From the Tohoku University Gene Research Center,
Sendai 981-8555, Japan, the ¶ Department of Animal Science,
College of Agriculture, Chonnam National University, Kwangju 500-600, Korea, the
Departments of Pathology, Ehime University School of
Medicine, Ehime 791-0295, Japan, the ** Laboratory of
Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and
Dental University, Chiba 282-0827, Japan, the
Department of Food and Human Health
Sciences, Graduate School of Human Life Science, Osaka City University,
Osaka 558-8585, Japan, the §§ Division of
Nephrology, Endocrinology, and Vascular Medicine, Department of
Medicine, Tohoku University Graduate School of Medicine, Sendai
980-8574, Japan and Yanagisawa Orphan Receptor Project, ERATO, Japan
Science and Technology Corporation, Tokyo 135-0064, Japan, and the
¶¶ Third Department of Internal Medicine, Fukui Medical
University, Fukui 910-1193, Japan
Received for publication, November 25, 2002, and in revised form, December 30, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone
accrual development. Recently, we demonstrated that LRP5 is also
required for normal cholesterol metabolism and glucose-induced insulin
secretion. To further define the role of LRP5 in the lipoprotein
metabolism, we compared plasma lipoproteins in mice lacking LRP5,
apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a
normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had ~60% higher plasma cholesterol levels compared
with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High
performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the
apoE;LRP5 double knockout mice. There were no apparent differences in
the pattern of apoproteins between the apoE knockout mice and the
apoE;LRP5 double knockout mice. The plasma clearance of
intragastrically loaded triglyceride was markedly impaired by LRP5
deficiency. The atherosclerotic lesions of the apoE;LRP5 double
knockout mice aged 6 months were ~3-fold greater than those in the
age-matched apoE-knockout mice. Furthermore, histological examination
revealed highly advanced arthrosclerosis, with remarkable accumulation
of foam cells and destruction of the internal elastic lamina in the
apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates
both apoE-dependent and apoE-independent catabolism of
plasma lipoproteins.
Genetic defects in the catabolism of plasma lipoproteins are
important causes of hypercholesterolemia and atherosclerosis in humans.
The prototypic diseases are familial hypercholesterolemia, caused by a
defect in the LDL1 receptor
(LDLR) (1), and familial type III hyperlipoproteinemia, caused by a
defect in one of the ligands for LDLR, apolipoprotein E (apoE) (2).
ApoE is hypothesized to mediate lipoprotein clearance by binding two
receptors: (i) LDLR and (ii) a hepatic chylomicron remnant receptor.
ApoE-deficient mice (3-5) and LDLR-deficient mice (6) exhibit
hypercholesterolemia, but the severity and manifestations differ
markedly. On a normal laboratory chow diet, the apoE knockout mice have
much more profound hypercholesterolemia and develop spontaneous
atherosclerosis (4).
LDL receptor-related protein 5 (LRP5) is a member of the LDL receptor
family that are characterized by the presence of cysteine-rich complement type ligand binding domains. LRP5 binds apoE-containing lipoproteins in vitro and is widely expressed in many
tissues including hepatocytes, adrenal gland, and pancreas (7).
LRP5 and its homologue, LRP6, are postulated to play as co-receptors
for Wnt receptors, Frizzled (8-13). The Wnt signaling pathway plays an
essential role in embryonic development (14, 15) and oncogenesis (16)
through various signaling molecules including Frizzled receptors (17),
LRP5 and LRP6 (8-13), and Dickkopf proteins (11, 12, 18). The Wnt
signaling is also involved in adipogenesis by negatively regulating
adipogenic transcription factors (19). Recent studies have revealed
that loss of function mutations in the LRP5 gene cause the autosomal
recessive disorder osteoporosis-pseudoglioma syndrome (20).
Consistent with human osteoporosis-pseudoglioma syndrome, LRP5
knockout mice generated by Kato et al. (21) exhibit a severe
low bone mass phenotype.
Recently, we demonstrated that LRP5-deficient mice develop high plasma
cholesterol levels after feeding a high fat diet (22). The hepatic
clearance of apoE-rich chylomicron remnants was also markedly decreased
in the LRP5 knockout mice. These data suggested that LRP5 plays a role
in the hepatic clearance of chylomicron remnants. In addition, we
showed that the LRP5-deficient mice fed a normal diet showed marked
impaired glucose tolerance. The LRP5-deficient islets had a marked
reduction in the levels of intracellular ATP and Ca2+ in
response to glucose; thereby, glucose-induced insulin secretion was
decreased (22). Together with the roles of LRP5 in the bone accrual
development (20, 23, 24) as well as in the Wnt signaling pathways
(8-11, 13), our data indicated that LRP5 is a multifunctional receptor
physiologically linked to common human disorders, including hypercholesterolemia and impaired glucose tolerance.
To further define the role of LRP5 in lipoprotein metabolism, we
produced double knockout mice that are deficient in apoE as well as in
LRP5 (apoE;LRP5 double knockout mice). In the current paper, we
describe that superimposition of an LRP5 deficiency onto apoE
deficiency increased plasma cholesterol beyond the level observed with
apoE deficiency alone. We also show that fat tolerance was markedly
impaired in the LRP5 knockout mice as well as in the apoE;LRP5 double
knockout mice. Consistent with extreme hypercholesterolemia, severe
atherosclerosis developed in the apoE;LRP5 double knockout mice. These
results provide further evidence for the role of LRP5 in the catabolism
of plasma lipoproteins.
Materials--
For the lipoprotein analysis, blood was collected
from the retroorbital plexus after 4 h of fasting. Plasma total
cholesterol levels were determined in individual mice at each time
point by enzymatic assay kits (Wako Pure Chemicals Co., Osaka, Japan).
For the detection of cholesterol and triglycerides with the high
performance liquid chromatography (HPLC) method (see below), we
obtained enzymatic reagents from Kyowa Medex Co. (Tokyo, Japan). The
reagent system for cholesterol detection consists of reagent 1 (R1-C)
and reagent 2 (R2-C) (R1-C: 20 mM MOPS, pH 7.0, 1.1 mM EMSE, 10 units/ml peroxidase, detergents, and
stabilizer; R2-C: 20 mM MOPS, pH 7.0, 1.5 mM
4-aminoantipyrine, 0.68 mM CaCl2, 0.3 units/ml
cholesterol esterase, 2 units/ml cholesterol oxidase, 10 units/ml
peroxidase, detergents, and stabilizer). The triglyceride reagent
system includes reagent 1 (R1-TG) and reagent 2 (R2-TG) (R1-TG: 50 mM PIPES, pH 6.2, 1.1 mM EMSE, 2 mM
MgSO4, 4.9 mM ATP, 3 units/ml glycerol kinase,
1.5 units/ml glycerol-3-phosphate oxidase, 5 units/ml peroxidase,
detergents, and stabilizer; R2-TG: 50 mM PIPES, pH 6.2, 1.5 mM 4-aminoantipyrine, 2 mM MgSO4, 3 units/ml lipoprotein lipase, 5 units/ml peroxidase, detergents, and
stabilizer. Equal amounts of R1 and R2 were mixed before use. After
mixing, the cholesterol reagent was used within 4 weeks, and the
triglyceride reagent was used within 2 weeks.
Lipoprotein Analysis by a Dual Detection HPLC System--
Plasma
lipoproteins were analyzed by an improved HPLC analysis according to
the procedure as described by Usui et al. (25). The HPLC
system consisted of an AS-8020 autoinjector, CCPS and CCPM-II pumps,
and two UV-8020 detectors (Tosoh, Japan) (26). An SC-8020 system
controller (Tosoh) was used for instrument regulation and data
collection. Lipoproteins were fractionated on two tandem connected
TSKgel LipopropakXL columns (300 × 7.8-mm; Tosoh) with 50 mM Tris acetate, pH 8.0, containing 0.3 M
sodium acetate, 0.05% sodium azide, and 0.005% Brij-35 at a flow rate
of 0.7 ml/min. The TSK column medium is composed of porous
polymermatrices with a nominal bead size of 10 µm and a pore size of
100 nm, which is expected to exclude most of chylomicron (CM) to the
void volume. Two TSK columns were connected in tandem and used to
obtain higher resolution within a relatively short analytical time. The
running buffer was filtered through a 0.22-µm filter (Millipore
Corp.) before use and continuously degassed with an SD-8022 on-line
degasser (Tosoh) during analysis. The column effluent was split equally into two lines by a Micro-Splitter P-460 (Upchurch Scientific Inc., Oak
Harbor, WA), one mixing with cholesterol reagent and the other with
triglyceride reagent, in order to achieve simultaneous profiles from a
single injection. The two enzymatic reagents were each pumped at a flow
rate of 0.35 ml/min for the TSK column. Both enzymatic reactions
proceeded at 37 °C in a reactor coil (Teflon, 15 m × 0.4 mm,
inner diameter). 10-µl samples diluted with saline were injected by
an AS-8020 autoinjector with a presuction volume of 25 µl at
intervals of 24 min. The enzymatic determination of cholesterol and
triglycerides involved the detection of hydrogen peroxide produced by
cholesterol oxidase and glycerol-3-phosphate oxidase, respectively.
Total cholesterol and triglyceride concentrations (in mg/dl) were
calculated by comparison with total area under the chromatographic
curves of a calibration material of known concentration.
SDS-Polyacrylamide Gel Electrophoresis--
Total lipoprotein
fractions (d < 1.215 g/ml) from pooled plasma of the
mice were isolated by ultracentrifugation, and the delipidated
apolipoproteins were boiled for 3 min in SDS sample buffer containing
2-mercaptoethanol and subjected to electrophoresis on an SDS/5-15%
polyacrylamide gel. Proteins were stained with Coomassie Blue.
Fat Tolerance Test--
6-month-old male mice were fasted for
16 h, and olive oil (1 ml/30 g body weight; Wako Pure Chemicals
Co.) was administered intragastrically as a bolus. Approximately 50 µl of blood was taken from the tail vein at the indicated times for
the measurement of triglyceride levels and HPLC analysis.
Mice--
LRP5 "knockout" mice (originally C57BL/6J-CBA
hybrids (22)), LRP5 Measurement of Atherosclerotic Lesions--
Mice were
euthanized, and thoracic and abdominal aorta were used for en
face staining with Oil Red O to visualize neutral lipid
(cholesteryl ester and triglycerides) accumulation. In brief, the aorta
was removed, cleaned, and cut open with the luminal surface facing up
and then immersion-fixed in 10% formalin in 10 mM
phosphate-buffered saline. After rinsing with phosphate-buffered saline, the aorta was thoroughly cleaned of adventitial fat using microforceps and spring iris scissors under a stereoscopic microscope. The inner aortic surface was stained with Oil Red O for 25 min at room
temperature. After rinsing with 60% isopropyl alcohol and distilled
water, the Oil Red O-stained area was quantified by NIH Image
1.62f software analysis of the digitized microscopic images.
Results are expressed as percentage of lipid-accumulating lesion
area of the total aortic area analyzed.
For light microscopy, the aortic tissue samples were fixed with 10%
formalin in 10 mM phosphate buffer (pH 7.2) and embedded in
paraffin. Sections 2-3 µm thick were taken longitudinally through the aortic lumen and stained with hematoxylin and eosin or
elastica-Masson. For Oil Red O staining, aortic tissue samples were
frozen in OCT compound (Miles Inc., Elkhart, IN). Cryostat tissue
sections were cut to a thickness of 5 µm and stained with Oil Red O. Nuclei were counterstained with hematoxylin.
Plasma Cholesterol and Lipoprotein Profile--
Fig.
1 compares the levels of total
cholesterol of mice of four different genotypes at the indicated ages.
Mice were fed a normal laboratory chow diet containing 4.5% (w/w) fat
and 0% cholesterol. Although there were no significant differences in
the total plasma cholesterol levels between the apoE knockout mice
(apoE
High resolution HPLC analysis (25) of plasma lipoprotein of 4-month-old
mice revealed that cholesterol levels in the VLDL and LDL fractions
were markedly increased in the apoE;LRP5 double knockout mice compared
with the apoE knockout mice (Fig. 1B and Table
I): the cholesterol levels in the VLDL
and LDL fractions in the apoE knockout were 180 ± 35 and 145 ± 7 mg/dl, respectively, and those in the apoE;LRP5 double knockout
mice were 244 ± 24 and 171 ± 21 mg/dl, respectively (Table
I). There were no significant differences in the levels of CM- and
HDL-cholesterol between the apoE knockout mice and the apoE;LRP5 double
knockout mice, although HDL-cholesterol levels in these mice were
~50% of those in the LRP5 knockout mice and normal controls. Despite
the severe hypercholesterolemia in the apoE knockout and apoE;LRP5
double knockout mice, there were no significant differences in the
total triglyceride levels among mice with the four different genotypes
(data not shown).
Fig. 2 shows the SDS-polyacrylamide gel
electrophoresis of apoproteins in pooled lipoprotein fraction from mice
of four different genotypes. Consistent with the previous work by
Ishibashi et al. (27), the amounts of apoB48 were markedly
increased in the apoE knockout mice as well as in the apoE;LRP5 double
knockout mice. Despite the severe hypercholesterolemia in the apoE;LRP5
double knockout mice, there were no apparent differences in the pattern of apoproteins between the apoE- and apoE;LRP5 double knockout mice.
Fat Tolerance Test--
In a pervious study, we showed that LRP5
plays a role in the hepatic uptake of dietary cholesterol. The LRP5
knockout mice displayed dietary derived hypercholesterolemia due to
decreased plasma clearance of chylomicron remnants (22). To further
define the role of LRP5, fat tolerance test was carried out using mice of four different genotypes. Mice were fasted for 16 h, and olive oil (1 ml/30 g body weight) was administered intragastrically. As shown
in Fig. 3, plasma levels of total
triglyceride increased and peaked at about 2 h and then declined
toward base line 6 h after loading in both apoE-knockout mice and
normal controls. In contrast, the increased levels of plasma
triglyceride were sustained for several h after loading in both LRP5
knockout and apoE;LRP5 double knockout mice, indicating that the plasma
clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. HPLC analysis of plasma lipoproteins revealed that
the majority of particles at 6 h after fat loading were in the
VLDL fraction.
In addition, we noticed that 16 h of fasting increased the levels
of VLDL-triglyceride in the apoE knockout, LRP5 knockout, and apoE;LRP5
double knockout mice. This result may indicate that both apoE and LRP5
mediate the plasma clearance of VLDL-triglyceride induced by fasting.
Atherosclerosis--
Aortic atherosclerotic lesions of the apoE
knockout and apoE;LRP5 double knockout mice were first analyzed by
en face lipid staining (Fig.
4A). At 4 months of age, the
area of the thoracic and abdominal aortas stained by Oil Red O of the
apoE;LRP5 double knockout mice was approximately the same as that in
the apoE knockout mice. In contrast, at 6 months of age, the lesions in
the apoE;LRP5 double knockout mice were ~3-fold larger than those in
the apoE knockout mice (Fig. 4B).
In histopathology under light microscopic examination, the lesions in
the apoE knockout mice at 6 months of age were relatively modest,
showing slightly atheromatous lesions with a fatty streak-like structure, which were localized on the surface of aortic intima but
were not associated with the destruction of internal elastic lamina or
the medial muscle layer (Fig. 4C). In contrast, the apoE;LRP5 double knockout mice developed multiple atheromatous lesions
manifesting a hump structure, which were associated with cholesterin
deposits, fibrosis, and elastosis (Fig. 4D). Some of them
showed the destruction of internal elastic lamina and the degenerative
change of medial muscle layers of the aorta (Fig. 4E). In
these lesions, severe deposition of neutral lipid was observed (Fig.
4F).
In the present study, we show extreme
hypercholesterolemia in mice lacking both apoE and LRP5. It has been
well established that both LDLR and apoE are critical in the plasma
clearance of cholesterol-carrying lipoproteins, including LDL and
apoE-containing intermediate density lipoprotein and chylomicron
remnants (1, 2). In contrast to the mice lacking apoE (3-5) or LDLR
(6), the lack of LRP5 alone did not increase the plasma levels of
cholesterol on a normal diet, whereas high fat feeding results in
hypercholesterolemia in the LRP5 knockout mice (22). Ishibashi et
al. (27) showed that the plasma cholesterol levels in the double
knockout mice lacking both apoE and LDLR were not significantly
different from the levels in the apoE knockout mice. The severe
hypercholesterolemia developed in the double knockout mice lacking both
apoE and LRP5 suggests the presence of an alternative pathway for
cholesterol catabolism mediated by LRP5, which appears to be
independent of the LDLR pathway.
Consistent with the previous work (22), the LRP5 knockout mice and the
apoE;LRP5 double knockout mice displayed markedly impaired fat
tolerance. In contrast, the plasma clearance of intragastrically loaded
triglyceride was not significantly impaired in the apoE-knockout mice.
These observations suggest that LRP5 modulates the plasma clearance of
diet-derived triglycerides in the absence of apoE by stimulating the
hydrolysis of triglycerides. In this context, it is important to
note that LRP5 and LRP6 can bind Dickkopf (Dkk), an antagonist
of Wnt proteins (12, 24). Dkk is involved in Xenopus head
formation and the impaired action of Dkk at LRP5 increases bone density
in humans (24). The Dkk sequence consists of two cysteine-rich domains.
The C-terminal domain has the typical cysteine pattern of colipase,
which is required by pancreatic lipase for the efficient lipid
hydrolysis (reviewed in Ref. 28). The C-terminal domain of
colipase binds to the C-terminal noncatalytic domain of pancreatic
lipase, which is thought to stabilize an active conformation of the
lipase, and is also conserved among various lipases, including hepatic
and lipoprotein lipases. Detailed sequence analysis and molecular
modeling of the Dkk sequence onto the colipase structure suggest that
Dkk and colipase have the same disulfide pattern and very similar
three-dimensional structures (28). This structural analogy implies a
common function (lipid interaction) and raises the possibility that Dkk
bound to LRP5 stimulates lipid hydrolysis by interacting with
hepatic lipase and/or lipoprotein lipase. Furthermore, the impaired fat
tolerance caused by the deficiency of LRP5 may lead to severe
hypercholesterolemia in the absence of apoE.
Another explanation for the impaired lipoprotein metabolism in the
apoE;LRP5 double knockout mice is that LRP5 may recognize other
lipoproteins in addition to apoE-containing lipoproteins. The candidate
apoproteins that may be recognized by LRP5 remain unidentified, since
the LRP5 deficiency did not significantly alter the pattern of
apoproteins in the plasma lipoproteins of the apoE knockout mice or
that of normal mice.
In addition to the role of LRP5 in embryonic development and bone
development, our current data provide further evidence that LRP5 also
plays a role in the metabolism of plasma lipoproteins. Furthermore,
consistent with the marked elevation of plasma cholesterol, severe
arthrosclerosis developed in the apoE;LRP5 double knockout mice. The
remarkable destruction of the internal elastic lamina seen in the
lesion of the double knockout mice is characteristic of highly advanced
atherosclerosis. The apoE;LRP5 double knockout mice manifesting extreme
hypercholesterolemia and highly advanced arthrosclerosis will provide a
useful animal model for the research and development of therapeutic
agents against hypercholesterolemia and atherosclerosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, have been continually mated with C57BL/6J; N6
and N7 generation descendents from this cross into the C57BL/6J
background were used. ApoE
/
mice (3) backcrossed 10 times on the
C57BL/6J background were obtained from the Jackson Laboratory (Bar
Harbor, ME). To obtain knockout mice that are homozygous for disruption of both the LRP5 and apoE loci, male apoE
/
mice were mated to female LRP5
/
mice. The resulting apoE+/
;LRP5+/
mice were
identified by PCR analysis and bred each other to produce
apoE
/
;LRP5
/
mice. Experiments were performed with those mice or
those with the same genotype from the next generation by breeding
apoE
/
;LRP5
/
with each other. Mice were maintained on 12-h
dark/12-h light cycles and had free access to a normal laboratory chow
diet (4.5% fat, 0% cholesterol, CE-2; CLEA, Tokyo, Japan) and water.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
;LRP5+/+) and the apoE;LRP5 double knockout mice
(apoE
/
;LRP5
/
) at 2 months of age, the cholesterol levels of the
double knockout mice older than 4 months were greatly increased (by
approximately 60%) beyond the levels observed with apoE deficiency
alone. In contrast, LRP5 deficiency alone had no significant effects on
the plasma cholesterol levels.
View larger version (20K):
[in a new window]
Fig. 1.
Age-dependent changes in plasma
cholesterol concentrations in mice with different genotypes fed a
normal diet. A, plasma levels of total
cholesterol of mice of each genotype at the indicated age were
determined enzymatically after 4 h of fasting. Data are mean ± S.D. of six mice. *, p < 0.01; Student's
t test. B, HPLC analysis of plasma lipoproteins.
Plasma samples from mice of each genotype at 4 months of age were
separated by HPLC, and cholesterol (red line) and
triglyceride (blue line) contents were determined
as described under "Experimental Procedures." Representative data
from six animals with the indicated genotype is shown. The CM, VLDL,
LDL, and HDL fractions are labeled C, V,
L, and H, respectively. Free glycerol is
indicated by an arrowhead. The cholesterol levels in the CM,
VLDL, LDL, and HDL fractions are shown in Table I.
Plasma cholesterol profiles in mice with different genotypes
View larger version (94K):
[in a new window]
Fig. 2.
SDS-polyacrylamide gel electrophoresis of
total lipoprotein fractions. Equal volumes (1 ml) of plasma were
pooled from four mice of different genotypes fed a normal diet and
total lipoprotein fractions (d < 1.215 g/ml) were
isolated by ultracentrifugation, and the delipidated apoproteins were
subjected to electrophoresis on an SDS/5-15% polyacrylamide gradient
gel. Proteins were stained with Coomassie Blue. Positions of migration
of apoB100, apoB48, apoA-VI, apoE, and apoA1 are denoted.
Representative data from four independent experiments is shown.
View larger version (26K):
[in a new window]
Fig. 3.
Effects of intragastric fat loading on plasma
triglyceride levels in mice with different genotypes. Six males (6 months old) of each genotype received an intragastrically
administration of olive oil (1 ml/30 g body weight). At the indicated
times, 50 µl of blood was taken from the tail vein and subjected to
HPLC analysis. Data are mean ± S.E. of six mice. *,
p < 0.01; Student's t test.
View larger version (74K):
[in a new window]
Fig. 4.
Atherosclerotic lesions in apoE and apoE;LRP5
double knockout mice. A, en face lipid
staining of aortas. Thoracic and abdominal aorta from the indicated
genotype was cut open with the luminal surface facing up, and the inner
aortic surface was stained with Oil Red O. Representative data of each
genotype are shown. Bar, 5 mm. B, quantitative
analysis of en face lipid staining. The inner aortic surface
area stained with Oil Red O was quantified by NIH Image 1.62f software
analysis of the digitized microscopic images. Results are expressed as
percentage of lipid-accumulating lesion area of the total aortic
area analyzed. Data are mean ± S.D. of six mice. *,
p < 0.01; Student's t test.
C-F, representative histopathological features of the
aorta. Bars, 100 µm. C, an apoE-knockout mouse
(aged 6 months) shows a slightly atheromatous lesion characteristic of
the accumulation of foam cells, which is not associated with the
destruction of internal elastic lamina (dark brown-colored) or the
degenerative change of muscle layer of the aorta (elastica-Masson
staining). D, one of the multiple atheromatous lesions
developed in an apoE;LRP5 double knockout mouse (aged 6 months)
manifests a hump structure associated with cholesterin deposits,
fibrosis (light green-colored), and elastosis (dark brown-colored).
Destruction of the internal elastic lamina adjacent with a degenerative
lesion of muscle layer of the aorta is remarkable (elastica-Masson
staining). E, an atheromatous lesion in an apoE;LRP5 double
knockout mouse (aged 6 months) reveals a remarkable accumulation of
foam cells, especially marked in the superficial region of atheroma,
and a crystal structure of cholesterin deposits (hematoxylin and eosin
staining). F, an atheromatous lesion in an apoE;LRP5 double
knockout mouse (aged 6 months) reveals severe deposition of neutral
lipid in the aortic wall, resulting in the destruction of lamellar
structure of the elastic fibers (Oil Red O staining).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We thank N. Suzuki for preparing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Japan Society for the Promotion of Science Grant RFTF97L00803.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Tohoku
University Gene Research Center, 1-1 Tsutsumidori-Amamiya, Aoba, Sendai 981-8555, Japan. Tel.: 81-22-717-8875; Fax: 81-22-717-8877; E-mail: tfujino@biochem.tohoku.ac.jp.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M211987200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LDL, low density lipoprotein; LDLR, LDL receptor; apoE, apolipoprotein E; CM, chylomicron; Dkk, Dickkopf; HDL, high density lipoprotein; HPLC, high performance liquid chromatography; LRP, LDL receptor-related protein; VLDL, very low density lipoprotein, EMSE, N-ethyl-N-(3-methylphenyl)-N'-succinylethylendiamine; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47[Medline] [Order article via Infotrieve] |
2. | Mahley, R. W., Weisgraber, K. H., Innerarity, T. L., and Rall, S. J. (1991) JAMA (J. Am. Med. Assoc.) 265, 78-83[Abstract] |
3. | Piedrahita, J. A., Zhang, S. H., Hagaman, J. R., Oliver, P. M., and Maeda, N. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4471-4475[Abstract] |
4. | Zhang, S. H., Reddick, R. L., Piedrahita, J. A., and Maeda, N. (1992) Science 258, 468-471[Medline] [Order article via Infotrieve] |
5. | Kashyap, V. S., Santamarina, F. S., Brown, D. R., Parrott, C. L., Applebaum, B. D., Meyn, S., Talley, G., Paigen, B., Maeda, N., and Brewer, H. J. (1995) J. Clin. Invest. 96, 1612-1620[Medline] [Order article via Infotrieve] |
6. | Ishibashi, S., Brown, M. S., Goldstein, J. L., Gerard, R. D., Hammer, R. E., and Herz, J. (1993) J. Clin. Invest. 92, 883-893[Medline] [Order article via Infotrieve] |
7. | Kim, D. H., Inagaki, Y., Suzuki, T., Ioka, R. X., Yoshioka, S. Z., Magoori, K., Kang, M. J., Cho, Y., Nakano, A. Z., Liu, Q., Fujino, T., Suzuki, H., Sasano, H., and Yamamoto, T. T. (1998) J Biochem. (Tokyo) 124, 1072-1076[Abstract] |
8. | Wehrli, M., Dougan, S. T., Caldwell, K., O'Keefe, L., Schwartz, S., Vaizel-Ohayon, D., Schejter, E., Tomlinson, A., and DiNardo, S. (2000) Nature 407, 527-530[CrossRef][Medline] [Order article via Infotrieve] |
9. | Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J. P., and He, X. (2000) Nature 407, 530-535[CrossRef][Medline] [Order article via Infotrieve] |
10. | Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J., and Skarnes, W. C. (2000) Nature 407, 535-538[CrossRef][Medline] [Order article via Infotrieve] |
11. | Bafico, A., Liu, G., Yaniv, A., Gazit, A., and Aaronson, S. A. (2001) Nat. Cell Biol. 3, 683-686[CrossRef][Medline] [Order article via Infotrieve] |
12. | Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., and Niehrs, C. (2001) Nature 411, 321-325[CrossRef][Medline] [Order article via Infotrieve] |
13. | Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H. R., Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L., and Wu, D. (2001) Mol. Cell 7, 801-809[CrossRef][Medline] [Order article via Infotrieve] |
14. | Nusse, R., and Varmus, H. E. (1992) Cell 69, 1073-1087[Medline] [Order article via Infotrieve] |
15. | Wodarz, A., and Nusse, R. (1998) Annu. Rev. Cell Dev. Biol. 14, 59-88[CrossRef][Medline] [Order article via Infotrieve] |
16. | Sparks, A. B., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998) Cancer Res. 58, 1130-1134[Abstract] |
17. | Bhanot, P., Brink, M., Samos, C. H., Hsieh, J. C., Wang, Y., Macke, J. P., Andrew, D., Nathans, J., and Nusse, R. (1996) Nature 382, 225-230[CrossRef][Medline] [Order article via Infotrieve] |
18. | Zorn, A. M. (2001) Curr. Biol. 11, R592-R595[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Ross, S. E.,
Hemati, N.,
Longo, K. A.,
Bennett, C. N.,
Lucas, P. C.,
Erickson, R. L.,
and MacDougald, O. A.
(2000)
Science
289,
950-953 |
20. | Gong, Y., Slee, R. B., Fukai, N., Rawadi, G., Roman-Roman, S., Reginato, A. M., Wang, H., Cundy, T., Glorieux, F. H., Lev, D., Zacharin, M., Oexle, K., Marcelino, J., Suwairi, W., Heeger, S., Sabatakos, G., Apte, S., Adkins, W. N., Allgrove, J., Arslan-Kirchner, M., Batch, J. A., Beighton, P., Black, G. C., Boles, R. G., Boon, L. M., Borrone, C., Brunner, H. G., Carle, G. F., Dallapiccola, B., De Paepe, A., Floege, B., Halfhide, M. L., Hall, B., Hennekam, R. C., Hirose, T., Jans, A., Juppner, H., Kim, C. A., Keppler-Noreuil, K., Kohlschuetter, A., LaCombe, D., Lambert, M., Lemyre, E., Letteboer, T., Peltonen, L., Ramesar, R. S., Romanengo, M., Somer, H., Steichen-Gersdorf, E., Steinmann, B., Sullivan, B., Superti-Furga, A., Swoboda, W., van den Boogaard, M. J., Van Hul, W., Vikkula, M., Votruba, M., Zabel, B., Garcia, T., Baron, R., Olsen, B. R., and Warman, M. L. (2001) Cell 107, 513-523[Medline] [Order article via Infotrieve] |
21. |
Kato, M.,
Patel, M. S.,
Levasseur, R.,
Lobov, I.,
Chang, B. H.,
Glass, D. A., 2nd,
Hartmann, C.,
Li, L.,
Hwang, T. H.,
Brayton, C. F.,
Lang, R. A.,
Karsenty, G.,
and Chan, L.
(2002)
J. Cell Biol.
157,
303-314 |
22. |
Fujino, T.,
Asaba, H.,
Kang, M. J.,
Ikeda, Y.,
Sone, H.,
Takada, S.,
Kim, D. H.,
Ioka, R. X.,
Ono, M.,
Tomoyori, H.,
Okubo, M.,
Murase, T.,
Kamataki, A.,
Yamamoto, J.,
Magoori, K.,
Takahashi, S.,
Miyamoto, Y.,
Oishi, H.,
Nose, M.,
Okazaki, M.,
Usui, S.,
Imaizumi, K.,
Yanagisawa, M.,
Sakai, J.,
and Yamamoto, T. T.
(2003)
Proc. Natl. Acad. Sci. U. S. A.
100,
229-234 |
23. | Little, R. D., Carulli, J. P., Del Mastro, R. G., Dupuis, J., Osborne, M., Folz, C., Manning, S. P., Swain, P. M., Zhao, S. C., Eustace, B., Lappe, M. M., Spitzer, L., Zweier, S., Braunschweiger, K., Benchekroun, Y., Hu, X., Adair, R., Chee, L., FitzGerald, M. G., Tulig, C., Caruso, A., Tzellas, N., Bawa, A., Franklin, B., McGuire, S., Nogues, X., Gong, G., Allen, K. M., Anisowicz, A., Morales, A. J., Lomedico, P. T., Recker, S. M., Van Eerdewegh, P., Recker, R. R., and Johnson, M. L. (2002) Am. J. Hum. Genet. 70, 11-19[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Boyden, L. M.,
Mao, J.,
Belsky, J.,
Mitzner, L.,
Farhi, A.,
Mitnick, M. A.,
Wu, D.,
Insogna, K.,
and Lifton, R. P.
(2002)
N. Engl. J. Med.
346,
1513-1521 |
25. |
Usui, S.,
Hara, Y.,
Hosaki, S.,
and Okazaki, M.
(2002)
J. Lipid Res.
43,
805-814 |
26. | Okazaki, M., Usui, S., and Hosaki, S. (2000) in Handbook of Lipoprotein Testing (Rifai, N. , Warnick, G. R. , and Dominiczak, M. H., eds), 2nd Ed. , pp. 647-669, American Association of Clinical Chemistry Press, Washington, D. C. |
27. | Ishibashi, S., Herz, J., Maeda, N., Goldstein, J. L., and Brown, M. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4431-4435[Abstract] |
28. | van Tilbeurgh, H., Bezzine, S., Cambillau, C., Verger, R., and Carrière, F. (1999) Biochim. Biophys. Acta 1441, 173-184[Medline] [Order article via Infotrieve] |