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(Circulation. 1998;97:1079-1086.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Expression of LDL Receptor, VLDL Receptor, LDL Receptor–Related Protein, and Scavenger Receptor in Rabbit Atherosclerotic Lesions

Marked Induction of Scavenger Receptor and VLDL Receptor Expression During Lesion Development

Timo P. Hiltunen, MD; Jukka S. Luoma, MD; Tapio Nikkari, MD, PhD; ; Seppo Ylä-Herttuala, MD, PhD

From the Department of Medical Biochemistry, University of Tampere Medical School (T.P.H., T.N.); the Department of Clinical Chemistry, Tampere University Hospital (T.P.H.); and the A.I. Virtanen Institute (J.S.L., S.Y.-H.) and Department of Medicine (S.Y.-H.), University of Kuopio, Finland.

Correspondence to Seppo Ylä-Herttuala, MD, PhD, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, Neulaniementie 2, FIN-70211 Kuopio, Finland. E-mail ylahertt{at}keula.uku.fi

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Atherosclerotic lesions contain foam cells that arise from monocyte-macrophages and smooth muscle cells (SMCs) by excessive uptake of lipoproteins. There are many candidate receptors for the lipid accumulation, such as LDL receptor (LDLR), VLDL receptor (VLDLR), LDL receptor-related protein (LRP), and scavenger receptors (SRs). However, little quantitative information exists on the expression of these receptors in normal and atherosclerotic arteries.

Methods and Results—Competitive reverse transcription–polymerase chain reaction and in situ hybridization were used for the studies in New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbit aortic intima-medias. NZW rabbits were fed a 1% cholesterol diet for 0 (control group), 3, 6, or 14 weeks. LDLR mRNA expression was low in aortic intima-medias of all groups. Of the analyzed receptors, LRP had the highest expression in the control group, and its mRNA was induced threefold in the 14-week group, the aortas of which had extensive lesions. SR expression was low and VLDLR expression moderate in the control group. Both receptors were highly induced during cholesterol feeding (SRs, 3-fold and 270-fold induction; VLDLR, 15-fold and 100-fold induction in the 3-week and 14-week groups, respectively). Comparable results were obtained from WHHL rabbits: high basal LRP mRNA in normal intima-medias; moderate induction of LRP and marked induction of SRs and VLDLR in fatty streaks and fatty plaques. In situ hybridization indicated that LRP and VLDLR were expressed in SMCs and macrophages. VLDLR expression was also observed in endothelial cells. SR expression was detected only in macrophages.

Conclusions—SR and VLDLR mRNAs were highly induced in atherosclerotic lesions. VLDLR and LRP may be involved in the formation of both SMC–and macrophage-derived foam cells, whereas SRs play an important role in lipid uptake in macrophages.

Key Words: proteins • lipoproteins • atherosclerosis • receptors

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Atherosclerotic lesions are characterized by accumulation of lipids and foam cells that arise from macrophages and SMCs.¹ There are several receptors that may contribute to the transfer of lipoproteins into the cell (for review, see Reference 2² ): LDLR, VLDLR, and LRP are capable of mediating uptake of native or minimally modified lipoproteins.^{3 4 5} Lipoprotein uptake by SRs requires modification, such as oxidation.^{5 6}

The role of LDLR in the regulation of plasma cholesterol level is well established.⁷ However, it is unlikely that LDLR plays an important role in atherogenesis in the arterial wall, because lesions develop rapidly in patients with homozygous familial hypercholesterolemia as well as in related animal models, WHHL rabbits,³ and LDLR knockout mice.⁸

SRs mediate the uptake of many negatively charged ligands, including oxidized LDL.^{3 5 6} The first characterized SRs were class A type I and II receptors.⁹ As opposed to LDLR, SRs are present in human and rabbit atherosclerotic lesions in the same areas as oxidized LDL.¹⁰

VLDLR is a recently characterized member of the LDLR gene family.⁴ LDLR and VLDLR have highly similar amino acid sequences and overall structures, but because of their additional eighth ligand-binding repeat, VLDLR effectively binds and internalizes VLDL and ß-VLDL but not LDL.⁴ In contrast to LDLR, VLDLR is not downregulated during ß-VLDL–induced foam cell formation.¹¹ There are divergent data on the expression of VLDLR in bovine and human arteries: VLDLR protein was detected in endothelial cells of bovine coronary arteries but not in aortic endothelium or in arterial SMCs,¹² whereas another study with human tissues found VLDLR mRNA and protein in endothelial cells, SMCs, and macrophage-derived foam cells.¹³

LRP is a multifunctional protease inhibitor and receptor molecule.⁵ It is expressed in normal and atherosclerotic arteries.¹⁴ Like VLDLR and SRs, the expression of LRP is not downregulated by increasing intracellular cholesterol concentration.⁵

In addition to mediating cellular lipoprotein uptake, lipoprotein receptors may have additional effects on atherogenesis: SRs can function as an adhesion receptor.¹⁵ Lipoproteins may carry various biologically active molecules, such as lipopolysaccharide, into arterial cells.¹⁶ Binding and internalization of lipoproteins may also initiate signal transduction and thus contribute to atherogenesis and its clinical manifestations.^{17 18}

In the present study, we quantified mRNA levels of LDLR, VLDLR, LRP, and SRs in arterial intima-medias of WHHL and cholesterol-fed NZW rabbits. We also used in situ hybridization to study cellular localization of the receptor expression. Our results suggest that the expression of SRs, VLDLR, and LRP in the arterial wall may contribute to atherogenesis, whereas the expression of LDLR is not involved in the lesion formation.

	Methods

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Animals
Two rabbit models of atherosclerosis were used for competitive RT-PCR studies as described.¹⁹ First, 3-month-old male NZW rabbits were fed a standard (control group, n=2) or 1% cholesterol diet for 3 (n=5), 6 (n=5), or 14 (n=4) weeks. Second, WHHL rabbits were maintained on a standard diet. Two of them were killed at the age of 3 months, one at 7 months, and one at 18 months. To get positive controls for LDLR quantifications, livers from a 10-month-old NZW rabbit and a 9-month-old WHHL rabbit were collected. The mean plasma cholesterol concentrations of control NZW rabbits, cholesterol-fed NZW rabbits, and WHHL rabbits were 1.3±0.8, 50.5±13.8, and 23.6±11.3 mmol/L, respectively.

The animals were killed under intravenous phentanyl-fluanisone (0.3 mL/kg, Jansen Pharmaceuticals) and midazolame (1 mg/kg, Hoffmann-La Roche) anesthesia. Aortas were opened, and the extent of atherosclerosis was recorded by naked-eye evaluation, after which intima-medias were dissected free of adventitia.¹⁹ NZW rabbits of the control and 3-week groups had no macroscopic lesions. After 6 weeks of cholesterol feeding, 20% of the aortic arches were covered with fatty streaks. The aortic arches of the rabbits in the 14-week group were almost completely covered with fatty streaks and fatty plaques. The intima-medias of NZW rabbit aortic arches were pooled according to the length of cholesterol feeding.

WHHL rabbit samples were pooled according to the extent of macroscopic atherosclerosis. Normal intima-medias were obtained from thoracic aortas of the two 3-month-old rabbits. Fatty streaks were obtained from aortic arches and orifices of intercostal arteries of the same animals. Fatty plaques were taken from aortic arches and thoracic aortas of the 7- and 18-month-old WHHL rabbits. All animal studies were approved by the Experimental Animal Committees of the University of Tampere and the University of Kuopio.

RNA Isolation and RT-PCR
Total RNA was isolated by a guanidinium thiocyanate method.^{19 20} The integrity of RNAs was checked on agarose gel electrophoresis. All RNA preparations and RT reagents were found to be free of DNA contamination by amplification of the samples with all PCR primer pairs used in this study.

RNA (3.5 µg/reaction) was reverse-transcribed to first-strand cDNA with avian myeloblastosis virus reverse transcriptase (5 U per reaction) and random hexamer primers (1 µg per reaction) (cDNA Cycle Kit, Invitrogen). Usually, 1/200 of the reverse-transcribed cDNA was used for a single PCR. With LDLR, the amount of cDNA was doubled. Negative controls were included in every assay.²¹

PCR primer sequences and lengths of the amplified fragments are given in the Table. All primers were checked against other lipoprotein receptors and all EMBL/GenBank sequences by use of the programs Gap and Fasta from the GCG program package (Genetics Computer Group). LDLR primers spanned a fragment (nucleotides 2092 to 2492) not mutated in WHHL rabbits.²² VLDLR primers spanned nucleotides 1735 to 2166 of the rabbit cDNA.⁴ Because the rabbit LRP sequence was not previously known, a fragment corresponding to nucleotides 2036 to 3013 of the human LRP sequence²³ was cloned from NZW rabbit lesions with PCR (pGEM-T, Promega). The sequence between primers (EMBL Data Library, accession number X82509) has a 92% identity with the human LRP. The rabbit LRP primers spanned a fragment corresponding to nucleotides 2220 to 2763 of the human sequence. SR primers were from a region (nucleotides 158 to 803) similar in type I and II receptors.²⁴ Rabbit -smooth muscle and ß-nonmuscle actin cDNA sequences²⁵ were used to design actin primers; their sequences and PCR conditions have been reported.¹⁹ All amplified fragments were confirmed by cloning and sequencing at least two independent clones.

View this table: [in this window] [in a new window]   Table 1. Primers and PCR Conditions

PCRs were performed in a 15-µL volume containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.8), 0.1% Triton X-100, and 1 µmol/L each of the gene-specific primers. Optimized PCR conditions for LDLR, LRP, VLDLR, and SRs were 0.3, 0.45, 0.6, or 0.75 U Dynazyme DNA polymerase (Finnzymes) per reaction; 200, 100, 100, or 200 µmol/L dNTPs (ACGU); and 1.5, 1.5, 1.5, or 3.5 mmol/L Mg,²⁺ respectively. The denaturation step was 3 minutes in the first cycle and 1 minute thereafter, and the annealing step was 1 minute at the temperatures indicated in the Table. The extension step was 1 minute at 72°C, except for the last cycle (15 minutes). The number of PCR cycles was selected so that the DNA bands were clearly but not very strongly visible in an ethidium bromide–stained agarose gel to decrease the generation of postexponential phase quantification errors.²⁶

Competitive PCR
Internal standards for competitive PCR were made from heterologous DNA (PCR Mimic Construction Kit, Clontech) so that they could be amplified with the same sets of primers as the corresponding target cDNAs (Table). The use of heterologous DNA as internal standard eliminates heterodimer formation during late PCR cycles. Therefore, the amplified fragments of standard and target can be readily separated with electrophoresis because of their size difference.

Competitive PCR was done by pipetting an equal amount of cDNA to each of the reactions, but different amounts of internal standard were added.¹⁹ A twofold dilution series of the standard was used in the final analysis that was performed two to four times per sample. Amplified fragments were electrophoresed in 2% agarose (Pharmacia) containing 100 µg/L ethidium bromide. The gel was photographed, and fluorescence of the amplified fragments was measured with SigmaScan/Image Software (Jandel Scientific). A restriction enzyme digestion of a plasmid showed that the fluorescence of DNA in the gel correlated linearly with the amount of DNA as long as the gel was not overloaded (data not shown). The ratio of standard to target fluorescences was plotted against the amount of added standard. The results were corrected according to the lengths of amplified fragments, actin expression, and difference in amplification efficiency of standard and target (see below). A regression line was drawn through the plot, and the amount of target cDNA in the samples was deduced from the point at which the ratio of fluorescences was 1. Mean interassay coefficients of variation for the quantification analyses were 18% for LDLR, 7% for VLDLR, 9% for LRP, 7% for SRs, and 9% for actin. Student's t test was used to compare the intima-medias of NZW rabbit control and WHHL rabbit normal groups with the other groups.

The validity of quantifications was verified for each gene by diluting the sample with the highest mRNA expression to the level of the sample with the lowest expression and quantifying that dilution. In addition, the relative amplification efficiencies of each standard-target pair were analyzed by using the average number of PCR cycles, pipetting varying ratios of standards and cloned targets to the reactions, and quantifying the amplified products. All standards of this study amplified 2.1 to 6.9 times more efficiently than the corresponding targets (Table), which translates into an average difference of 2.3% to 7.1% per cycle. The ratios in the Table were used to correct the quantification results. Similar results, showing that shorter fragments amplify more efficiently in quantitative PCR, have been reported earlier.²⁷ The analysis of amplification efficiencies prevented the underestimation of LDLR mRNA levels in this study: a denaturation temperature of 94°C was initially used for LDLR PCRs, but after poor amplification efficiency of the target became evident, denaturation was performed at 96°C and amplification became efficient. The inefficient amplification of the LDLR target under the initial conditions was probably due to a high GC content of the LDLR gene (69% for the amplified fragment), which makes denaturation of the double-stranded DNA at 94°C incomplete.

In Situ Hybridization
Because VLDLR and LRP have many stretches of similar cDNA sequence with other members of the same gene family, antisense oligonucleotide probes were designed for in situ hybridizations. VLDLR probes V47 and V49 comprised nucleotides 1275 to 1321 and 1419 to 1467 of the rabbit VLDLR sequence.⁴ The 47-mer LRP probe, which was also used in a previous study for human samples,¹⁴ corresponds to nucleotides 644 to 690 of the rabbit LRP cloned in this study and has only three incompatible bases. The probes were checked against all EMBL/GenBank sequences with Fasta. In addition, the following lipoprotein receptors were individually examined with the program Gap: rabbit LDLR,²² rabbit VLDLR,⁴ human LRP,²³ partial rabbit LRP (from this study), rat LRP-2/gp330,²⁸ partial human LRP-2/gp330,²⁹ human apo E receptor-2,³⁰ chicken LR8B,³¹ rabbit LR11,³² rabbit SRs types I and II,²⁴ mouse MARCO,³³ hamster SR-BI,³⁴ human CLA-1,³⁵ and human CD36.³⁶ Random homologies (obtained by iteratively scrambling the gene under inspection) would have been 53%, and the obtained values were <60%. Thus, cross-hybridization of the probes with these mRNAs is highly unlikely. The oligonucleotide probes were end-labeled by terminal transferase (MBI Fermentas) with ³⁵ S-ATP (1000 to 1500 Ci/mmol, Amersham International).

SR in situ hybridizations were done with a pBluescript plasmid (Stratagene) containing nucleotides 56 to 1158 of rabbit SR type II cDNA.²⁴ Because 90% of the insert is similar to SR type I, it can be used for the detection of both SR types. Antisense and sense riboprobes were synthesized by use of T3 and T7 RNA polymerases (Promega) with ³⁵ S-UTP (1200 Ci/mmol) (New England Nuclear).

Samples for in situ hybridization and immunocytochemistry studies were collected from aortic arches and thoracic and abdominal aortas of four 5- to 36-month-old WHHL and five NZW rabbits that were kept on a 1% cholesterol diet for 3 to 16 weeks. Either tissue samples were immersion-fixed or the animals were perfusion-fixed with formal-sucrose (4% paraformaldehyde, 15% sucrose, 1 mmol/L EDTA, and 50 µmol/L butylated hydroxytoluene, pH 7.4) for 10 minutes. For frozen sections, aortic tissue was immediately removed, embedded in OCT, frozen, and kept at -70°C until it was analyzed.^{14 37} For paraffin-embedded sections, the samples were further immersion-fixed for 4 hours, kept overnight in 15% sucrose containing 1 mmol/L EDTA and 50 µmol/L BHT, and embedded in paraffin.

In situ hybridization studies were done on a set of serial sections (5 to 7 µm) as described.^{14 37} Briefly, paraffin-embedded tissue sections were deparaffinized and rehydrated. Sections were treated with proteinase K, acetylated, dehydrated, and dried in vacuo. Frozen sections were thawed, fixed with formal-sucrose for 10 minutes, dehydrated, and dried in vacuo. The sections were hybridized at 52°C for 14 hours in 50 µL of hybridization solution containing 6x10⁶ cpm/mL of the labeled probe, 50% formamide (Fluka), 2xSSC, 20 mmol/L Tris (pH 7.4), 1xDenhardt's solution (0.02% Ficoll and 0.02% BSA) (Sigma), 1 mmol/L EDTA, 10% dextran sulfate (Pharmacia), 1 mmol/L dithiothreitol, and 0.5 mg/mL yeast tRNA (Boehringer Mannheim). 1xSSC contained 150 mmol/L NaCl and 15 mmol/L sodium citrate (pH 7.0) (Sigma). After hybridization, the sections were washed at 37°C in 4xSSC (once for 30 minutes and twice for 5 minutes), 2xSSC and 1xSSC (15 minutes each), and at 55°C in 0.1xSSC (30 minutes; riboprobes) or at 42°C in 1xSSC (15 minutes; oligonucleotide probes). The sections were then dehydrated, dried, dipped in autoradiographic emulsion (NTB-2, Eastman-Kodak Co), and developed after 2 to 10 weeks' exposure time. Hematoxylin-eosin was used for counterstaining. Nonhybridizing sense riboprobes were used as controls.

Immunocytochemistry
Serial paraffin-embedded sections (5 to 7 µm) were used for immunostainings. Monoclonal antibodies were used for the identification of macrophages (RAM-11, Dako), SMCs (HHF-35, Enzo Diagnostics), and endothelial cells (CD31, Dako). An avidin-biotin–horseradish peroxidase system (Vector Laboratories) was used for the immunostainings.^{37 38} Hematoxylin was used for counterstaining. Immunocytochemistry was controlled by class- and species-matched irrelevant immunoglobulins and by omitting the primary antibody.

	Results

Top Abstract Introduction Methods Results Discussion References

 
LDLR mRNA Expression
The expression of LDLR mRNA was low, from 1600 to 6300 molecules/µg total RNA, in all aortic samples (Figs 1A and 2). In NZW and WHHL rabbit livers, which were used as positive controls, LDLR mRNA expression was 23 000 and 60 000 molecules/µg total RNA, respectively.

View larger version (42K): [in this window] [in a new window]   Figure 1. Examples of lipoprotein receptor quantification by competitive RT-PCR in aortic intima-medias of control and cholesterol-fed (14 weeks) NZW rabbits. LDLR mRNA expression is low in both groups (A), VLDLR mRNA is greatly induced in cholesterol-fed rabbits (B), LRP mRNA is highly expressed in controls and moderately induced in cholesterol-fed rabbits (C), and SR mRNA expression is low in control group but greatly induced in cholesterol-fed rabbits (D). Numbers below agarose gel lanes indicate amount of added standard, and ratios of standard to target fluorescences are plotted. Samples were analyzed as described in "Methods."

View larger version (31K): [in this window] [in a new window]   Figure 2. Results of LDLR, VLDLR, LRP, and SR mRNA quantifications in aortic intima-medias of NZW rabbits after indicated times of cholesterol feeding (A) and in normal intima-medias, fatty streaks, and fatty plaques of WHHL rabbits (B). Each sample was quantified two to four times as described in "Methods." SD bars are omitted because of logarithmic scale. *P<.05 vs controls (A) or normal intima-medias (B).

Although LDLR expression was low, there was a slight but statistically insignificant descending trend in LDLR mRNA expression in the NZW rabbit samples during cholesterol feeding (Fig 2A). In WHHL rabbit lesions, the expression of LDLR mRNA (which does not lead to the synthesis of functional receptor²² ) was stable and slightly lower than in the NZW rabbit intima-medias (Fig 2B). Thus, no induction of LDLR mRNA expression could be detected in either of these two animal models during the progression of atherosclerosis.

VLDLR mRNA Expression
VLDLR mRNA was moderately expressed in normal aortic intima-medias (Figs 1B and 2). In the cholesterol-fed NZW rabbits, the expression was greatly increased: 15- and 100-fold induction in the 3- and 14-week groups, respectively (Figs 1B and 2A). VLDLR mRNA expression in the 14-week group was highest of all the lipoprotein receptors, 6 million molecules/µg total RNA. A strong, 3- and 19-fold induction was also observed in the WHHL rabbit fatty streaks and fatty plaques (Fig 2B).

LRP mRNA Expression
LRP had the highest basal mRNA expression in the aortic intima-medias (Figs 1C and 2). In the cholesterol-fed NZW rabbits, there was no major induction of LRP mRNA during the first 6 weeks of the study (Fig 2A). When the feeding had continued for 14 weeks, a threefold induction was detected (Figs 1C and 2A). In WHHL rabbit fatty streaks and plaques, LRP mRNA expression was twofold compared with normal intima-medias (Fig 2B).

SR mRNA Expression
Basal expression of SR mRNA was low in the intima-medias of control NZW rabbits (Figs 1D and 2A). During cholesterol feeding and lesion development, SR mRNA was greatly induced: threefold in the 3-week group and 270-fold in the 14-week group (Fig 2A). In normal WHHL rabbit intima-medias, SR mRNA expression was slightly higher than in control NZW rabbits (Fig 2). Compared with the normal intima-medias, the expression was increased 5- and 70-fold in WHHL fatty streaks and fatty plaques, respectively.

In Situ Hybridization Analyses
SR mRNA was expressed in macrophage-rich areas of WHHL and cholesterol-fed NZW rabbit atherosclerotic lesions. As an example, a fatty plaque from the thoracic aorta of a WHHL rabbit shows SR mRNA expression in the upper subendothelial part of the lesion (Fig 3A), where most of the cells are macrophages (Fig 3B). We could not detect SR expression in SMCs. Control hybridizations with the sense riboprobe (Fig 3C) and control immunostainings were negative (Fig 3D).

View larger version (86K): [in this window] [in a new window]   Figure 3. Expression of SR, LRP, and VLDLR mRNAs in rabbit atherosclerotic lesions. A through D, Serial sections from aortic lesion of 24-month-old WHHL rabbit showing SR mRNA expression in macrophages. A, In situ hybridization with 35 S-UTP–labeled rabbit SR type I antisense riboprobe. B, Immunostaining with monoclonal antibody against macrophages (RAM-11, dilution 1:1000). C, In situ hybridization control with rabbit SR type I sense riboprobe. D, Immunocytochemistry control (primary antibody omitted). E and F, LRP mRNA is expressed in lesion and media of NZW rabbit fed 1% cholesterol diet for 16 weeks. E, In situ hybridization with 35 S-ATP–labeled LRP antisense oligonucleotide probe; lesion and whole depth of media are shown. F, Bright-field illumination from E with higher magnification showing LRP mRNA as dark spots in lesion (uppermost third of picture) and in underlying media. G and H, Serial sections from 5-month-old WHHL rabbit abdominal aorta. G, In situ hybridization with 35 S-ATP–labeled antisense oligonucleotide probe (V47) shows that VLDLR mRNA is expressed in endothelium and in deeper parts of lesion. H, Immunostaining with monoclonal antibody against SMCs (HHF-35, dilution 1:200). A, C, E, and G were photographed with polarized light epiluminescence showing expression of mRNA as bright spots. Hematoxylin-eosin counterstain, except for B, D, and H, which were counterstained with hematoxylin. Magnification: A through D, x11; E and H, x22; F, x45; G, x12.

LRP mRNA was expressed in SMCs of normal (data not shown) and atherosclerotic aortas. Figs 3E and 3F show an example of LRP expression in the thoracic aorta of a cholesterol-fed NZW rabbit lesion in which both macrophages and SMCs express LRP mRNA (immunostainings for cell types not shown). The results are similar to the data published earlier by our group.¹⁴

VLDLR mRNA was expressed in normal and atherosclerotic arteries. In lesion-free sections, the expression was found primarily in the endothelium, but some hybridization was also seen in medial SMCs (data not shown). In the lesions of both WHHL and NZW rabbits, VLDLR mRNA was expressed in the endothelium, macrophages, and SMCs. An example is shown from a fibrous plaque of the abdominal aorta of a 5-month-old WHHL rabbit, in which the expression is seen in the endothelium and in the subendothelial, shoulder, and deeper parts of the lesion (Fig 3G). An immunostaining of a serial section with RAM-11 showed that most of the VLDLR-expressing cells in the deeper part of the lesion were macrophages (data not shown). Also, some but not all SMCs were positive for VLDLR mRNA (Fig 3H).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study describes analysis of four lipoprotein receptor mRNAs in normal and atherosclerotic aortas in two experimental models of atherosclerosis: WHHL rabbits and cholesterol-fed NZW rabbits. Because mRNA levels are not directly proportional to the production of functional proteins, the mRNA-based results should be interpreted cautiously. However, it is likely that sizable differences in mRNA concentrations are also reflected in the amounts of functional proteins. In this study, competitive RT-PCR was used for mRNA quantifications. The method was verified by (1) diluting the sample with the highest expression to the level of the sample with the lowest expression and (2) comparing amplification efficiencies of the target and standard fragments. Because of the low LDLR expression in arterial intima-medias, (3) livers were used as positive controls to confirm an efficient first-strand synthesis of LDLR cDNA. (4) The specificity of the PCRs was verified by cloning and sequencing, which is necessary because of the novel lipoprotein receptors that have recently been identified.^{30 31 32 39} The applied controls ensure reliable detection of moderate differences in mRNA expression between various samples.

Earlier quantitative data on lipoprotein receptor expression in arterial samples include only LDLR: in a pioneering report of quantitative RT-PCR, 13 000 and 4000 LDLR mRNA molecules/µg total RNA were found in a normal human coronary artery and in an atherosclerotic carotid artery, respectively.⁴⁰ Analyzed with the same standard, human livers contained 50 000 to 350 000 molecules,⁴¹ but only 860 molecules/µg total RNA were found in bovine liver.⁴² The amplification efficiencies of the standard and target were not compared in any of those studies. We found 1600 to 6300 LDLR mRNA molecules/µg total RNA in rabbit arterial samples and 23 000 and 60 000 molecules in NZW and WHHL rabbit livers, respectively. Thus, the present results are in line with earlier observations. Compared with LDLR mRNA, VLDLR, LRP, and SR mRNAs were much more abundant in the lesions of NZW and WHHL rabbits (Fig 2). The quantitative data on LDLR mRNA expression are consistent with our earlier results obtained with immunocytochemistry and in situ hybridization: LDLR protein was detected in only two of nine human arteries in medial SMCs near the adventitial border.¹⁴ In normal or atherosclerotic human intimas, LDLR mRNA or protein was not found.^{10 14}

In normal rabbit arteries, VLDLR mRNA was clearly expressed according to both RT-PCR and in situ hybridization. The basal expression was localized primarily to the endothelium, as reported earlier for VLDLR protein in bovine arteries,¹² but some in situ hybridization signal was also detected in medial SMCs, as in the study with human samples.¹³ Competitive RT-PCR indicated that VLDLR mRNA was induced as early as after 3 weeks of cholesterol feeding, when the NZW rabbit aortas were still macroscopically normal but contained some infiltrated monocyte-macrophages. VLDLR mRNA expression continued to increase to a very high level in the 14-week group. As has been reported for human atherosclerotic carotid arteries,¹³ rabbit lesions contained VLDLR mRNA expression in endothelial cells, SMCs, and macrophages.

According to both competitive RT-PCR and in situ hybridization, there was a high basal expression of LRP mRNA in normal rabbit arteries, which is consistent with the many functions of LRP.⁵ We have reported LRP mRNA and protein expression in SMCs and macrophages of normal and atherosclerotic arteries.¹⁴ Because LRP is a monocyte differentiation antigen,⁴³ the twofold to threefold increase in LRP mRNA in the lesions is probably derived from macrophages, whereas the basal expression is derived from SMCs.

SR mRNA expression was very low in normal aortic intima-medias but was induced in NZW rabbits as early as after 3 weeks and further after 6 and 14 weeks of cholesterol feeding. Studies with human lesions have shown SR mRNA and protein expression in macrophages.^{10 14 44} It was recently reported that lesions from hypercholesterolemic NZW rabbits contained some SR expression in SMCs located near the arterial lumen, even though most of the SR-positive cells were macrophages.⁴⁵ In balloon-injured arteries, a significant portion of SR expression was associated with SMCs.⁴⁵ We detected SR mRNA expression only in macrophages, but the studies do not exclude the possibility of SR expression in proliferating intimal SMCs. We did not analyze type I and II SRs separately. It has been reported that the expression of type I is induced, whereas type II expression remains constant during macrophage differentiation and foam cell formation.⁴⁶ It is probable that other SR types are also expressed in atherosclerotic lesions.^{2 47}

A continuous increase in VLDLR, LRP, and SR mRNAs was observed in both the NZW and WHHL rabbit samples during the progression of atherosclerosis. This might be thought to be a general phenomenon associated with the increased cellularity and/or activation of the lesion cells. In a previous study, however, we found that the high 15-lipoxygenase mRNA expression in WHHL rabbit fatty streaks decreased to 1 in 10 in fatty plaques,¹⁹ which suggests a divergent regulation of genes during the progression of atherosclerosis. A related phenomenon is that lesion macrophages do not always express LRP and SRs uniformly.^{10 14}

Several potential ligands for lipoprotein receptors are present in the arterial wall. These include apo E–containing ß-VLDL–like particles, IDL, LDL, and oxidized LDL.^{48 49 50} It is conceivable that the uptake of these particles via SRs, VLDLR, and LRP may enhance foam cell formation, although we cannot exclude a possibility that atherosclerosis could develop in the absence of these receptors. VLDLR and LRP can also internalize lipoproteins enriched or complexed with apo E and LPL,^{51 52 53} both of which are expressed in atherosclerotic lesions.^{54 55 56} In addition, oxidized LDL that is complexed with LPL, apo E, and proteoglycans may be internalized by LRP (Ylä-Herttuala et al, unpublished data, 1997). VLDLR and LRP also have nonlipoprotein ligands potentially related to atherogenesis and its complications, such as ₂-macroglobulin–protease complexes and urokinase–plasminogen activator inhibitor-1 complexes.^{5 57}

This study provides, for the first time, quantitative assessment of the expression of lipoprotein receptors in normal and atherosclerotic arteries. We conclude that VLDLR and LRP may be involved in the formation of SMC- and macrophage-derived foam cells. SRs provide macrophages an effective route for lipid accumulation by the uptake of oxidized lipoproteins. Conversely, the expression of LDLR in the arterial wall does not play a significant role in atherogenesis. Thus, it is likely that no single receptor system is responsible for the increased lipid uptake in atherosclerotic lesions but rather that several mechanisms contribute to the uptake and degradation of lipoproteins in lesion cells.

	Selected Abbreviations and Acronyms

 

LDLR = LDL receptor LRP = LDL receptor–related protein NZW = New Zealand White RT-PCR = reverse transcription–polymerase chain reaction SMC = smooth muscle cell SR = scavenger receptor VLDLR = VLDL receptor WHHL = Watanabe heritable hyperlipidemic

	Acknowledgments

 
This study was supported by grants from the Finnish Foundation for Cardiovascular Research, Finnish Academy, Sigrid Juselius Foundation, Astra Finland, and the Research and Science Foundation of Farmos Finland. The authors thank Dr Mason Freeman and Dr Perry Bickel for providing partial rabbit SR cDNA. The technical assistance of Merja Lehtinen is acknowledged.

Received June 3, 1997; revision received October 24, 1997; accepted October 30, 1997.

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