Unique Cellular Events Occurring during the Initial Interaction of Macrophages with Matrix-retained or Methylated Aggregated Low Density Lipoprotein (LDL)

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.

The Uptake and Degradation of Matrix-bound Lipoproteins by Macrophages Require an Intact Actin Cytoskeleton, Rho Family GTPases, and Myosin ATPase Activity

“…In the first study, we showed that macrophages plated on top of SMase-aggregated and matrix-retained LDL internalized and degraded this material over a 24-h period and accumulated large amounts of intracellular, ACAT-derived CE (21). In the second study, we focused on the very earliest events that occur when macrophages first engage matrix-retained lipoproteins (22). During this period, there is prolonged cell-surface contact …”

“…with relatively little LDL-protein degradation but much more LDL-CE hydrolysis (22). The actual quantity of cholesterol internalized during this period of "selective lipid uptake" is probably not large.…”

“…9). In Phase I, there is initial prolonged cell-surface contact between the macrophage cell surface and the retained lipoproteins, leading to partial selective CE uptake and hydrolysis (22). In Phase II, the macrophages internalize and degrade the lipoproteins, and this critical process partially involves LRP and actinmyosin cytoskeletal processes as discussed above.…”

“…In this regard there are probably unique cellular processes that occur in vivo during foam cell formation that would clearly be missed in the typical experimental model, which emphasizes receptor-mediated endocytosis. Indeed, using an experimental system in which macrophages are plated on top of matrix-retained and SMaseaggregated LDL (21), we have shown previously that there is an initial period of prolonged contact between macrophages and matrix-retained lipoproteins during which LDL-CE hydrolysis exceeds LDL-protein degradation (22). This process clearly does not occur during typical receptor-mediated endocytosis, which involves rapid uptake of whole lipoprotein particles and parallel degradation of the CE and protein moieties of the lipoproteins (22,23).…”

The Cytoplasmic Domain of the Low Density Lipoprotein (LDL) Receptor-related Protein, but Not That of the LDL Receptor, Triggers Phagocytosis

“…LDL was isolated from human plasma by preparative ultracentrifugation at d ϭ 1.063 g/dl (17) and was labeled with 125 I using Iodogen-coated tubes (Pierce) and Na[ 125 I] as described previously (18); the labeled LDL had a specific activity of 150 -300 cpm/ng protein and was used within 3 weeks of iodination. Lipoprotein-depleted serum (LPDS) was prepared from fetal bovine serum by preparative ultracentrifugation at d ϭ 1.21 g/dl.…”