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...

BackgroundThe metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas.MethodsWe classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis.ResultsGroup-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported.ConclusionsIntratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1469-5) contains supplementary material, which is available to authorized users.

Background-Low-density lipoprotein (LDL) receptor-related protein (LRP) is highly expressed in vascular smooth muscle cells (VSMCs) of both normal and atherosclerotic lesions. However, little is known about LRP regulation in the vascular wall. Methods and Results-We analyzed the regulation of LRP expression in vitro in human VSMCs cultured with native LDL (nLDL) or aggregated LDL (agLDL) by semiquantitative reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, and Western blot and in vivo during diet-induced hypercholesterolemia by in situ hybridization. LRP expression in human VSMCs is increased by nLDL and agLDL in a time-and dose-dependent manner. Maximal induction of LRP mRNA expression was observed after 24 hours of exposure to LDL. However, agLDL induced higher LRP mRNA expression (3.0-fold) than nLDL (1.76-fold). LRP mRNA upregulation was associated with an increase on LRP protein expression with the greatest induction by agLDL. VSMC-LRP upregulation induced by nLDL or agLDL was reduced by an inhibitor of sterol regulatory element binding protein (SREBP) catabolism (N-acetyl-leucyl-leucyl-norleucinal). In situ hybridization analysis indicates that there is a higher VSMC-LRP expression in hypercholesterolemic than in normocholesterolemic pig aortas. Conclusions-These results indicate that LRP expression in VSMCs is upregulated by intravascular and systemic LDL.

Abstract-Versican-like proteoglycans are the main component of the intimal extracellular matrix interacting with low density lipoprotein (LDL). The aim of this study has been to investigate the receptors involved in versican-modified LDL uptake by human vascular smooth muscle cells (VSMCs). We have found that versican-LDL interaction leads to the following: (1) monomeric LDL particles that are similar in size and electrophoretic mobility to native LDL but that have a higher capacity to induce intracellular cholesteryl ester (CE) accumulation and (2) V ascular proteoglycans (PGs) have a common structure of a core protein to which glycosaminoglycan (GAG) chains are covalently attached. The content of versican-like PG, the main PG structuring the extracellular matrix, is high in regions prone to lesion development and increases with lesion progression. 1-3 Interestingly, it has been demonstrated that LDL binds and is retained by versican-like PGs secreted by human vascular smooth muscle cells (VSMCs) 3-5 and macrophages. 6,7 The binding between LDL and versican or biglycan seems to be enhanced in the presence of lipoprotein lipase 8 or phospholipase A 2 . 9,10 The relevance of the interaction between LDL and PG in vivo has been demonstrated (1) by the isolation of complexes apoB-100 -chondroitin sulfate (CS), which is rich in negatively charged GAGs from human arteries 1 ; (2) by the colocalization of apoB in the intima with CS-rich regions 11,12 ; and (3) by the formation of complexes ex vivo between LDL and CS-PGs extracted from arterial wall. 13,14 Versican is one of the PGs with the highest binding affinity for plasma LDL. It has been described that GAGs accelerate proteolytic and oxidative modification of the particles 5 and can also induce LDL aggregation and fusion of the LDL particle under certain incubation conditions. 15 Fused LDLs, remarkably similar to those found in the arterial wall, 16,17 can be obtained by vortexing. 18 We have recently demonstrated that modified LDL particles generated by vortexing are taken up through the LDL receptor-related protein (LRP) in human VSMCs. 19 LRP is also the receptor that mediates the binding and internalization of other modified lipoproteins, such as apoE-enriched VLDL, 20 lipoprotein lipase-triglyceride-rich lipoprotein complexes, 21,22 Lp(a), 23 and chylomicron remnants. 24 Although LRP is expressed in normal vessels and atherosclerotic lesions, LRP expression increases in rabbit atherosclerotic lesions; furthermore, LRP seems to play a role in the development of atherosclerotic lesions. 25,26 In the present study, we report on the characteristics of changes induced in the LDL particle by the interaction with versican, the effect of versican-LDL on the cholesteryl ester (CE) content of VSMCs, and the involvement of LRP in the uptake of versican-LDLs in human VSMCs. Our results indicate that versican interaction with LDL leads to the following: (1) monomeric LDL particles (similar to native LDL [nLDL] in electrophoretic mobility and electron microscopy size) th...