor appear necessary for the excess LDL-CE hydrolysis; and (c) LDL-CE hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis
Matrix-retained lesional lipoproteins are often aggregated and fused (16, 20 –25). The importance of these issues in foam cell formation is demonstrated by the finding that prolonged incubation of macrophages with either aggregated LDL or matrix-retained and aggregated LDL leads to massive CE accumulation (26 –31). In view of this background, it occurred to us that the cellular processes involved in the initial interaction of macrophages with lipoproteins that are retained and aggregated in a threedimensional matrix may differ substantially from the processes involved in the initial interaction of macrophages with monomeric lipoproteins dissolved in tissue culture medium
Summary
Materials—Tissue culture media and reagents were purchased from Life Technologies, Inc., tissue culture plates were from Corning, and defined fetal bovine serum was from HyClone Laboratories, Inc. (Logan, UT). Cells—J774.A1 macrophages (from the American Type Culture Collection) [39] were maintained in spinner culture in DMEM, 10% (v/v) fetal bovine serum containing penicillin (50 units/ml), streptomycin (50 units/ml), and glutamine (2 mM). The cells were plated in 16-mm wells (24-well dishes) in DMEM, 10% (v/v) fetal bovine serum, containing penicillin, streptomycin, and glutamine, and allowed to grow until confluent. The day prior to the experiment, the cells were washed three times with warm PBS and incubated with 1 ml of DMEM, 0.2% (w/v) fatty acid-free BSA per well. Experimental System Involving Retained and Aggregated LDL—For the experimental system depicted in Fig. 1A (below), monolayers of endothelial or smooth muscle cells were incubated for 6 h at 37 °C with DMEM, 0.2% BSA, containing 10 g of lipoprotein lipase/ml. Absent error bars signify S.D. values smaller than the graphics symbol
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