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(Circulation. 2002;106:3104.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Low-Density Lipoprotein Upregulates Low-Density Lipoprotein Receptor-Related Protein Expression in Vascular Smooth Muscle Cells

Possible Involvement of Sterol Regulatory Element Binding Protein-2-Dependent Mechanism

Vicenta Llorente-Cortés, PhD; Marta Otero-Viñas, MS; Sonia Sánchez, MS; Cristina Rodríguez, PhD; Lina Badimon, PhD, FESC

From the Cardiovascular Research Center, IICB (CSIC)-ICCC, Barcelona. Spain.

Correspondence to Professor Lina Badimon, Cardiovascular Research Center, C) Jordi Girona 18-26, 08034 Barcelona Spain. E-mail lbmucv{at}cid.csic.es

	Abstract

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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.

Key Words: arteriosclerosis • lipoproteins • receptors • hypercholesterolemia • vasculature

	Introduction

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Low-density lipoprotein receptor-related protein (LRP) is a type I membrane protein (600 kDa) that has a short C-terminal subunit (LRP-85) and a large N-terminal subunit (LRP-515) that contains most of the extracellular portion of the molecule and binds to all known LRP ligands.^1–3 LRP has been shown to act as an endocytosis-mediating receptor for several ligands, including trombospondin,⁴ protease-anti-protease complexes,⁵ tissue factor pathway inhibitor (TFPI),⁶ and plasma lipoproteins such as apolipoprotein E-enriched VLDL,^7,8 lipoprotein lipase, hepatic lipase and lipoprotein lipase-triglyceride-rich lipoprotein complexes,^9,10 chylomicrons remnant,¹¹ and lipoprotein a.¹² We recently demonstrated that in human vascular smooth muscle cells (VSMCs), LRP mediates the internalization of aggregated LDL (agLDL) generated either by vortexing¹³ or by incubation with versican,¹⁴ one of the main proteoglycans interacting with LDL in the arterial wall.¹⁵ LRP-mediated agLDL uptake could be one of the main mechanisms for intracellular lipid accumulation in VSMCs. Indeed, an increase in LRP expression has been described in advanced atherosclerotic plaques¹⁶ and in patients with coronary obstruction.¹⁷ These results indicate that LRP might play a role in atherosclerosis progression, although little is known about how LRP is upregulated. In macrophages, it has been described that LRP mRNA levels are increased by colony stimulating factor-1 and insulin,^18,19 whereas they were decreased by transforming growth factor-ß and LPS.^20,21 Despite the strong sequence homology of LRP and LDL receptor,²² they have little similarity in the promoter region.²³ LDL receptor, like other genes involved in lipid metabolism, contains the consensus sterol regulatory elements (SRE) in the promoter, allowing its regulation through SRE-1 binding proteins (SREBPs).²⁴ SRE-1 can bind 3 different transcription factors: SREBP-1a, SREBP-1c, and SREBP-2.²⁵ Although LRP does not contain an SRE in the promoter, an intriguing SRE-1 site has been found in the unusually long 5'-untranslated region of LRP.²⁶

The aim of this work was to analyze the effect of agLDL on LRP expression in vitro in cultured human VSMCs and in vivo in a hypercholesterolemic porcine model. Here, we show that agLDL upregulates LRP expression in a dose- and time-dependent manner in human VSMCs. In agreement, LRP expression was significantly enhanced in the vascular wall of hypercholesterolemic animals.

	Methods

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VSMC Culture
Primary cultures of human VSMCs were obtained from nonatherosclerotic areas of macroscopically healthy ascending aortas of explanted hearts removed from nonischemic cardiomyopathy patients transplanted at the Hospital de la Santa Creu i Sant Pau with the permission of the ethics committee of the Hospital. Histological analysis (Masson Trichromic stain) of these arteries did not show atherosclerotic changes, although prearteriosclerotic molecular abnormalities in these human arteries cannot be excluded. VSMCs were obtained by a modification of the explant technique as we described previously.^13,14 VSMCs were identified morphologically using light microscopy and by their growth behavior and were characterized by immunological staining of cytoskeleton proteins. Primary and subcultured VSMCs with a low in vitro age had a spindle-shaped appearance. Confluent VSMC cultures showed the characteristic "hill-and-valley" growth pattern. Mouse monoclonal antibodies specific for human -SM actin (clone 1A4), human von Willebrand factor (clone F8/86), and human fibroblast surface protein (clone 1B10) were used. VSMCs were used between passages 2 and 4. Cells showed positive immunostaining with anti-SMC -actin. No staining was observed with antibodies against a human fibroblast cell-surface antigen or von Willebrand factor. Cells were arrested 48 hours in M199 supplemented with 0.2% FCS and incubated with different concentrations of nLDL and agLDL for the indicated times (0 to 48 hours). In some experiments, cells were treated for 24 hours with N-acetyl-leu-leu-norleucinal (ALLN) (25 µmol/L).

Animals
Female pigs (body weight at initiation, 32±4 kg) were divided into two groups: normocholesterolemic animals (n=6), which were fed a normal chow diet, and hypercholesterolemic animals (n=10), which were fed a cholesterol-rich diet (2% cholesterol, 1% cholic acid, 20% beef tallow) for 100 days.²⁷ After 100 days, the animals were killed with a thiopental overdose. Plasma cholesterol levels and hematological parameters were measured at baseline and at death. Because the porcine model of atherosclerosis initially develops lesions in the abdominal aorta, rings from this vessel were collected and fixed in 4% paraformaldehyde in PBS 0.1 mol/L (pH 7.4), cryoprotected in 30% saccharose in PBS 0.1 mol/L (pH 7.4), embedded in OCT, and frozen on dry ice. All procedures were in accordance with institutional guidelines and followed the American Physiological Society guidelines for animal research.

Plasma total cholesterol was determined with an automatic analyzer (Kodack Ektachem DT System). Plasma LDL-cholesterol was analyzed using the validated methods of the Lipid Research Clinic Program²⁸ and quantified spectrophotometrically (Kontron Instruments).

LDL Preparation and Determination of the Free Cholesterol and Cholesteryl Esters Content
Human LDLs (d_1.019-d_1.063 g/mL) were obtained and modified as previously described.^13,14 LDL preparations were <48 hours old, nonoxidized (<1.2 mmol malondialdehyde/mg protein LDL), and without detectable levels of endotoxin (Limulus Amebocyte Lysate test, Bio Whittaker).

Arrested cells were incubated with nLDL or agLDL (100 µg/mL) for 6, 12, 24, and 48 hours. In some experiments, cells were pretreated for 24 hours with ALLN (25 µmol/L) before adding LDL (50, 100 µg/mL). Cells were then exhaustively washed and harvested into 1 mL of 0.10 N NaOH. Lipid extraction and TLC were performed as previously described.^13,14

Semiquantitative and Real-Time PCR
Arrested cells were incubated with nLDL or agLDL for 6, 12, 24, and 48 hours. RNA and protein were isolated by using the Tripure isolation Reagent (Roche Molecular Biochemicals) according to the manufacturer. LRP and LDL receptor mRNA levels were analyzed by semiquantitative RT-PCR as previously described.¹⁴ The specific oligonucleotides for SREBPs were SREBP-1 forward primer: 5'-atgtagtcgatggccttgcg-3'; SREBP-1 reverse primer: 5'tgtgacc-tcgcagatccagc-3'; SREBP-2 forward primer: 5'-tgggaccattctgaccacaa-3'; and SREBP-2 reverse primer: 5'-tgtgacctcgcagatccagc-3'. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize results. Amplification was carried out by 20 (GAPDH), 20 (LRP), 25 (LDL receptor), 20 (SREBP-1), and 22 (SREBP-2) cycles. PCR products (10 µL) were resolved by electrophoresis in 1.5% to 2% agarose gels and transferred onto nylon membranes by the standard capillary technique. Blots were UV cross-linked. Detection of digoxigenine (DIG)-labeled nucleic acids was performed with an anti-DIG antibody linked to alkaline phosphatase, and CSPD (Disodium 3-(4-methoxispiro (1,2-dioxetane-3,2'-15'-chloro) tricyclo [3.3.1.1^3,7] decan)-4-yl) phenylphosphate (Roche Molecular Biochemicals) was used as substrate.

TaqMan fluorescent Real-Time PCR primers and probes (6'FAMMGB) for LRP and LDL receptor were designed by use of Primer Express software from PE biosystems and were as follows: LRP forward: 5'-gagctgaaccacgcctttg-3'; LRP reverse: 5'-ggtagacactg-ccactccgatac-3'; LRP probe: 5'-ttgccatggtgacacag-3'; LDL receptor forward: 5'- tgacaatgtctcaccaagctctg-3'; LDL receptor reverse: 5'-ctcacgctactgggcttcttct-3'; and LDL receptor probe: 5'- ctgccagcaacgtcg-3'. Human gapdh (4326317E) was used as endogenous control. The specificity and the optimal primer and probe concentrations were tested. Taqman real-time PCR was performed with 2-µL/well of RT products (1 µg total RNA) in 25 µL of TaqMan PCR Master Mix (PE Biosystem) with the primers at 300 nmol/L and the probe at 200 nmol/L. PCR was performed at 95°C for 10 minutes (for AmpliTaq Gold activation) and then run for 40 cycles at 95°C for 15 seconds and 60°C for 1 minute on the ABIPRISM 7000 Detection System. The threshold cycle (Ct) values were determined and normalized to the housekeeping gene gapdh. Because the targets have similar amplification efficiency as endogenous controls, we have used the comparative Ct method (delta delta Ct) to perform relative quantification of LRP and LDL receptor.

Western Blot Analysis
SDS/PAGE was run as previously described.¹⁴ Blots were incubated with monoclonal antibodies against human LRP (ß-chain; Research Diagnostics, clone 8B8 RDI 61067, dilution 1:40). To test equal protein loading for the different samples, blots were also incubated with monoclonal antibodies against human -actinin (Chemicon International, MAB 1682, dilution 1:10000).

LRP In Situ Hybridization
Abdominal aorta sections (n=3) from the same segment of normocholesterolemic and hypercholesterolemic animals were analyzed. Conventional histology (Masson Trichrome) was performed to identify the lesion type.

For riboprobe synthesis, human LRP cDNA corresponding to nucleotides 2221 to 2764 (544 mer) was cloned in plasmid vectors using standard techniques. Antisense and sense riboprobes were synthesized using T3 and T7 RNA polymerase (Promega), respectively, and DIG-labeled nucleotides (Roche Molecular Biochemicals). In situ hybridization studies were performed on a set of 20-µm-thin sections. Sections were fixed before being permeabilized by proteinase K. Proteinase K was then deactivated and the sections were treated with TEA, postfixed again, and blocked with 2 mg/mL glycine in PBS. After wash with SSCx2, sections were prehybridized with a solution of 50% formamide, SSCx5, 50 µg/mL heparin, 5% dextran sulfate, 0.01%SDS, Denhardt’s solution x5, 100 µg/mL polyAmRNA, 25 µg/mL tRNA, 25 µg/mL salmon sperm DNA. Sections were then incubated with the probe at 1 µg/mL in the prehybridization solution overnight at 53°C. Digoxigenin labeled nucleotides were detected by an alkaline phosphatase conjugated anti-digoxigenin antibody provided by the Dig-labeled immunodetection kit, using NBT/BCIP as substrate (Vector Laboratory). Results were evaluated with an Olympus Vanox fluorescence microscope. Images were digitalized using a Sony 3CCD camera. Controls were performed by the incubation of the section with the sense riboprobe and with the empty-plasmid riboprobes.

Data Analysis
Data were expressed as mean±SEM. A statview (Abacus Concepts) statistical package for the Macintosh computer system was used for all analysis. Multiple groups were compared by nonparametric tests, Wilcoxon, or Mann-Whitney U, as needed. Statistical significance was considered when P<0.05.

	Results

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nLDL and agLDL Downregulate LDL Receptor and Upregulate LRP mRNA Expression in a Time- and Dose-Dependent Manner
Differences in cholesteryl ester (CE) levels between VSMCs incubated with nLDL or agLDL were highly significant (P<0.05) (Figure 1). VSMC-CE accumulation derived from agLDL increased from undetectable levels to 103±5.2 µg/mg protein at 48 hours. On the contrary, VSMC-CE accumulation from nLDL was slightly increased (from undetectable levels to 32.2±3.16 µg/mg protein at 48 hours), reaching a plateau after 12 hours of incubation. Free cholesterol content was not changed by either agLDL or nLDL.

View larger version (50K): [in this window] [in a new window]   Figure 1. Thin layer chromatography of the VSMC intracellular cholesterol accumulation induced by nLDL and agLDL. VSMCs were incubated with nLDL (triangles) or agLDL (circles) (100 µg/mL) for increasing times. Filled symbols indicate Cholesteryl esters (CE); open symbols, free cholesterol (FC). Results are expressed as microgram of cholesterol per milligram of protein and are shown as mean±SEM (n=2).

LRP was slightly upregulated by nLDL and strongly by agLDL in a time-dependent (Figure 2A) and dose-dependent (Figure 2C) manner. AgLDL was able to upregulate LRP expression at the lowest concentration tested (50 µg/mL). Maximal LRP induction by both nLDL and agLDL (100 µg/mL) was observed after 24 hours; however, agLDL induced a much higher LRP expression than nLDL (agLDL, 3±0.12 versus nLDL, 1.76±0.32; P<0.05). In absence of LDL (control), LRP expression remained unaltered along the tested times.

View larger version (26K): [in this window] [in a new window]   Figure 2. Time and dose response of LRP and LDL receptor to nLDL and agLDL. VSMCs were incubated in the absence (squares) or presence of nLDL (triangles) or agLDL (circles) (100 µg/mL) for increasing times (A and B) or incubated with increasing concentrations of nLDL (triangles) or agLDL (circles) for 24 hours (C, D). A and C, Real-time PCR quantification of LRP mRNA. B and D, Real-time PCR quantification of LDL receptor mRNA. Data were processed with a specially designed software program based on Ct values of each sample and normalized to gapdh mRNA (n=3).

Both nLDL and agLDL downregulated LDL receptor mRNA expression in a time-dependent (Figure 2B) and dose-dependent (Figure 2C) manner. The lowest concentration tested of both nLDL and agLDL (50 µg/mL) completely reduced LDL receptor expression. The time-course assays performed with the highest LDL concentration (100 µg/mL) showed a complete LDL receptor downregulation after 6 hours with nLDL and after 12 hours with agLDL. A slight reversion of the LDL downregulation was observed after 24 and 48 hours of LDL incubation. In absence of LDL (control), LDL receptor expression remained unaltered along the tested times.

Western blot analysis showed that both nLDL and agLDL (100 µg/mL, 24 hours) were able to induce LRP protein synthesis. However, agLDL induced higher LRP protein expression than nLDL (agLDL: 8.3±1.2 versus nLDL: 3.3±0.5; P<0.05) (Figure 3).

View larger version (29K): [in this window] [in a new window]   Figure 3. Western blot showing LRP (ß-chain) and -actinin protein levels in VSMCs incubated with nLDL or agLDL (100 µg/mL). Results are expressed as a percentage of control cells (incubated in absence of LDL) and are shown as mean±SEM (n=3).

nLDL and agLDL Downregulated SREBP-2 mRNA Levels in a Time- and Dose- Dependent Manner
We evaluated SREBPs mRNA expression levels in VSMCs exposed to increasing concentrations of nLDL and agLDL, and although SREBP-1 remained unaltered, SREBP-2 levels were downregulated in a dose-dependent (Figure 4A) and time-dependent (Figure 4B) manner. The lowest concentration tested of both nLDL and agLDL (50 µg/mL) decreased SREBP-2 mRNA expression by 50%, and a complete SREBP-2 downregulation was observed at 100 µg/mL. The time-course assays performed with the highest LDL concentration (100 µg/mL) showed that, as observed with the LDL receptor mRNA expression, there was a delay on the SREBP-2 downregulation induced by agLDL (12 hours) compared with that induced by nLDL (6 hours). In addition, similarly to the LDL receptor expression, a slight reversion of the SREBP-2 downregulation was observed after 24 and 48 hours of incubation with nLDL or agLDL.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of nLDL and agLDL on SREBP mRNA levels. A, VSMCs were incubated with increased concentrations of nLDL (triangles) or agLDL (circles) for 24 hours, and SREBP-1 (open symbols) or SREBP-2 (filled symbols) mRNA levels were determined by RT-PCR. B, Time-course regulation of SREBP-2 by nLDL and agLDL (100 µg/mL). Results were normalized by gapdh mRNA level and are expressed as percentage of controls (n=2).

ALLN Prevents the LDL Receptor Downregulation and the LRP Upregulation Caused by nLDL and agLDL
To analyze the possible involvement of SREBPs in LDL-mediated upregulation of LRP expression, we analyzed the effect of ALLN (25 µmol/L), an inhibitor of SREBP catabolism, on LRP mRNA levels. ALLN blocked LRP upregulation induced by LDL not only at mRNA (Figure 5B) but also at the protein level (Figure 5D). As expected, ALLN almost completely prevented the downregulation on LDL receptor mRNA expression induced by nLDL and agLDL (100 µg/mL, 24 hours) (Figure 5C). ALLN also decreased the CE accumulation derived from agLDL by 36±4% at 50 µg/mL agLDL and by 46±6.8% at 100 µg/mL agLDL (Figure 5E), indicating that LRP function is affected by ALLN. Taken together, these results suggest the involvement of SREBP-2 on the LRP upregulation caused by nLDL and agLDL. ALLN did not show any effect on SREBP-2 mRNA expression in control or LDL-incubated VSMCs (Figure 5A).

View larger version (46K): [in this window] [in a new window]   Figure 5. Effect of ALLN on LRP upregulation induced by nLDL and agLDL. VSMCs were incubated with nLDL or agLDL (100 µg/mL) and ALLN (25 µmol/L) during 24 hours. A, Autoradiography showing LRP and LDL receptor mRNA expression levels obtained by semiquantitative RT-PCR. B, Real-time PCR quantification of LRP mRNA in ALLN absence (open bars) or presence (closed bars). C, Real-time PCR quantification of LDL receptor mRNA in ALLN absence (open bars) or presence (closed bars). Data were processed with a specially designed software program based on Ct values of each sample and normalized to gapdh mRNA (n=2). D, Western blot showing LRP (ß-chain) and -actinin protein levels. E, Thin-layer chromatography showing the FC and CE bands of VSMC. Bar graphs showing the quantification of CE bands in absence (open bars) or presence (closed bars) of ALLN. Results are expressed as microgram of cholesterol per milligram of protein and are shown as the mean of two experiments performed in duplicate (deviations <5% of the mean do not appear in the computer-originated graphs).

LRP mRNA Levels Were Higher in VSMCs of Hypercholesterolemic Pigs
To determine whether hypercholesterolemia influences LRP expression in VSMCs in vivo, we performed in situ hybridization analysis. As shown in the Table, animals fed the hypercholesterolemic diet showed higher cholesterol plasma levels. Masson Trichromic staining shows very incipient lesions in hypercholesterolemic pigs (Figure 6B). In situ hybridization analysis revealed that there is an increase in LRP expression (arrows) in the intima-media layer (just below developing plaques) of hypercholesterolemic (Figure 6D) compared with normocholesterolemic pigs (Figure 6C). Controls with sense riboprobes were negative for normocholesterolemic (Figure 6E) and hypercholesterolemic (Figure 6F) pigs.

View this table: [in this window] [in a new window]   Plasma Lipid Profile in Normolipemic and Hyperlipemic Pigs

View larger version (55K): [in this window] [in a new window]   Figure 6. In situ hybridization analysis of LRP expression in normocholesterolemic and hypercholesterolemic pigs. Masson trichromic stain of samples from abdominal aorta of normocholesterolemic (A) and hypercholesterolemic (B) pigs. In situ hybridization with LRP antisense riboprobe in normocholesterolemic (C) and hypercholesterolemic (D) pigs. Controls with LRP sense riboprobe in normocholesterolemic (E) and hypercholesterolemic (F) pigs. Arrows indicate LRP mRNA expression. Magnification x100.

	Discussion

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In human VSMCs, LRP is the lipoprotein receptor that internalizes agLDL obtained by vortexing¹³ or incubation with versican.¹⁴ Because vortexing generates agLDL similar to that isolated from the arterial wall¹⁵ and versican is one of the main proteoglycans interacting with the LDL in the arterial intima,¹⁶ the LRP-mediated agLDL uptake could have a crucial role in VSMC-lipid deposition in atherosclerotic plaques.

In this work, we demonstrate that agLDL strongly upregulates LRP at transcriptional level. The increase on mRNA LRP transcription leads to a large increase in LRP protein expression. In these cells, both nLDL and agLDL completely downregulate LDL receptor expression. Consequently, by inducing LRP expression in human VSMC, agLDL could lead to a progressive intracellular accumulation of cholesterol in these cells. The equal capacity of nLDL and agLDL to downregulate LDL receptor expression can be explained by their identical capacity to downregulate SREBP-2 mRNA levels. LDL receptor downregulation is prevented by ALLN, an inhibitor of SREBP-2 catabolism, confirming the expected response of cells to cholesterol loading. Interestingly, nLDL and agLDL were unable to upregulate LRP expression in ALLN-treated VSMCs. Furthermore, LRP upregulation, like LDL receptor downregulation, seems to be dependent on SREBP-2 downregulation. However, other factors besides SREBP-2 must be involved on LRP upregulation, because agLDL has much more capacity than nLDL to increase LRP expression levels, whereas both lipoproteins have equal capacity to completely downregulate SREBP-2 levels. These results suggest that the large amount of cholesterol internalized from agLDL into cells could modulate other transcription factors likely involved in the LRP upregulation. Although the LRP gene does not have SRE-1 sequences in its promoter, an SRE-1 site in the unusually long 5'-untranslated region has been described.²⁶ Other genes such as microsomal triglyceride transfer protein²⁹ or 7-hydroxylase³⁰ have been described to be upregulated through SREBPs downregulation. Our results obtained in human VSMCs are in agreement with those obtained in macrophages, because LRP upregulation has been observed in cells incubated with cholesterol and 25-hydroxycholesterol.²³ In addition, the LRP upregulation observed in vitro has been corroborated in vivo; in situ hybridization analysis revealed that LRP expression is upregulated in the vessel wall of hypercholesterolemic animals. LRP upregulation in hypercholesterolemic aortas is concomitant with the SREBP-2 downregulation previously described by our group.³¹ Our results in vivo are in agreement with those obtained in blood mononuclear cells, in which dietary cholesterol has been shown to increase LRP mRNA levels,³² and in aortas of Watanabe rabbits, although the main cellular component at both early and late stages of atherosclerosis in this animal model are infiltrated macrophages.³³

To our knowledge, this is the first demonstration that exposure to high LDL concentration and cellular accumulation of CE increases LRP expression in VSMCs. Our results suggest that hypercholesterolemia might increase the capacity of VSMCs to take up LDL from the intima by regulating cellular LRP levels. In addition, LRP upregulation may influence other pathways involved in atherothrombosis, because LRP mediates the degradation of molecular complexes involved in thrombogenesis and fibrinolysis.^4,5,34

	Acknowledgments

 
This study was made possible by funds provided by Marató TV3-1995. Support for studies on atherosclerosis has been partially provided by PN-SAF 2000/0174, FIC-Catalana Occidente, FIS 01/0354, and MSD. The authors are indebted to M. Baldellou, PhD, for assistance. M. Otero and S. Sanchez are predoctoral fellows of the Fundación de Investigación Cardiovascular.

Received July 12, 2002; revision received September 18, 2002; accepted September 23, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Ann Rev Bioche. 1994; 63: 601–637.[Medline] [Order article via Infotrieve]
   
2. Willnow TE. The low density lipoprotein receptor gene family: multiple roles in lipid metabolism. J Mol Med. 1999; 77: 306–315.[CrossRef][Medline] [Order article via Infotrieve]
   
3. Neels JG, Horn IR, Van den Berg BMM, et al. Ligand-receptor interactions of the low density lipoprotein receptor-related protein, a multi-ligand endocytic receptor: fibrinolysis 38. Proteolysis. 1998; 12: 219–240.
   
4. Mikhailenko I, Kounnas MZ, Strickland DK. Low density lipoprotein receptor-related protein/₂-macroglobulin receptor mediates the cellular internalization and degradation of thrombospondin. J Biol Chem. 1995; 270: 9593–9549.
   
5. Willnow TE, Goldstein JL, Orth K, et al. Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. J Biol Chem. 1992; 267: 26172–26180.[Abstract/Free Full Text]
   
6. Warshawsky I, Herz J, Broze GJ, et al. The low density lipoprotein receptor-related protein can function independently from heparan sulfate proteoglycans in tissue factor pathway inhibitor endocytosis. J Biol Chem. 1996; 271: 25873–25879.[Abstract/Free Full Text]
   
7. Kowal RC, Herz J, Goldstein JL, et al. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived form apolipoprotein E-enriched lipoproteins. Proc Natl Acad Sci U S A. 1989; 86: 5810–5814.[Abstract/Free Full Text]
   
8. Beisiegel U, Weber W, Ihrke G, et al. The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature. 1989; 341: 162–164.[CrossRef][Medline] [Order article via Infotrieve]
   
9. Chappel DA, Fry GL, Waknitz LE, et al. Lipoprotein lipase induces catabolism of normal triglyceride-rich lipoproteins via the low density lipoprotein receptor-related protein/₂-macroglobulin receptor in vitro. J Biol Chem. 1992; 268: 14168–14175.
   
10. Kounnas MZ, Chappell DA, Wong H, et al. The cellular internalization and degradation of hepatic lipase is mediated by the low density lipoprotein receptor-related protein and requires cell surface proteoglycans. J Biol Chem. 1995; 270: 9307–9312.[Abstract/Free Full Text]
    
11. Fujioka Y, Cooper AD, Fong LG. Multiple processes are involved in the uptake of chylomicron remnant by mouse peritoneal macrophages. J Lipid Res. 1998; 39: 2339–2349.[Abstract/Free Full Text]
    
12. Reblin T, Niemeier A, Meyer N, et al. Cellular uptake of lipoprotein [a] by mouse embryonic fibroblasts via the LDL receptor and the LDL receptor-related protein. J Lipid Res. 1997; 38: 2103–2110.[Abstract]
    
13. Llorente-Cortés V, Martínez-González J, Badimon L. LDL receptor-related protein mediates uptake of aggregated LDL in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2000; 20: 1572–1579.[Abstract/Free Full Text]
    
14. Llorente-Cortés V, Otero-Viñas M, Hurt-Camejo E, et al. Human coronary smooth muscle cells internalize versican-modified LDL through both the low density lipoprotein receptor-related protein and the LDL receptor. Arterioscler Thromb Vasc Biol. 2002; 22: 387–393.[Abstract/Free Full Text]
    
15. Tirziu D, Jinga VV, Serban G, et al. The effects of low density lipoprotein modified by incubation with chondroitin 6-sulfate on human aortic smooth muscle cells. Atherosclerosis. 1999; 147: 155–166.[CrossRef][Medline] [Order article via Infotrieve]
    
16. Luoma J, Hiltunen T, Särkioja T, et al. Expression of ₂-macroglobulin receptor/low density lipoprotein receptor-related protein and scavenger receptor in human atherosclerotic lesions. J Clin Invest. 1994; 93: 2014–2021.[Medline] [Order article via Infotrieve]
    
17. Handschug K, Schulz S, Schnurer C, et al. Low density lipoprotein receptor-related protein in atherosclerotic development: up-regulation of gene expression in patients with coronary obstruction. J Mol Med. 1998; 76: 596–600.[CrossRef][Medline] [Order article via Infotrieve]
    
18. Hussaini IM, Srikumar K, Quesenberry PJ, et al. Colony stimulating factor-1 modulates -macroglobulin receptor expression in murine bone marrow macrophages. J Biol Chem. 1990; 265: 19441–19446.[Abstract/Free Full Text]
    
19. Misra UK, Gawdi G, González-Gronow M, et al. Coordinate regulation of the ₂-macroglobulin signaling receptor and the low density lipoprotein receptor-related protein/₂ macroglobulin receptor by insulin. J Biol Chem. 1999; 274: 25785–25791.[Abstract/Free Full Text]
    
20. Hussaini IM, LaMarre J, Lysiak JJ, et al. Transcriptional regulation of LDL receptor-related protein by IFN- and the antagonistic activity of TGF-ß₁ in the RAW 264.7 macrophage-like cell line. J Leukocyte Biol. 1996; 59: 733–739.[Abstract]
    
21. LaMarre J, Wolf BB, Kittler ELW, et al. Regulation of macrophage ₂-macroglobulin/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-. J Clin Invest. 1993; 91: 1219–1224.[Medline] [Order article via Infotrieve]
    
22. Herz J, Kowal RC, Goldstein JL, et al. Proteolytic processing of the 600 KD low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment. EMBO J. 1988; 9: 1769–1776.
    
23. Kütt H, Herz J, Stanley KK. Structure of the low-density lipoprotein receptor-related protein (LRP) promoter. Biochim Biophys Acta. 1989; 1009: 229–236.[Medline] [Order article via Infotrieve]
    
24. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997; 89: 331–340.[CrossRef][Medline] [Order article via Infotrieve]
    
25. Sakai J, Nohturfft A, Goldstein JL, et al. Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein: evidence from in vivo competition studies. J Biol Chem. 1998; 273: 5785–5793.[Abstract/Free Full Text]
    
26. Gaeta BA, Borthwick I, Stanley KK. The 5'-flanking region of the ₂ MR/LRP genes contains and enhancer-like cluster of Sp1 binding sites. Biochim Biophys Acta. 1994; 1219: 307–313.[Medline] [Order article via Infotrieve]
    
27. Alfon J, Royo T, Garcia-Moll X, et al. Platelet deposition on eroded vessel wall at stenotic shear rate is inhibited by lipid-lowering treatment with atorvastatin. Arterioscler Thromb Vasc Biol. 1999; 19: 1812–1817.[Abstract/Free Full Text]
    
28. Lipid Research Clinic Program. Manual of Laboratory Operation. Washington, DC: US Government Printing Office; 1974.DHEW publication No. NIH75-628.
    
29. Sato R, Miyamoto W, Inoue J, et al. Sterol regulatory element-binding protein negatively regulates microsomal triglyceride transfer protein gene transcription. J Biol Chem. 1999; 274: 24714–24720.[Abstract/Free Full Text]
    
30. Peet DJ, Turley SD, Ma W, et al. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR . Cell. 1998; 93: 693–704.[CrossRef][Medline] [Order article via Infotrieve]
    
31. Rodríguez C, Martínez-González J, Sánchez-Gómez S, et al. LDL downregulates CYP51 in porcine vascular endothelial cells and in the arterial wall through a sterol regulatory element binding protein-2-dependent mechanism. Circ Res. 2001; 88: 268–274.[Abstract/Free Full Text]
    
32. Boucher P, Lorgeril M, Salen P, et al. Effect of dietary cholesterol on low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase and low density lipoprotein receptor-related protein mRNA expression in healthy humans. Lipids. 1998; 33: 1177–1186.[CrossRef][Medline] [Order article via Infotrieve]
    
33. Wanatable Y, Inaba T, Shimano H, et al. Induction of LDL receptor-related protein during the differentiation of monocyte-macrophages: possible involvement in the atherosclerosis process. Arterioscler Thromb. 1994; 14: 1000–1006.[Abstract/Free Full Text]
    
34. Biessen EA, van Teijlingen M, Vietsch H, et al. Antagonists of the mannose receptor and the LDL receptor-related protein dramatically delay the clearance of tissue plasminogen activator. Circulation. 1997; 95: 46–52.[Abstract/Free Full Text]
    

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