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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2687.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Expression of LR11, a Mosaic LDL Receptor Family Member, Is Markedly Increased in Atherosclerotic Lesions

Tatsuro Kanaki; Hideaki Bujo; Satoshi Hirayama; Itsuko Ishii; Nobuhiro Morisaki; Wolfgang Johann Schneider; Yasushi Saito

From the Second Department of Internal Medicine, School of Medicine (T.K., H.B., S.H., N.M., Y.S.), and the Laboratory of Clinical Pharmacology, Faculty of Pharmaceutical Sciences (I.I.), Chiba University, Japan; and the Department of Molecular Genetics, Biocenter and University of Vienna, Vienna, Austria (S.H., W.J.S.).

Correspondence to Hideaki Bujo, Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Japan. E-mail hbujo{at}ruby.famille.ne.jp

	Abstract

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Abstract—Receptors belonging to the LDL receptor (LDLR) family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. Here, we report that the expression of a 250-kDa mosaic LDLR family member, which we called LR11 for the presence of 11 ligand-binding repeats, is markedly induced during the process of atherogenesis in 2 animal models. Analysis by reverse transcription–polymerase chain reaction and RNase protection assays revealed that LR11 transcript levels rise in rabbit aortas displaying atheromatous lesions after the rabbits have been fed a high-cholesterol diet. Immunohistochemistry demonstrated that the highest induction of LR11 occurs in intimal smooth muscle cells (SMCs), followed by medial SMCs close to the intimal border of the atheromatous lesions. Experimental intimal hyperplasia by endothelial denudation showed that LR11 mRNA levels were also increased in the arteries after balloon injury, with the transcripts localized primarily in the hyperplastic intimal layer. In agreement with the correlation of LR11 induction during increased cell proliferation, cultured SMCs showed an increase in LR11 expression in the proliferative phase. Furthermore, Northern and Western blot analyses showed that medium conditioned by the monocyte-macrophage cell line THP-1 enhanced LR11 expression in cultured SMCs. These findings suggest that upregulation of LR11 might be contributing to the pathological roles of intimal and medial SMCs during arteriosclerotic lesion development and provide the first insight into the as yet unknown functional significance of this intriguing LDLR family member.

Key Words: LDL receptor • atherosclerosis • smooth muscle cell • THP-1

	Introduction

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Members of the LDL receptor (LDLR) gene family are believed to play important roles in lipoprotein metabolic pathways, and derangements of these processes may lead to accelerated atheroma formation in the arterial wall.^{1 2 3 4} Atherosclerotic lesions are characterized mostly by accumulation of excessive lipid in the intima, and foam cells arise from macrophages migrating through endothelial cells and from smooth muscle cells (SMCs) in the medial layer.⁵ Recent extensive histochemical studies have shown that receptors belonging to the LDLR family, as well as scavenger receptors mediating the incorporation of modified lipoproteins such as oxidized LDL, show marked induction of their expression during the formation of atherosclerotic lesions.^{6 7 8 9 10} For instance, 1 LDLR family member, the so-called VLDL receptor (VLDLR/LR8), has been shown to be highly expressed in SMCs, macrophages, and endothelial cells in rabbit atherosclerotic lesions.^{7 8 9 10} Furthermore, VLDLR/LR8 has been shown not to be downregulated during ß-VLDL–induced foam cell formation in vitro, and overexpression of the receptor in fibroblasts causes excessive lipid droplet accumulation in the transformed cells.¹¹

The expression of LDL receptor–related protein/₂-macroglobulin (₂M) receptor (LRP) is also induced in atheromata; however, this large LDLR family member is present at high basal levels in normal intima-media,^{6 10} probably in support of the proposed multifunctionality of LRP. For LRP and certain other LDLR relatives, nonlipoprotein ligands potentially related to atherosclerosis and vascular remodeling, such as ₂M-protease complexes, urokinase plasminogen activator–plasminogen activator inhibitor-1 complexes, lipoprotein lipase, thrombospondin, and clusterin/apolipoprotein J, have been identified (for reviews, see References ² and ^{12 13 14 15 16} ).

We recently discovered and molecularly characterized a novel, unusually complex, and highly conserved member of the LDLR gene family.^{17 18} The predominant domain of this type I membrane protein consists of a cluster of 11 LDLR ligand-binding repeats; according to our preferred nomenclature, we called the new receptor LR11.¹⁹ Another feature of LR11 is the presence of an extracellular domain homologous to the yeast vacuolar protein sorting receptor, Vps10p²⁰ ; thus, Jacobsen and colleagues²¹ named the receptor sorLA-1 (where LA stands for LDLR class A repeats). Another intriguing characteristic of LR11 is the presence of 6 fibronectin type III repeats following the 11 LDLR-binding repeats. The similarity with the repeats in neural cell adhesion molecules, murine L1, and their homologues suggests a potential role of LR11 in cell-cell interaction. Furthermore, the unique expression pattern of the LR11 mRNA in brain and other organs active in morphogenesis during murine development is compatible with roles in neural development and organ formation.^{22 23}

In the present investigation, we studied, by means of immunohistochemistry, in situ hybridization, RNase protection assay, and reverse transcription–polymerase chain reaction (RT-PCR) analyses, the regional changes in mRNA and protein levels of LR11 during atheroma formation. We used rabbit and rat arteries as models for experimental atherosclerosis induced by cholesterol feeding and balloon injury, respectively. We also assessed regulatory features of expression of the mosaic receptor in cultured SMCs in vitro. Taken together, the expression pattern of LR11 in the SMCs indeed suggests a possible role, particularly in the pathogenesis of SMCs involved in the process of atherosclerosis, as well as in the growth and differentiation of embryonic tissues.

	Methods

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Animals and Experimental Atherosclerosis
Male Japan White rabbits, average 2.5 kg body weight, and male Wistar rats, 250 to 300 g body weight, were used. The animals were housed in temperature-, humidity-, and light-controlled rooms with free access to water and standard chow or an atherogenic diet containing 1% cholesterol. Adult rabbits (2 to 3 kg) were used for primary cultures of vascular SMCs. Rats (250 to 300 g) were subjected to endothelial denudation of the left common carotid artery with an arterial embolectomy catheter (Fogarty 2F catheter; American Edwards Laboratories) as described previously.²⁴ The right common carotid artery was exposed but not subjected to endothelial denudation and was used as a control. Both common arteries were removed 3 or 14 days after endothelial denudation. Aortas and common carotid arteries from rabbits fed with standard rabbit chow or atherogenic diet containing 1% cholesterol for 12 weeks and common carotid arteries from rats after endothelial denudation were used for immunohistochemistry, in situ hybridization, RNase protection assays, and RT-PCR.

Cell Culture
To analyze receptor gene expression during the course of cell proliferation or after conditioned medium from THP-1 cells was added, primary cultures of rabbit SMCs were prepared from the media of rabbit aorta by the explant method as previously described.²⁴ Briefly, after the removal of the adventitia, explants were cultured at 37°C under 5% CO₂ in DMEM (glucose, 5.5 mmol/L) supplemented with 10% FBS (Irvine Scientific) and 40 µg/mL gentamicin. After 2 weeks, cells that had migrated out of the explant were removed by trypsinization and seeded in T-75 flasks. Cells were characterized as SMCs by morphological criteria (spindle shape and hill-and-valley pattern) and by their expression of smooth muscle -actin. SMCs were subcultured at a 1:2 split ratio at weekly intervals. Cells from the third to fourth passages were used for experiments. Three different preparations of SMCs were used, with similar results. Cell counts were performed on triplicate wells with a model D industrial Coulter counter (Coulter Electronics).

Cells (1x10⁵) seeded in T-75 flasks were incubated for 48 hours in DMEM supplemented in the absence of FBS. To quantify the LR11 mRNA level during cell proliferation, cell numbers were counted 1, 2, 3, 4, 6, 9, and 12 days after 10% FBS was added, and total RNA was extracted from the cells at each point for RNase protection assay. To analyze the effects of conditioned medium on the LR11 expression levels, the normal medium was replaced with conditioned medium (in the presence of 10% FBS) from THP-1 cells. The THP-1 cell–conditioned medium (including 10% FBS) was prepared by incubation of nearly confluent THP-1 cells (preincubated in the presence of 100 nmol/L PMA for 24 hours followed by thorough washes) for 48 hours. mRNA and cell extracts were prepared from the cells at 24 and 48 hours after medium replacement for Northern and Western blot analyses, respectively.

Cloning of Rat LR11 cDNA
A 255-bp fragment corresponding to nucleotides 5328 to 5582 of the mouse LR11 sequence was cloned from a rat brain cDNA library (Clontech) with PCR using the primers 5'-TGGCTACGTGGT-GAACCTTTTCTG-3' (sense) and 5'-CACTCCACTGCAGTCT-GATTGATG-3' (antisense), corresponding to nucleotides 5328 to 5351 and 5559 to 5582 of the mouse LR11 sequence, respectively, encoding a portion of the fibronectin type III repeats (domain V) of LR11.²² The 255-bp amplified product was subcloned into pGEMT (Promega) and sequenced with the Cy5 Autoread Sequencing kit (Pharmacia). The sequence between the primers has a 98.8% identity with the corresponding mouse LR11 sequence.²² The plasmid containing the cDNA insert specific for rat LR11 was used for the preparation of the hybridization probe for in situ hybridization. The primers used for RT-PCR analysis are described under Northern Blot Analysis and RT-PCR.

Northern Blot Analysis and RT-PCR
Total RNA was extracted from the indicated rabbit aortas and cultured SMCs, and poly(A)⁺ RNA was isolated as described previously.²⁵ For Northern blot analysis, poly(A)⁺ RNA prepared from the indicated cultured cells was denatured with glyoxal-dimethyl sulfoxide, separated by electrophoresis on a 1.0% agarose gel, and blotted onto Hybond-N⁺ membrane (Amersham) by standard methods.²⁶ The above-described membranes were probed with HB 404,¹⁷ corresponding to nucleotides 151 to 554 (specifying part of domain I). Hybridizations were at 65°C in 1% BSA, 7% SDS, 500 mmol/L sodium phosphate (pH 6.8), 1 mmol/L EDTA, and ³²P-labeled probe. Membranes were washed in 1% SDS, 40 mmol/L sodium phosphate (pH 6.8), and 1 mmol/L EDTA at 65°C. Filters were exposed to a Fuji imaging plate for Bioimaging analyzer (Fuji Bas 2000). The relative amount of each signal was determined by densitometric scanning with NIH Image software. The amounts of LR11 mRNA were normalized with the amounts of cyclophilin B mRNA used as reference.

For RT-PCR, single-stranded cDNA was synthesized from 1 µg of poly(A)⁺ RNA with SuperScript reverse transcriptase (Life Technologies) and random hexamer primers. One tenth of the cDNA was subjected to PCR with sense (S) and antisense (AS) primers as follows: for rabbit LR11, 5'-GTGCAGGGCGACCCGCGC-GAGCTG-3' and 5'-CCATAGTCATAAGACACGTACACA-3', corresponding to nt 328 to 351 (S) and 579 to 602 (AS) of rabbit LR11, encoding portions of domains I and II of rabbit LR11¹⁷ ; for rat LR11, 5'-TGGCTACGTGGTGAACCTTTTCTG-3' (S) and 5'-CACTCCACTGCAGTCTGATTGATG-3' (AS), corresponding to nucleotides 5328 to 5351 and 5559 to 5582 of murine LR11, encoding a portion of domain V of murine LR11²² ; for rat cyclin E,²⁷ 5'-ATGGAGGTGTGTGAAGTCTAT-3' and 5'-TGGAACCAT-CCACTTGACACA-3', corresponding to nt 646 to 666 (S) and 1168 to 1188 (AS); for rat cyclin B,²⁷ 5'-GTGCCCAAGAAGAT-GCTGCAGCTG-3' and 5'-GAGTGCTGATCTTAGCATGCT-3', corresponding to nt 716 to 739 (S) and 1224 to 1244 (AS); and for cyclophilin B,²⁸ 5'-GGAAAGACTGTTCCAAAAACAGTG-3' and 5'-GTCTTGGTGCTCTCCACCTTCCG-3', corresponding to nt 211 to 234 (S) and 553 to 575 (AS) of murine cyclophilin B. The reaction mixture (100 µL) containing the cDNA, 100 pmol of each of the primers, and 2.5 mmol/L dNTP was heated to 95°C for 10 minutes and then immediately cooled on ice. One unit of Taq DNA polymerase was added, followed by 20, 25, or 30 cycles of reannealing at 65°C for 1 minute, elongation at 72°C for 2 minutes, and denaturation at 94°C for 1 minute. The PCR products were then analyzed on a 2.0% agarose gel and photographed. The relative amount of each signal was determined by densitometric scanning with NIH Image software. Amounts of LR11 mRNA were normalized with the amounts of cyclophilin B mRNA used as reference. The RT-PCR generated the expected fragments of 275, 255, 543, 529, and 375 (375) nucleotides for rabbit LR11, rat LR11, rat cyclin E, rat cyclin B, and rabbit (rat) cyclophilin B, respectively. The amplified products were subcloned into pGEMT and sequenced by use of the Cy5 Autoread Sequencing kit.

In Situ Hybridization
For the detection of rat LR11 mRNA on cryostat sections, digoxigenin-labeled cRNA probes were generated by in vitro transcription of the plasmid pGEMT containing a 255-bp cDNA insert specific for rat LR11 (as described under Cloning of Rat LR11 cDNA). To generate a template for the sense or antisense probe, pGEMT was linearized with NotI (S) or SphI (AS). The templates were transcribed in vitro by T7 and SP6 RNA polymerase (DIG RNA Labeling Kit, Boehringer Mannheim), respectively, as previously described.²² Common carotid arteries from rats at 14 days after endothelial denudation were fixed with 4% paraformaldehyde in PBS for 3 hours at 4°C, rinsed with PBS, and incubated with 30% sucrose in PBS for 3 hours at 4°C. The fixed arteries were embedded in OCT compound (SRL) and immediately frozen with ethanol that had been precooled in dry ice. Cryostat sections 5 µm thick were prepared, transferred to glass slides pretreated with 2% 3-aminopropyltriethoxysilane, and stored at -70°C. For in situ hybridization, the sections were dried, serially deparaffinized in 100% to 70% ethanol, and incubated with 0.3% Triton X-100 in PBS for 10 minutes at 23°C. After 2 washings in PBS, the sections were treated with 1 µg/mL proteinase K in PBS for 15 minutes at 37°C, fixed with 4% paraformaldehyde in PBS for 5 minutes, incubated with glycine buffer, and acetylated with acetylation buffer (In-Situ Hybridization Reagents, Nippon Gene). Prehybridization was performed for 2 hours at 42°C in a solution containing 50% formamide and 2xSSC.²⁵ Sections were hybridized overnight at 42°C in hybridization buffer (Nippon Gene) containing 200 pg/mL digoxigenin-labeled antisense or sense cRNA probes, washed twice in prehybridization solution at 42°C for 30 minutes, and incubated with 20 µg/mL RNase A in NTE buffer (Nippon Gene) at 37°C for 30 minutes. Sections were rinsed in 0.1xSSC and equilibrated with buffer 1 (100 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5) for 10 minutes, followed by blocking for 1 hour in buffer 2 (1% Boehringer blocking reagent in buffer 1), and incubated for 1 hour at 23°C with anti–digoxigenin–alkaline phosphatase antibodies at a dilution of 1:250 in buffer 1 (DIG DNA Labeling and Detection Kit, Boehringer Mannheim). Sections were then washed twice for 10 minutes in buffer 1, equilibrated for 10 minutes in buffer 3 (100 mmol/L Tris-HCl, 100 mmol/L NaCl, 50 mmol/L MgCl₂, pH 9.5), and developed overnight in the dark with buffer 3 containing 0.34 mg/mL 4-nitroblue tetrazolium chloride (Boehringer Mannheim) and 0.175 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer Mannheim). The reaction was terminated by washing with buffer 4 (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0), after which sections were air-dried and mounted. Photographs were taken with an Olympus BX50F4 light microscope.

RNase Protection Assay
The plasmid for the preparation of the rabbit LR11 cRNA probe was generated as described under In Situ Hybridization. In addition, the 375-bp PCR-amplified fragment carrying rabbit cyclophilin B cDNA (as described under Northern Blot Analysis and RT-PCR) was also ligated with pGEMT vector and subsequently linearized by digestion with NcoI. Radiolabeled RNA transcripts were synthesized by use of the linearized template DNA according to the manufacturer’s recommendations (DIG RNA Labeling Kit). Digestion with 1 U of RQ1 RNase-free DNase was performed to remove the template DNA. The cRNA probes (2x10⁵ cpm) were hybridized in 1xtranscription buffer (RPA II, Ambion). RNase was used to digest unhybridized RNA, and the remaining RNA/RNA hybrid was analyzed on a 4% polyacrylamide gel containing 8 mol/L urea.²⁹ Autoradiography was performed in a Fuji Bioimaging analyzer. The relative amount of each signal was determined by densitometric scanning with NIH Image software. The amounts of LR11 mRNA were normalized with the amounts of cyclophilin B mRNA used as reference.

Western Blot Analysis
Membranes from cultured cells were prepared and solubilized as previously described.¹⁹ Protein concentrations were determined with the BCA Protein Assay Reagent (Pierce). Where indicated, aliquots (400 µL) of the detergent extract were mixed with 100 µL of wheat germ lectin Sepharose 4B (50% gel suspension). After being mixed at 4°C for 1 hour, the gels were washed 5 times with 20 mmol/L Tris-HCl (pH 7.4) and 0.15 mol/L NaCl containing 0.5% Triton X-100 and then incubated with 100 µL of 0.4 mol/L N-acetylglucosamine in 20 mmol/L Tris-HCl (pH 7.4), 0.15 mol/L NaCl, and 0.5% Triton X-100 at 4°C for 2 hours. The N-acetylglucosamine eluates were then analyzed by 5% SDS-PAGE. Electrophoresis was performed at 30 mA for 60 minutes under reducing conditions (30 mmol/L 2-mercaptoethanol) and after heating of the samples to 95°C for 5 minutes. Calibration was with Rainbow colored protein molecular weight markers (Amersham). Electrophoretic transfer of the proteins to polyvinylidene difluoride membranes (Millipore, pore size 0.45 µm) was performed in transfer buffer (100 mmol/L Tris, 192 mmol/L glycine, 20% methanol) for 1 hour at room temperature and 110 mA with the Atto Horizeblot System AE-6670. Western blotting was performed with the IgG fraction purified from the supernatant of cultured 43405 cells¹⁷ at 1:100 dilution, followed by peroxidase-conjugated anti-mouse IgG (heavy and light chain, Promega) and the chemiluminescence detection method (ECL system, Amersham). This murine monoclonal antibody is directed against the synthetic peptide GIIQCRDGSDEDPAFAGCS, corresponding to residues 1297 to 1315 (domain IV) of rabbit LR11.¹⁷ The membranes were exposed for 5 minutes on Hyperfilm-ECL (Amersham). The relative intensities of the signals were determined by densitometric scanning with NIH Image software.

Immunohistochemistry
Serial paraffin-embedded sections (10 µm) were used for immunostaining. For immunostaining, we used Pathostain ABC-POD (M) kit (Wako) according to the manufacturer’s instructions.¹⁷ The sections were incubated for 10 minutes in 10% H₂O₂ to inactivate endogenous peroxidase. The same monoclonal antibody as used for Western blot analysis was applied in immunostaining the heat-denatured and hydrated sections. Adjacent sections were used for control incubations with a monoclonal anti–glutathione S-transferase antibody; development was with peroxidase-labeled streptavidin and incubation for 30 minutes in the peroxidase substrate provided. Monoclonal antibodies against rabbit macrophages (RAM11, Dako) and rat smooth muscle -actin (1A4, Dako), respectively, were used for the identification of rabbit macrophages and rabbit and rat SMCs to determine the cell composition of atheromatous plaques. Hematoxylin was used for counterstaining.

	Results

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LR11 Expression in Atherosclerotic Arteries in Rabbits Fed a High-Cholesterol Diet
To elucidate the possible pathological roles of LR11 in atheroma formation, we analyzed the expression levels of LR11 mRNA in arteries from rabbits fed an atherogenic diet containing 1% cholesterol for 12 weeks. Intimal hyperplasia of aortas in these animals was observed, whereas that of carotid arteries was not obvious macroscopically, as described previously.³⁰ Figure 1A shows the results of LR11 mRNA expression analysis obtained by RT-PCR with primer combinations corresponding to LR11 domains I and II. Amplified mRNA of the expected size was detected both in aortas (lane 1) and in carotid arteries (lane 3) from rabbits fed standard chow. In atherosclerotic aortas (lane 2) but not in carotid arteries (lane 4) from rabbits fed the high-cholesterol diet, there was a dramatic increase in the amount of amplified LR11 domains I plus II. When primers corresponding to other domains of LR11 cDNA were used, we obtained identical results (data not shown).

View larger version (53K): [in this window] [in a new window]   Figure 1. LR11 mRNA expression in aortas and carotid arteries of rabbits fed standard chow or a cholesterol-rich diet for 14 weeks. A, Single-stranded cDNA was synthesized by use of poly(A)+ RNA (1 µg) from pooled arteries of 5 control or 5 cholesterol-fed rabbits. One tenth of the cDNA was PCR-amplified for 25 cycles with specific primers for rabbit LR11 or cyclophilin B (Cyclo.) as described in Methods. One tenth (LR11, 275 bp) or 1/50 (cyclophilin B, 375 bp) of amplified fragments were used for electrophoresis on a 2.0% agarose gel. Lane 1, aortas from standard chow–fed rabbits; lane 2, aortas from cholesterol-rich chow–fed rabbits; lane 3, carotid arteries from standard chow–fed rabbits; lane 4, carotid arteries from cholesterol-rich chow–fed rabbits. Similar results were obtained in 2 experiments. B, Total RNA (10 µg) from pooled artery samples was hybridized with LR11 or cyclophilin B cRNA probe in solution. Hybridized samples were digested with RNase and loaded onto a 3.5% polyacrylamide gel containing 8 mol/L urea, as described in Methods. Lane 1, RNA probe used was loaded onto the gel for comparison; lane 2, tRNA was used instead of pooled samples; lane 3, aortas from standard chow–fed rabbits; lane 4, aortas from cholesterol-rich chow–fed rabbits; lane 5, carotid arteries from standard chow–fed rabbits; lane 6, carotid arteries from cholesterol-rich chow–fed rabbits. Similar results were obtained in 2 experiments.

On the basis of these results obtained by RT-PCR, we next performed a quantitative procedure using total RNA from the same pooled samples of aortas and carotid arteries as used in the above-described experiments. A solution hybridization RNase protection assay produced the results demonstrated in Figure 1B. LR11 mRNA levels in aortas from rabbits fed a high-cholesterol diet increased 6.8-fold compared with those from normal chow–fed rabbits (lanes 3 and 4). In contrast, LR11 mRNA levels in carotid arteries from rabbits fed a high-cholesterol diet increased 1.7-fold compared with those from normal chow–fed rabbits (lanes 5 and 6). Thus, the solution-hybridization assay produced results entirely compatible with those obtained by RT-PCR shown in Figure 1A, strongly suggesting increased expression of LR11 mRNA in atherosclerotic arteries of rabbits. The signal intensity for cyclophilin B mRNA did not show significant differences among the samples.

Immunohistochemical Analysis of LR11 in Aortas From High-Cholesterol Diet–Fed Rabbits
To examine the localization of LR11 protein in atheromatous lesions, we next performed immunohistochemical analysis using sections of aortas from rabbits fed the high-cholesterol diet and from control animals. In aortic areas without atherosclerotic lesions in the control rabbits, no significant positive labeling for LR11 was seen with the specific antibodies directed against domain IV of the receptor (Figure 2C). In aortas from the rabbits fed the high-cholesterol diet, clear positive signals for LR11 were detected in the region of intimal thickening (Figure 2A); for comparison, we performed immunostaining with a monoclonal anti-GST antibody as negative control (Figure 2B).

View larger version (152K): [in this window] [in a new window]   Figure 2. Immunohistochemical analysis of LR11 in rabbit atherosclerotic arteries. Paraffin-embedded sections (10 µm) of aortas from a standard chow–fed rabbit (C) or a cholesterol-rich chow–fed rabbit (A, B, and D through F) were subjected to immunohistochemistry. Sections were stained with specific antibodies to LR11 (A, C, and D), GST from Schistosoma japonicum (B), SMC marker (E), or macrophages (F), as described in Methods, after the hematoxylin staining. Data are representative of 5 atherosclerotic arteries and 2 normal arteries. Magnifications: A through C, x32; D through F, x160.

The LR11 signals in the intima (Figure 2D) mostly overlapped with those revealed by a cytochemical marker for SMCs (Figure 2E), suggesting that the LR11 protein is localized over SMCs in the intima. In addition, LR11 expression, although at lower levels than in intima, was also detected in medial SMCs close to the intimal border (Figure 2, A and D). Immunohistochemical analysis of adjacent sections with a macrophage-specific marker (Figure 2F) showed a rather different localization of its antigen, suggesting that the LR11 protein is not significantly expressed in intimal macrophages. These results indicate that the increased levels of LR11 transcripts in atherosclerotic aortas from rabbits fed a high-cholesterol diet observed in Figure 1 were caused mainly by increased expression of LR11 in intimal SMCs.

LR11 mRNA Expression in Atherosclerotic Lesions by Endothelial Denudation
The above results prompted us to examine LR11 expression in arterial lesions with hyperplastic intima, as well as in atheromata. It has been shown that atheromatous lesions formed by balloon-injury endothelial denudation have higher intimal SMC content than those induced by high-cholesterol-diet feeding.³¹ Intimal hyperplasia in experimental vascular remodeling results primarily from an excessive proliferative response of SMCs, which also undergo dedifferentiation and migration. Figure 3 shows the expression of LR11 transcripts during atheroma formation after balloon injury, analyzed by RT-PCR. Under our conditions, 30 cycles of amplification did not result in detectable signals in rat normal carotid arteries (lanes 1 to 3). Faint products of the expected size were seen 3 days after endothelial denudation (lanes 4 to 6), and the amount of amplified products showed a marked increase at 14 days after denudation (lanes 7 to 9). At the same time, amplified cDNA of cyclins E and B also became detectable, in agreement with increased cell proliferation in the lesions. Increased mRNA expression of LR11 and of cyclins after denudation suggests that LR11 might be expressed in proliferative intimal cells, such as SMCs.

View larger version (52K): [in this window] [in a new window]   Figure 3. Effect of endothelial denudation on LR11 expression in rat carotid arteries. Poly(A)+ RNA from rat carotid arteries before or 3 or 14 days after endothelial denudation (3 rats each) was prepared and used for cDNA synthesis as described in Methods. RT-PCRs for 30 cycles were performed for LR11, cyclophilin B, and cyclins E and B, respectively. One tenth (LR11, 255 bp), or 1/50 (cyclophilin B, 375 bp; cyclin E, 543 bp; cyclinB, 529 bp) of amplified fragments were used for electrophoresis on a 2.0% agarose gel. Lane numbers refer to the corresponding rat number used for the experiment (1 to 3, before denudation; 4 to 6, 3 days after denudation; 7 to 9, 14 days after denudation).

To determine the localization of intimal LR11 expression in a different animal model, we performed in situ hybridization analysis of LR11 mRNA using sections of rat carotid arteries obtained 14 days after denudation (Figure 4). In control rat carotid areas, only faint positive labeling for LR11 mRNA was seen in the medial layer with the antisense probe corresponding to LR11 domains I and II (Figure 4C). In rat arteries after denudation, immunohistochemical analysis with a SMC-specific marker revealed that these hyperplastic lesions are mostly accompanied by proliferating SMCs (Figure 4D). LR11 mRNA hybridization signals were clearly detected in most regions of intimal hyperplasia, together with the faint signals in medial SMCs (Figure 4A). Hybridization with an LR11 sense probe did not show any significant staining using the adjacent section (Figure 4B). These results strongly suggest that as in the rabbit model, LR11 transcripts are elevated in the intimal SMCs in the endothelial denudation rat model.

View larger version (127K): [in this window] [in a new window]   Figure 4. In situ hybridization analysis of LR11 transcripts in sections of rat arteries after endothelial denudation. Cryostat sections (5 µm) of carotid arteries after (A and B) or before (C) denudation were subjected to in situ hybridization with digoxigenin-labeled antisense (A and C) or sense (B) cRNA probe (255 nt) corresponding to rat LR11 cDNA as described in Methods. D, Immunohistochemical analysis of a paraffin-embedded section in the same hyperplastic lesion using an antibody to a SMC-specific marker as described in Figure 2. Data are representative of 4 atherosclerotic arteries and 2 normal arteries. Magnification x94.

LR11 Expression in Cultured SMCs
To verify the presence of LR11 mRNA in vascular SMCs and to examine whether the increase of receptor gene expression level that occurs in vivo can be observed in vitro, regulation of LR11 mRNA was studied during proliferation of cultured rabbit SMCs. Cultured SMCs in quiescence showed low but significant levels of LR11 mRNA, as assessed by the solution hybridization RNase protection assay (Figure 5A). On culture, LR11 mRNA rose 2.4-fold within 1 day and increased gradually for 4 days after addition of serum, consistent with its being related to cell proliferation activities (Figure 5B and 5C). The maximum amount of mRNA expression was 21-fold that before serum was added. Thereafter, the expression of LR11 mRNA decreased again; at day 12, when cell numbers had reached a plateau, the level of LR11 transcript still was 5.6-fold that before serum addition.

View larger version (67K): [in this window] [in a new window]   Figure 5. LR11 mRNA expression in cultured SMCs during the process of cell proliferation. A, Total RNA (10 µg) from cultured SMCs at each time point (0, 1, 2, 3, 4, 6, 9, or 12 days) after serum addition was prepared and subjected to RNase protection analysis. RNA was hybridized with specific cRNA probes for LR11 or cyclophilin B (Cyclo.) as described in Figure 1B. In lane C, tRNA was used instead of samples. Data are representative of 3 experiments. B, Quantitative analyses of the RNase protection assay shown in A. Relative amounts of the signals were determined by densitometric scanning as described in Methods. The amounts of LR11 mRNA were normalized relative to the amounts of cyclophilin B mRNA. C, Cell numbers of proliferating SMCs on each day. Cell counting was performed on triplicate wells.

Finally, to examine possible regulatory factors for the receptor in proliferating SMCs, we investigated the effects on LR11 expression of medium that had been preconditioned with cultured macrophages (THP-1 cells). Northern blot analysis revealed that at 24 hours after cell proliferation was induced by addition of serum, expression of LR11 mRNA was induced 2.2-fold above basal level (Figure 6A, lanes 1 and 2), consistent with the above results (Figure 5). However, the addition of THP-1–conditioned medium to quiescent SMCs resulted in an 8.6-fold increase in LR11 mRNA (Figure 6A, lane 3) compared with the level in cells that had received 10% FBS (lane 2). To test whether these transcriptional effects of THP-1–conditioned medium also could be observed at the protein level, we performed Western blot analysis with the same monoclonal antibody as used in Figure 2. LR11 in quiescent cultured SMCs was visualized under our conditions (Figure 6B, lane 4) as a 250-kDa protein, as described previously in various tissues¹⁷ and confirmed here in rabbit brain (lanes 2 and 3). The LR11 protein in SMCs was increased slightly 24 hours after addition of 10% FBS (lane 5). Significantly, incubation with THP-1–conditioned medium for 24 and 48 hours resulted in 4.4- and 6.2-fold increases in LR11 protein abundance, respectively (lanes 6 and 7), over that obtained by addition of 10% FBS. These effects on LR11 protein expression, together with the results obtained by Northern blot analysis (Figure 6A), strongly suggest that LR11 expression in cultured SMCs responds to addition of macrophage-conditioned medium.

View larger version (67K): [in this window] [in a new window]   Figure 6. Effects of THP-1–conditioned medium on LR11 expression in cultured SMCs. A, Rabbit SMCs were cultured in serum-free medium (lane 1), medium with 10% serum (lane 2), or THP-1–conditioned medium (lane 3). Poly (A)+ RNA was prepared and subjected to Northern blot analysis (10 µg/lane) as described in Methods. Rabbit brain poly(A)+ RNA was used as control (lane 4; 2 µg/lane). Cyclo indicates cyclophilin B. Data are representative of 3 independent experiments. B, Rabbit SMCs were cultured in serum-free medium (lane 4), medium with 10% serum (lane 5), or THP-1–conditioned medium for 24 (lane 6) or 48 (lane 7) hours. Membrane fractions were prepared and subjected to immunoblot analysis (20 µg/lane) without (lanes 1 and 2) or with (lanes 3 through 7) prior partial purification with wheat germ lectin Sepharose 4B (see Methods). Rabbit brain membrane extract was used as control (lane 1 to 3). In lane 1, the LR11-specific antibodies were omitted. Data are representative of 3 independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates marked induction of the mosaic receptor LR11, which belongs to the LDL receptor supergene family, in 2 experimental models of atherogenesis: cholesterol-fed rabbits and balloon-injured rats. Taken together, the results from the arteries of both models show that the expression of LR11 is predominantly localized to SMCs in the intima. The analysis using cultured SMCs revealed that LR11 expression is induced during conditions of rapid proliferation and by the addition of medium conditioned with cultured macrophages, THP-1 cells.

The migration of medial SMCs from the media to the intima and the proliferation of intimal cells to form a thickened neointima are sequential events that characterize tissue remodeling in the arterial wall after balloon denudation.³¹ SMCs in the media are entirely surrounded by a basal lamina and embedded in various extracellular matrices; thus, the migration requires SMCs to traverse major extracellular barriers, including the internal elastic lamina and a dense meshwork of interstitial proteoglycans and collagens.³² It has been shown that various kinds of proteases, such as plasminogen activator and matrix metalloproteases, are important for SMC migration in the process of vascular remodeling.³² In addition, because of the low mitogenic activity of SMCs in the media, during the early stages of arterial wall injury or atherosclerosis, arterial SMCs may undergo transition from a contractile to a synthetic phenotype and begin migration and proliferation in response to various growth factors, causing intimal thickening of the arterial wall. In this context, extensive studies on the ligand specificities of the LDL receptor gene family members have shown that most of them may play roles beyond those as receptors for uptake of plasma lipoproteins (for reviews, see References 2, 3^{2 3} , and ^{12 13 14 15} ). VLDLR/LR8, LRP, and gp330/megalin have broad and overlapping ligand specificities, including tissue plasminogen activator, urokinase plasminogen activator, and lipoprotein lipase. VLDLR/LR8 and LRP also take up ₂M and lactoferrin (for reviews, see References 3 and 15^{3 15} ). In addition, LRP and gp330/megalin have been proposed to be receptors for the multifunctional glycoproteins thrombospondin and apolipoprotein J/clusterin, respectively.^{33 34 35} Thus, several potential ligands for the multifunctional receptor family, particularly some proteases and their complexes, are present in the arterial wall and are believed to play key roles in proliferation and migration of SMCs during vascular remodeling.

To date, the only identified ligands of LR11 are the apoE-rich lipoprotein ß-VLDL¹⁷ and the 39-kDa receptor-associated protein,²¹ in agreement with the notion that all previously described LDLR family members bind both ligands (for reviews, see References 2, 3^{2 3} , and ^{12 13 14 15} ). Recently, Hermans-Borgmeyer et al²³ and Franke et al³⁶ reported that the putative LR11 homologue of the coelenterate Hydra is a specific receptor for the neuropeptide "head activator," which stimulates head-specific growth and differentiation processes in this invertebrate. If analogy holds, the function of the peptide in mammals might be the stimulation of proliferation of neural precursor cells, stabilization of nerve cell survival, and enhancement of neural outgrowth.^{37 38} At present, in the absence of a transgenic animal model, we cannot determine which ligand(s) may be the true physiological partner(s) of LR11. However, the structural feature of homology with neural adhesion molecules possibly suggests that at least part of the function of LR11 might be in the process of cell-cell interaction and differentiation of various cells, similar to the roles of neural cell adhesion molecule and L1.¹⁷ Conversely, the homology to the putative ligand-binding regions of a yeast carboxypeptidase Y sorting receptor is compatible with LR11 being involved in the action of certain proteases reported to be important for cell growth and migration. In agreement with these features of LR11, transcripts are detected during periods of active morphogenesis, suggesting a role for this receptor in the proliferation that occurs before differentiation of many embryonic cells and tissues.^{22 23}

So far, the physiological significance, if any, of the expression of LDLR gene family members in arterial walls has not been fully elucidated. Certain LDLR relatives are expressed in the arterial wall, implying possible roles in the progression of atherosclerotic plaques. For instance, LRP has been demonstrated in lesion macrophages and SMCs,^{6 10} and VLDLR/LR8 in endothelial cells, macrophages, and SMCs^{7 8 9 10} ; in contrast, the LDLR is not abundant in arterial walls.^{6 10} LRP has been demonstrated to be involved in the migration and proliferation of various cells related to atherosclerosis and cancer invasion. Embryonic fibroblasts genetically deficient in LRP show increased activity of the urokinase receptor system and accelerated migration on vitronectin.³⁹ Recent studies on the involvement of LRP in the progression of various cancer types have produced intriguing results in vitro, which show that LRP is decreased or absent in certain malignant tumors,^{40 41} whereas in others there is overexpression of the receptor.^{42 43} Moreover, Li et al⁴⁴ showed that the upregulation of LRP expression in a breast cancer line was to a large degree dependent on increasing cell density.

Here we demonstrate high levels of expression of LR11 in intimal SMCs and lower levels in medial SMCs close to the intimal border in atherosclerotic lesions. This expression pattern of LR11 in the arterial walls suggests that it may be involved in SMC (patho)biology, such as migration and proliferation into the intima from the media. The characteristic expression during rapid proliferation and induction by macrophage-conditioned medium suggests roles of LR11 in growth and differentiation of vascular SMCs, as well as certain functions in other cells during embryogenesis.^{22 23} Modulation of the SMC phenotype accompanying the migration of medial SMCs through the internal elastic lamina into the intima has been proposed by several observations.^{45 46} The modulation process can be triggered by intimal macrophages, which degrade the heparan sulfate proteoglycan component of the SMC basal lamina via membrane-bound or secreted proteases and endoglycosidase.^{47 48} Macrophages also produce a number of growth factors for SMCs, such as platelet-derived growth factor.⁴⁹ The enhanced expression of LR11 by macrophage-conditioned medium in vitro possibly reflects its induction in rabbit SMCs of atherosclerotic arteries; the expression of LR11 might be regulated by factors secreted from the macrophages in the hyperplastic intima. Preliminary experiments show that neither interleukin-1 nor granulocyte-macrophage colony–stimulating factor affects the expression levels of LR11 in cultured SMCs. Thus, an important challenge is the identification of the factors stimulating LR11 expression in intimal SMCs during atherogenesis and vascular remodeling.

Note: The nucleotide sequences identified in this study have been deposited in GenBank/EMBL (accession No. AB026993).

	Acknowledgments

 
These studies were supported by grants from the Japanese Ministry of Education, Science, and Culture to Y.S. and H.B. and from the Austrian Science Foundation to W.J.S. (FWF-F-0608).

Received January 19, 1999; accepted April 9, 1999.

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