LDL Receptor–Related Protein Mediates Uptake of Aggregated LDL in Human Vascular Smooth Muscle Cells

Abstract

Abstract-Foam cell formation is a key event in the onset and progression of atherosclerotic lesions. We have previously reported that internalization of aggregated low density lipoproteins (agLDLs) by vascular smooth muscle cells (VSMCs) produces cholesteryl ester (CE) accumulation in these cells. The aim of this study was to analyze whether the low density lipoprotein receptor-related protein (LRP) mediates the uptake of agLDL by VSMCs. First, immunocytochemistry and fluorescence microscopic analysis with the use of anti-LRP antibodies indicated that there was a high expression of LRP in VSMCs. Confocal microscopic analysis with the use of agLDLs labeled with fluorochrome 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine and anti-LRP antibodies showed the colocalization of agLDL and LRP.The second approach was to analyze the effect of LRP ligands on agLDL internalization; lactoferrin strongly inhibited CE accumulation from agLDLs (85.0Ϯ5.7% at 25 g/mL) by impairing agLDL binding. Coincubation of agLDL with anti-LRP antibodies decreased in a dose-dependent manner agLDL-derived CE accumulation (from 20% at 12.5 g/mL to 80% at 50 g/mL). The third approach was to evaluate whether antisense LRP oligodeoxynucleotides were able to block agLDL internalization. Treatment of VSMCs with 5 mol/L antisense LRP oligodeoxynucleotides reduced agLDL-derived CE accumulation by 84Ϯ2%. In conclusion, these results from immunologic, biochemical, and molecular interventions demonstrate that LRP mediates the binding and internalization of agLDL in human VSMCs. Because LRP is highly expressed in VSMCs and the uptake of 1 LDL aggregate amounts to the deposition of several hundreds of LDL particles, the uptake of agLDL through LRP could have a crucial role for lipid deposition in VSMCs. O ne of the main events in the atherogenic process is the accumulation of lipids, mainly cholesteryl esters (CEs). 1,2 Vascular smooth muscle cells (VSMCs) synthesize proteoglycans, extracellular matrix components involved in the focal deposition of cholesterol-rich particles. 3,4 The uptake of matrix-retained lipoproteins by VSMCs and macrophages produces CE accumulation and leads to foam cell formation. Macrophages become foam cells through the uptake of diversely modified LDLs, whereas the aggregation of LDLs seems to be a key condition for lipid accumulation in VSMCs. [5][6][7] Recently, we have shown that accumulation of CEs in VSMCs from in vitro-aggregated LDL (agLDL) is dependent on agLDL concentration and the degree of aggregation. 8 AgLDLs obtained by vortexing LDL in vitro share structural characteristics with LDL aggregates present in atherosclerotic lesions. 9 -11 In macrophages, phagocytosis and/or scavenger receptors mediate cholesterol accumulation from different types of modified LDL. [12][13][14][15][16] In human lesions, scavenger receptors are present at high levels in macrophages but not in VSMCs. 17,18 VSMCs express scavenger receptors only after stimulation with certain growth factors, 19,20 and a direct involvement of these...

Aggregated Low-Density Lipoprotein Uptake Induces Membrane Tissue Factor Procoagulant Activity and Microparticle Release in Human Vascular Smooth Muscle Cells

“…AgLDL was prepared as described previously. 17,18 Levels of thiobarbituric acid-reactive substances (Ͻ1.2 mmol of malonaldehyde per milligram of protein LDL) remained similar to those in nLDL after LDL aggregation.…”

“…17,18 Non-LRP-expressing VSMCs were obtained by treatment of the cells with antisense LRP oligodeoxynucleotides (ODNs; 10 mol/L) as described previously. [17][18][19] …”

“…Lipid extraction was performed as described previously. 17,18 The chromatographic conditions were as previously described, 23 with minor modifications.…”

“…15,16 We have recently demonstrated that agLDLs induce high intracellular cholesteryl ester accumulation through LDL receptor-related protein (LRP) uptake in human VSMCs. [17][18][19] Additionally, agLDL upregulates LRP expression in a time-and dose-dependent manner. 20 The aim of this study was to analyze the role of agLDL, compared with nLDL, in cellular TF expression, TF release, and TF activity in human VSMCs.…”

Macrophages of genetically characterized familial hypercholesterolaemia patients show up‐regulation of LDL‐receptor‐related proteins

“…Macrophages attempt to clear the particles and become filled with esterified cholesterol droplets. We and others have demonstrated that LRPs play a crucial role in the uptake of agLDL either in vascular smooth muscle cells or in macrophages 33, 73. Moreover, our group has reported a higher efficacy of LRP5 than LRP1 for promoting CE accumulation in human macrophages 22 which was related to an early saturation of LRP1 lipid uptake activity.…”