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
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.
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.
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
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
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
Competitive PCR
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
In Situ Hybridization
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
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
VLDLR mRNA Expression
LRP mRNA Expression
SR mRNA Expression
In Situ Hybridization Analyses
LRP mRNA was expressed in SMCs of normal (data not shown) and
atherosclerotic aortas. Figs 3E
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
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
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
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.
Received June 3, 1997;
revision received October 24, 1997;
accepted October 30, 1997.
© 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
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.
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
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
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.
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. 
. 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 a new window]
Table 1. Primers and PCR Conditions 
. 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
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. ), 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.
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).
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.
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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 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). ). 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 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 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).
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.
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):
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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. 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 ). 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. ). 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 2-macroglobulin–protease complexes and
urokinase–plasminogen activator
inhibitor-1 complexes.5 57
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.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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