Overexpression of Human Apolipoprotein A-II in Mice Induces
Hypertriglyceridemia Due to Defective Very Low Density Lipoprotein
Hydrolysis*
Elisabeth
Boisfer
§,
Gilles
Lambert
§,
Véronique
Atger¶,
Nhuan Quang
Tran
,
Danièle
Pastier
,
Claire
Benetollo
,
Jean-François
Trottier
,
Isabelle
Beaucamps
,
Micheline
Antonucci
,
Michel
Laplaud
,
Sabine
Griglio
,
Jean
Chambaz
, and
Athina-Despina
Kalopissis
**
From
Unité 505 INSERM, Institut des Cordeliers,
15, rue de l'Ecole de Médecine, 75006 Paris, the
¶ Laboratoire de Biochimie Hôpital Broussais, 96, rue Didot,
75014 Paris, and
Unité 321 INSERM, Hôpital de la
Pitié-Salpêtrière, 83, boulevard de
l'Hôpital, 75013 Paris, France
 |
ABSTRACT |
Two lines of transgenic mice, hAIItg-
and
hAIItg-
, expressing human apolipoprotein (apo)A-II at 2 and 4 times
the normal concentration, respectively, displayed on standard chow
postprandial chylomicronemia, large quantities of very low density
lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly
reduced high density lipoprotein (HDL). Hypertriglyceridemia may result
from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent Vmax of the
reaction decreasing proportionately to human apoA-II expression. Human
apoA-II was present in appreciable amounts in the VLDL of transgenic
mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a
dose-dependent decrease in LPL and hepatic lipase
activities. In conclusion, overexpression of human apoA-II in
transgenic mice induced the proatherogenic lipoprotein profile of low
plasma HDL and postprandial hypertriglyceridemia because of decreased
VLDL catabolism by LPL.
 |
INTRODUCTION |
Low plasma HDL1 levels
are negatively correlated with the risk of atherosclerosis. Although a
number of metabolic functions of HDL have been identified, no direct
link has been established between HDL functions and its antiatherogenic
effect (1). In vitro studies have shown that apolipoprotein
(apo)A-I, the major HDL apolipoprotein, activates reverse cholesterol
transport from extrahepatic tissues to the liver (2). However,
conflicting results have been reported concerning the role of apoA-II,
the second most abundant HDL apolipoprotein (3, 4). Studies of
transgenic mice overexpressing human apoA-I and apoA-II reported that
apoA-I protected more against aortic lesions than apoA-II (3).
Furthermore, HDL from transgenic mice overexpressing mouse apoA-II lost
the ability of HDL to protect against low density lipoprotein (LDL)
oxidation (5) and was even proinflammatory (6). Deficiency of either
apoA-I (7, 8) or apoA-II (9) obtained by gene targeting technology
resulted in very low plasma HDL, showing the critical importance of
both apolipoproteins in maintaining the normal structure and metabolism
of HDL. At present, apoA-II has been linked with HDL metabolism only,
but its exact role remains to be elucidated.
Studies of transgenic mice expressing human (10-13) or murine (14, 15)
apoA-II, alone or in combination with human apoA-I, apoC-III, and
cholesterol ester transfer protein (16), have provided interesting
information. When human apoA-II was expressed at normal levels, overall
lipoprotein metabolism was not markedly modified, except for the
appearance of a smaller HDL population containing solely human apoA-II
(10). At greater expression levels of human (12) or murine (15)
apoA-II, plasma HDL was increased in mice overexpressing murine apoA-II
(14, 15) but decreased in mice overexpressing human apoA-II (12, 16).
Surprisingly, mouse apoA-II overexpression resulted in increased
atherosclerotic lesions under standard chow (14), whereas human apoA-II
overexpression increased fatty streaks only after long term feeding
with an atherogenic diet (13). Two of the above studies reported mildly
increased plasma triglyceride (TG), which was associated with the VLDL
fraction when mice expressed high levels of human (12) or murine (15) apoA-II. However, comparisons among the various studies are rendered difficult by the different nutritional status of transgenic mice, namely whether they were fasted overnight (10-15) or fed ad
libitum (16).
In the present work we were interested in studying the metabolic
effects of human apoA-II overexpression under ad libitum feeding conditions. The two transgenic lines we generated were characterized by marked hypertriglyceridemia that was proportional to
the level of human apoA-II expression (2 and 4 times the normal in
human and mouse serum, respectively). To elucidate the mechanisms underlying this drastic VLDL increase, we focused on the two possible causes for VLDL accumulation in the circulation: greater hepatic VLDL
production and/or decreased catabolism by lipoprotein lipase (LPL).
 |
EXPERIMENTAL PROCEDURES |
Generation of Transgenic Mice--
Transgenic mice were
generated by microinjection of the 3-kilobase genomic clone of the
human apoA-II gene (
911/+2045), subcloned into the HindIII
site of pUC19 (17), into one-cell embryos of (C57BL/6J × CBA/2J)
F1 female mice (IFFA-CREDO, Lyon, France). The two founder mice were
then backcrossed to strain C57BL/6J, and the majority of the transgenic
mice used in this study were obtained after four or five backcrosses.
Southern blot analysis of genomic DNA was performed using as a probe a
1,273-base pair fragment obtained by PstI digestion, which
is specific for the human apoA-II gene. Approximately 7 and 25 copies
of the human apoA-II gene were integrated, respectively, into the
genome of lines hAIItg-
and -
.
Animals--
The animals were housed in animal rooms with
alternating 12-h periods of light (7 a.m.-7 p.m.) and dark (7 p.m.-7
a.m.) and were fed a chow diet (UAR, Villemoisson-sur-Orge). The
presence of the transgene was detected by polymerase chain reaction of tail-derived DNA, and human apoA-II was also measured in plasma by
immunonephelometry using an antibody (IMMUNO-AG) specific for human
apoA-II and not recognizing mouse apoA-II. All transgenic mice were
hemizygous for the transgene and were more than 8 weeks old. Because
most studies were performed with pooled plasma, we discarded
nontransgenic littermates to exclude misidentifications and used as
controls C57BL/6J mice (IFFA-CREDO) of the same age and maintained
under the same nutritional conditions. Male and female mice were used
in equal proportions. In all experiments, mice with free access to food
and water were bled between 9 and 12 a.m., either from the
retroorbital venous plexus or from the abdominal vein.
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from several tissues using RNA Instapure kit (Eurogentech).
RNA samples (15 µg) were separated in 3% formaldehyde-containing
agarose gels and transferred to nylon membranes (Hybond N+,
Amersham Pharmacia Biotech). The membranes were hybridized to the human
apoA-II cDNA probe. The 18 S RNA probe was used as an internal
standard. Autoradiograms were scanned and analyzed with the NIH Image program.
Lipoprotein Analysis--
Blood was collected into
EDTA-containing tubes on ice and plasma supplemented with 0.005%
gentamycin, 1 mM EDTA, and 0.04% sodium azide and protease
inhibitors. Chylomicrons were prepared by ultracentrifugation at
10,000 × g for 30 min at 20 °C and washed once by
ultracentrifugation for 18 h at 100,000 × g and
10 °C to eliminate albumin. Then, sequential ultracentrifugations
were performed at 100,000 × g and 10 °C to isolate
VLDL, intermediate density lipoprotein (IDL), and LDL (for 18 h at
densities 1.006, 1.020, and 1.063 g/ml, respectively) and HDL (for
40 h at density 1.21 g/ml). LDL and HDL were dialyzed against
phosphate-buffered saline. The protein content of lipoproteins was
measured (18), and triglyceride (Biotrol A 01548), total cholesterol
(Biotrol A 01368), free cholesterol (Biotrol A 01371), and phospholipid (BioMérieux PAP 150) contents were determined with commercial kits.
Fast protein liquid chromatography was performed on 0.2 ml of pooled
plasma, using two Superose 6 columns operating in series. The elution
rate was 0.4 ml/min. 0.2-ml fractions were collected into 96-well
microplates, and the TG and total cholesterol contents were determined
in each tube.
The size distribution of HDL was analyzed by nondenaturing gradient gel
electrophoresis in 4-15% polyacrylamide gels (precast, Bio-Rad). The
HMW Calibration kit (Amersham Pharmacia Biotech) was used for
calibration of HDL particle size (19).
Apolipoprotein Analysis--
Apolipoproteins were analyzed by 5 and 15% SDS-PAGE (20) and 4-20% SDS-PAGE (precast gels, Bio-Rad),
under nonreducing conditions to detect the dimeric form of human
apoA-II. The gels were scanned, analyzed with the NIH Image program,
and the percentages of the various apolipoproteins were calculated.
The isoforms of human apoA-II and the proportions of apoC-II and
apoC-III were determined by isoelectric focusing (IEF) electrophoresis. Gels with a pH range of 3-5 containing 8 M urea
(Ultrapure, Bio-Rad), 7.5% acrylamide, 0.2% bisacrylamide, and 2%
Ampholines 3-5 (Amersham Pharmacia Biotech) were run at 4 °C, with
voltage increasing in a stepwise fashion: 200 V for 10 min, 400 V for
10 min, and 600 V for 4 h 30 min.
The presence of human apoA-II in lipoproteins from transgenic mice was
verified by Western blotting after SDS-PAGE and IEF. After protein
transfer to polyvinylidene difluoride membranes (0.2 µm, Bio-Rad),
human apoA-II was detected by a rabbit antihuman apoA-II antiserum that
does not recognize mouse apoA-II (courtesy of Dr. A. Mazur, INRA,
Theix). ApoC-II and apoC-III were detected by immunoblotting IEF gels
with rabbit antiserum directed against total mouse apoCs (Interchim).
Bands were visualized with an alkaline phosphatase substrate system
(Bio-Rad).
In Vivo TG Production--
Control and transgenic mice fed
ad libitum were studied between 10:00 a.m. and noon. An
equal number of control, hAIItg-
, and hAIItg-
mice were studied
on any given day. Mice were injected into the jugular vein with Triton
WR 1339 (500 mg/kg, as described in Ref. 21) and 5 min later, with 4 µCi of [1-14C]oleic acid complexed to albumin, to label
newly synthesized VLDL-TG (22). Four blood samples were taken from each
mouse: at 0 min (before Triton) and 30, 50, and 70 min after Triton. Plasma VLDL clearance in mice is essentially completely inhibited under
these conditions, so that the accumulation of [14C]TG in
plasma between 0 and 70 min post-Triton reflects VLDL-TG production
rate. Plasma samples were extracted for lipids (23), and lipid classes
were separated by TLC and counted (22). Total TG was determined with a
commercial kit.
LPL and Hepatic Lipase (HL) Activities in Postheparin Plasma and
Tissue Homogenates--
Heparin (Choay, France) was injected into the
jugular vein of fed transgenic and control mice (500 units/kg). Blood
was collected 5 min later into EDTA-containing tubes on ice, and plasma
was stored at
80 °C up until the assays. Lipolytic activities were assayed in triplicate using a radiolabeled triolein emulsion (24). Total and LPL activities were measured in the absence of 1 M NaCl and in the presence of human heat-inactivated serum
as a source of apoC-II. HL was measured separately on another triolein
emulsion optimal for this enzyme (24) in the presence of 1 M NaCl to inhibit LPL activity. LPL was calculated by
subtraction. One milliunit of activity is defined as the amount that
generates 1 nmol of free fatty acids/min at 37 °C. Plasma LPL and HL
activities were expressed as milliunits/ml of plasma.
LPL activity was measured directly in muscle (heart, diaphragm, and
gastrocnemian muscle) and adipose tissues (brown and interscapular), which were immediately frozen in liquid nitrogen and stored at
80 °C up until the assays. Tissues were homogenized at 4 °C in 0.223 M Tris, 0.2% sodium deoxycholate, 0.008% Nonidet
P-40, 0.25 M sucrose, 0.05% heparin, 1% albumin, pH 8.3, centrifuged at 13,000 rpm for 15 min at 4 °C, and LPL activity was
measured on supernatants as described above (24).
Inhibition of LPL and HL Activities of Postheparin Plasma from
Control Mice by Purified Human ApoA-II--
5 µl of pooled
postheparin plasma from control mice was incubated with the
LPL-specific or the HL-specific emulsions, as described above.
Postheparin plasma was preincubated at 4 °C for 30 min without
apoA-II or with the specified amounts of purified human apoA-II (Sigma).
In Vivo Labeling of VLDL-TG/in Vitro Lipolysis by Commercial
LPL--
Mice were injected in the jugular vein with 5 µCi of
[1-14C]oleic acid (ICN, Irvine, CA) complexed to albumin,
and blood was collected 60 min later. Plasma was ultracentrifuged to
isolate chylomicrons, VLDL, and IDL. Lipoproteins were analyzed by TLC (22) to ensure that 85-90% of the label was in the TG fraction. Lipoproteins were incubated at 0.03, 0.05, 0.075, 0.1, 0.2, 0.3, 0.5, 0.6, and 0.8 mM TG concentrations with 10 µg of LPL
(recombinant, Sigma; specific activity 8,600 milliunits/ml) for 20 min
in 100 mM Tris/HCl and 4% fatty acid-free albumin, pH 8.2. Blank samples without LPL were performed for each concentration, and
the difference was regarded as the hydrolyzed amount of TG. Reactions
were stopped and free fatty acids extracted as described in Ref. 24.
Rates of lipolysis under these experimental conditions were linear for all lipoprotein preparations (not shown). Apparent Michaelis constants (Km) and maximal enzyme activities
(Vmax) for LPL were calculated from
Lineweaver-Burk plots.
Statistical Analysis--
Results are given as the mean ± S.E., and differences were determined by the t test for
nonpaired samples after analysis of variance, using GraphPad Prism.
Linear regression analyses for post-Triton TG production rates were
performed using GraphPad Prism and the slopes of the regression lines
were compared by unpaired t test.
 |
RESULTS |
Expression of Human ApoA-II--
Fig.
1 illustrates Northern blot analysis of
mRNA prepared from several tissues. Expression of human apoA-II was
restricted to liver, as expected (10, 12). Despite similar relative
amounts of human apoA-II mRNA to 18 S (2.6 and 1.9 in lines
and
, respectively), the human apoA-II concentration in plasma was
56 and 116 mg/dl in mice
and
, respectively, suggesting that the
higher plasma human apoA-II in line
resulted from
post-transcriptional regulation or decreased clearance. The two
transgenic lines permitted us to study in the fed state a gradient of
human apoA-II expression corresponding to 2 and 4 times the apoA-II
concentration in normal human plasma (30 mg/dl) or in plasma of control
mice (25 mg/dl, according to Ref. 8).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Northern blot detecting human apoA-II
mRNA in tissues. Panel A, control mice;
panel B, hAIItg- mice; panel
C, hAIItg- mice; L, liver; I,
intestine; H, heart; K, kidney; M,
muscle.
|
|
Occurrence of Hypertriglyceridemia under Ad Libitum Feeding--
A
strong correlation between plasma apoA-II and TG concentrations was
found in fed transgenic mice over a wide range of values (r = 0.81; p < 0.01). To establish
whether the hypertriglyceridemia depended on nutritional status, we
measured plasma apoA-II and TG levels in the same animals fed ad
libitum or fasted for 18 h. Although hypertriglyceridemia
occurred essentially in the fed state, higher fasting plasma TG
persisted in the higher expressing hAIItg-
mice (Table
I).
View this table:
[in this window]
[in a new window]
|
Table I
Plasma concentrations of human apoA-II and triglyceride in the same
mice under ad libitum feeding or after an 18-h fast
The same six animals from each group were bled from the retroorbital
plexus at two separate occasions. For the first bleeding, animals were
fed ad libitum; the second bleeding took place 3 weeks
later, with food withdrawn for 18 h (between 16:00 p.m. and 10:00
a.m.). The statistical significance of the results was calculated by an
unpaired t test after analysis of variance.
|
|
All plasma lipids were increased in both transgenic lines,
proportionately to human apoA-II concentration (Table
II). At the same time, the molar ratio of
esterified to total cholesterol was reduced 2-3-fold.
View this table:
[in this window]
[in a new window]
|
Table II
Plasma lipids in control and human apoA-II transgenic mice.
Three distinct plasma pools (2 ml/group) were characterized for each
genotype. Results are expressed as the mean ± S.E. The
statistical significance of the results was calculated by an unpaired
t test after analysis of variance. TG, triglyceride; TC,
total cholesterol; FC, free cholesterol; PL, phospholipid; CE,
cholesterol ester.
|
|
Fast protein liquid chromatographic analysis showed very large
increases in the VLDL fractions of both TG and cholesterol and the
presence of intermediate sized particles up to the LDL fraction,
especially in the higher expressing line
(Fig.
2).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Fast protein liquid chromatographic analysis
of plasma lipoproteins. Note the different scales between the
lipoprotein profiles of transgenic and control mice. Because of the
high TG values, HDL-cholesterol appears very low in hAIItg- and
nonexistent in hAIItg- mice.
|
|
HDL Decrease in Transgenic Mice--
HDL decreased substantially
in both transgenic lines (Fig. 2). The HDL-cholesterol peak is not
apparent in hAIItg-
mice because of the large scale used due to
their high VLDL-TG values.
Lipoprotein isolation by sequential ultracentrifugation confirmed the
3-fold decrease in plasma HDL of transgenic mice (Table III). Surprisingly, this HDL had an
increased TG/protein ratio, whereas the ratios of free cholesterol,
cholesterol ester, and phospholipid to HDL-protein were 2 times lower.
The net result was a lower ratio of HDL-total lipid/HDL-protein in
transgenic mice, indicative of decreased particle size. Indeed,
nondenaturing gradient gel electrophoresis showed that HDL of our
transgenic mice comprised two populations, both smaller than control
mouse HDL (Fig. 3A), as
reported for other human apoA-II-expressing transgenic mouse lines (10,
12). Mouse apoA-I accounted for 80% of total apolipoproteins in
control HDL and drastically decreased down to 12 and 1.5% in HDL from
and
mice, respectively (Fig. 3B). The presence of
dimeric human apoA-II in HDL from transgenic mice, migrating as a
triplet, was confirmed by immunoblotting (Fig. 3C).
View this table:
[in this window]
[in a new window]
|
Table III
Lipoprotein profile in control and human apoA-II transgenic mice
Lipoproteins were isolated by sequential ultracentrifugation from the
same three plasma pools. Statistical differences are as in Table I,
between transgenic and control mice. CM, chylomicrons; ND, not
determined. Other abbreviations are as in Table II.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Electrophoretic analysis of HDL. Three
separate plasma pools from each group of mice were ultracentrifuged to
isolate HDL, and characteristic gels are shown. Panel
A, nondenaturing gradient gel electrophoresis using 4-15%
gels. Lane 1, high molecular weight standards
(Amersham Pharmacia Biotech), with the corresponding sizes of the
markers in nm; lane 2, human HDL; lanes 3-5, HDL
from control, hAIItg- , and hAIItg- mice, respectively.
Panel B, 15% SDS-PAGE of HDL apolipoproteins.
Lane 1, low molecular weight standards (Bio-Rad),
with the corresponding sizes of the markers in kDa; lane
2, human HDL; lanes 3-5, HDL from control,
hAIItg- , and hAIItg- mice, respectively. Panel
C, Western blot after 15% SDS-PAGE of HDL apolipoproteins
probed with a specific rabbit anti-human apoA-II antiserum.
Lane 1, human HDL; lanes
2-4, HDL from control, hAIItg- , and hAIItg- mice,
respectively.
|
|
Apparent Chylomicronemia--
The milky appearance of several
plasma samples from transgenic animals led us to isolate a
chylomicron-like fraction in both transgenic lines, which was absent
from control mouse plasma (Table III). Chylomicrons are synthesized in
the intestine and carry TG of dietary origin. Because all mice were fed
standard chow containing 5% lipid, very few chylomicron particles
should have been produced. Apolipoprotein analysis (Fig.
4, A and B) showed
that the chylomicron-like lipoproteins from transgenic mice carried
approximately equal amounts of apoB-48 and apoB-100, which is solely
synthesized in the liver, thus demonstrating that they were actually
very large VLDL of mainly hepatic origin. The very low cholesterol/TG
ratio also indicates that these large VLDL were little catabolized.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Electrophoretic analysis of lipoproteins
isolated by sequential ultracentrifugation. Panel
A, 5% SDS-PAGE. Lane 1, chylomicrons
of mice; lanes 2 and 3, VLDL from
control and mice, respectively; lane 4, LDL
from mice; lane 5, human LDL.
Panel B, 4-20% SDS-PAGE, with constant protein
amounts loaded on the gels to compare relative apolipoprotein contents
between transgenic and control mice. Lane 1, low
molecular weight standards (Bio-Rad), with the corresponding sizes of
the markers in kDa; lane 2, chylomicrons from mice; lanes 3 and 4, VLDL from control
and mice, respectively; lane 5, human VLDL;
lanes 6 and 7, HDL from control and
mice, respectively; lane 8, human HDL.
Panel C, Western blot after 15% SDS-PAGE of VLDL
and LDL apolipoproteins probed with a specific rabbit anti-human
apoA-II antiserum. Lanes 1 and 2, VLDL
from and mice, respectively; lanes 3 and
4, LDL from and mice, respectively; lane
5, purified human apoA-II (Sigma). Apolipoproteins from
VLDL, LDL, and HDL from control mice did not react with anti-human
apoA-II antiserum (not shown).
|
|
Large amounts of human dimeric apoA-II were present in all
apoB-containing lipoproteins of transgenic mice (as confirmed by immunoblotting in Fig. 4C), whereas apoA-II was absent from
VLDL of control mice (Fig. 4B). All lipoproteins from
mice transported consistently more apoA-II than those from
mice.
Regarding apolipoprotein composition, control VLDL transported 48%
apoBs, 30% apoE and 22% apoCs. VLDL from
mice comprised 45%
apoBs, 11% apoE, 33% human apoA-II, and 11% apoCs. On the other
hand, more apoB-48 than apoB-100 was present in VLDL from transgenic
compared with control mice (ratios of apoB-48/B-100 of 1.7 and 0.9, respectively).
The concomitant increase of all VLDL and LDL constituents in transgenic
mice (Table III) indicated an increased particle number, whereas their
greater TG/protein ratios, suggestive of larger size, further suggested
accumulation caused by decreased lipolysis. However, the ratio of
cholesterol to TG decreased in VLDL of
mice only, suggesting that
decreased lipolysis may not be the only process leading to VLDL accumulation.
Influence of Human ApoA-II Overexpression on VLDL
Production--
VLDL production was assessed in vivo after
Triton WR 1339 injection, which blocks VLDL-TG hydrolysis by LPL (21).
Thus, VLDL-TG accumulation in plasma after Triton serves as a measure of VLDL production rate. Total plasma TG of hAIItg-
and -
mice was already dramatically elevated before Triton injection, and the
increase after Triton did not allow adequate estimation of total
VLDL-TG secretion (Fig. 5), except in
control mice (2.40 mg TG/ml/h). Therefore, mice were injected with
[1-14C]oleic acid to label newly synthesized TG in the
liver, which is partly incorporated into VLDL-TG and exported into the
circulation from 20 min onward (22). This method allows estimation of
secretion rates of newly formed VLDL, the majority of which originates
from the liver under standard, high carbohydrate feeding (25). We thus
showed that production of [14C]TG was similar in
transgenic and control mice.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Post-Triton VLDL-TG production in fed
transgenic and control mice. Blood was drawn at 0 min (before
Triton WR 1339) and 30, 50, and 70 min after Triton WR 1339 injection
(500 mg/kg of body weight). [1-14C]Oleic acid was
injected 5 min after Triton. Linear regression analysis showed that
total TG production was linear in control mice only, whereas
14C-TG production was linear in all groups.
Panel A, total TG production (mg/ml plasma);
panel B, [14C]TG production (cpm/ml
of plasma). , control mice; , mice; , mice.
|
|
LPL and HL Activities--
LPL and HL activities measured in
postheparin plasma using an emulsion-based assay were decreased by at
most 30% in transgenic mice (Table IV).
Because LPL activity in postheparin plasma is assessed indirectly from
the difference between total lipase and HL activities, we measured LPL
activity in homogenates directly from five tissues in which LPL is most
abundant and HL is absent. Table IV shows that muscle and adipose
tissue LPL activities were very comparable with those of control mice,
and it is usually assumed that tissue LPL activity is proportional to
the enzyme mass synthesized in tissues. Thus, transgenic mice probably
synthesized LPL at normal rates.
View this table:
[in this window]
[in a new window]
|
Table IV
Enzyme activities of LPL and HL in postheparin plasma and tissues of
control and human apoA-II transgenic mice
Values are mean ± S.E. Numbers in parentheses indicate number of
mice per group. Statistical analysis is as in Table I.
|
|
In Vitro VLDL Hydrolysis by Exogenous LPL--
To account for the
hypertriglyceridemia of transgenic mice, we hypothesized that their
VLDL might not be a good substrate for LPL. This hypothesis was tested
by incubating in vivo labeled TG-rich lipoproteins of
transgenic and control mice with commercial LPL. In control mice,
chylomicrons were absent, and 90% of the injected 14C
label was in the VLDL fraction. In transgenic mice, approximately 8%
of the label was present in the chylomicron-like fraction, 72% in
VLDL, and 20% in IDL.
Hydrolysis of chylomicron-TG and VLDL-TG of hAIItg-
and -
mice
was considerably slower than hydrolysis of control mouse VLDL-TG (Table
V). IDL of transgenic mice gave results
similar to those obtained with chylomicrons and VLDL (not shown). No
significant differences in apparent Km were
observed, whereas the apparent Vmax of the
reaction was decreased 2 and 4 times in lines hAIItg-
and -
,
respectively, proportionately to human apoA-II expression.
View this table:
[in this window]
[in a new window]
|
Table V
Apparent kinetic parameters of triglyceride-rich lipoproteins for
LPL-mediated lipolysis in vitro
In vivo labeled TG-rich lipoproteins were tested as
lipolytic substrates of exogenous LPL. Three distinct plasma pools from
each group of mice were used for lipoprotein preparation. Numbers in
parentheses represent different incubations of each lipoprotein
fraction with exogenous LPL. Apparent Michaelis constants
(Km) and maximal enzyme activities
(Vmax) were calculated from Lineweaver-Burk plots.
Statistical analysis was as in Table I. FFA, free fatty acids.
|
|
Human ApoA-II Isoforms and ApoC Content of VLDL--
The decreased
catabolism of VLDL from transgenic mice by an exogenous LPL led us to
analyze their apolipoprotein composition with respect to human apoA-II
and mouse apoCs, because LPL activity necessitates the presence of
apoC-II on VLDL.
IEF electrophoresis followed by immunoblotting revealed three major
isoforms of human apoA-II in VLDL and HDL of transgenic mice, the more
basic isoform corresponding to apoA-II of human HDL (Fig.
6). These isoforms were also reported in
humans and were designated apoA-II-2 to 4, apoA-II-2 being the major
one (26). On the other hand, clear bands of apoC-II and C-III were present in VLDL from transgenic mice, whereas apoC-II was almost undetectable in VLDL from control mice (Fig. 6B, lanes
5-10).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 6.
Identification of human apoA-II isoforms and
mouse apoCs in VLDL and HDL from transgenic mice by IEF followed by
immunoblotting. At least four different lipoprotein preparations
from control and transgenic mice were used, and characteristic gels are
shown. Human VLDL and HDL apolipoproteins were run in parallel to spot
the positions of mouse apoC-II and apoC-III relative to the isoforms of
human apoA-II. Panel A, IEF separation of VLDL
and HDL apolipoproteins (50 µg/lane) using a pH gradient from 5 (top) to 3 (bottom). Lanes
1 and 2, VLDL from control and mice,
respectively; lanes 3 and 4, human
VLDL and HDL; lanes 5-7, HDL from control, ,
and mice, respectively; lane 8, purified
human apoA-II. In lane 3, human apoCs are, from
top to bottom: C-III0, C-II,
C-III1, and C-III2. Panel
B, Western blot after IEF electrophoresis using a 3-5 pH
gradient. Lanes 1-4, immunoblotting with
anti-human apoA-II antiserum; lanes 5-10,
immunoblotting with antiserum directed against total mouse apoCs;
lane 1, purified human apoA-II; lane
2, VLDL from mice; lanes 3 and
4, HDL from and mice, respectively; lanes
5-7, VLDL from control, , and mice, respectively;
lanes 8-10, HDL from control, , and mice,
respectively. Note that the more acidic apoA-II-3 and 4 isoforms were
present in greater amounts in our transgenic mice (lanes 2 to 4 in panel B) than in humans (26). Mouse
apoC-II was consistently found between human apoA-II-2 and 3 isoforms
(in accord with Ref. 26) and was slightly more basic than human
apoC-II. ApoC-III-2 was the only form observed in mice and was slightly
more acidic than its human counterpart and than human apoA-II-4
(26).
|
|
Human ApoA-II Inhibits LPL and HL Activities in Vitro--
The
observation that postheparin LPL activity was significantly lower in
transgenic compared with control mice, whereas tissue LPL activities
were very similar, suggested that apoA-II transported in VLDL in
postheparin plasma of transgenic mice may have interfered with LPL
activity in the enzymatic assay. We therefore measured lipolytic
activity of pooled postheparin plasma from control mice in the absence
or presence of increasing amounts of purified human apoA-II, up to the
concentration of human apoA-II in the plasma of hAIItg-
mice. The
addition of increasing apoA-II amounts resulted in concomitant
inhibition of LPL activity, down to 21% of controls in the presence of
7.5 µg of apoA-II in the assay medium (Fig. 7). HL activity likewise decreased as a
function of human apoA-II addition into the assay medium, down to 23%
of controls.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibition of LPL and HL activities by human
apoA-II. Postheparin plasma from control mice was incubated with
the LPL-specific or the HL-specific emulsion as described under
"Experimental Procedures." Lipase activities were expressed as a
percent of those obtained without the addition of apoA-II.
|
|
ApoA-II may inhibit LPL activity either directly, or indirectly by
altering the apolipoprotein composition of the lipoproteins present in
postheparin plasma, in turn changing the activator properties of the
emulsion. A direct inhibitory effect of apoA-II on LPL can only be
shown using purified components (apoC-II/LPL/HL).
 |
DISCUSSION |
Hypertriglyceridemia is a common metabolic disorder caused by an
array of nutritional and genetic factors, most of which are not
identified clearly. In this paper we present an unusual mouse model of
hypertriglyceridemia resulting solely from overexpression of human
apoA-II, which is normally an apolipoprotein component of HDL.
Hypertriglyceridemia was proportional to human apoA-II expression in
two transgenic lines fed standard chow diet and occurred concomitantly
with a decrease in HDL. Excess apoA-II was carried mostly in VLDL of
transgenic mice and hindered the action of LPL so that VLDL accumulated
in plasma for several hours.
The hypertriglyceridemia of transgenic mice was very marked under
ad libitum feeding and was abolished after an overnight fast
in line
but persisted at a lower level in the higher expressing line
. The plasma apoA-II concentration was lowered concomitantly by
fasting. The near normalization of plasma TG after fasting indicates
that VLDL in our transgenic animals has a longer residence time in
plasma but is ultimately catabolized and the remnants taken up by
tissues. This is supported further by the absence of overt
hypercholesterolemia, which is the hallmark of defective lipoprotein
uptake resulting from apoE (27, 28) or LDL receptor deficiency
(29).
In our study, an increase in plasma apoA-II was consistently
accompanied by proportional increases in the TG and apoA-II content of
VLDL. A careful survey of the literature shows slight increases in
plasma TG proportionately to apoA-II expression in other transgenic lines that also exhibited increased VLDL and LDL levels, even after an
overnight fast (12, 15). However, apoA-II has never been detected in
the VLDL fraction of these transgenic mice.
Conversely, apoA-II overexpression resulted in markedly decreased
plasma HDL, which contained little apoA-I, in accord with the study by
Marzal-Casacuberta et al. (12). These authors attributed the
HDL decrease to an apoA-I displacement by human apoA-II, as shown in
Ref. 30. In our study, the very low apoA-I content in HDL from line
and the near absence of apoA-I in HDL from line
also suggest a
dose-dependent displacement of mouse apoA-I from the
surface of HDL by human apoA-II. The present study suggests a second
mechanism whereby the decreased availability of VLDL surface components
caused by inhibition of LPL activity may result in reduced HDL
formation in the circulation, as proposed originally by Tall and Small
(31). Indeed, plasma LPL activity correlates with HDL-cholesterol
levels in humans.
The presence of chylomicron-like lipoproteins in transgenic, but not in
control mice, was unexpected because all animals were fed a standard
chow diet containing only 5% lipid so that very few chylomicrons would
be produced by the intestine. Analysis of the apoB content of these
lipoproteins clearly showed the presence of apoB-100 as well as
apoB-48. Because apoB-100 is exclusively synthesized in the liver (32),
the chylomicron-like fraction corresponded to very large hepatic VLDL.
The mainly hepatic origin of VLDL under high carbohydrate feeding has
long been known from experiments in rats (25).
The large increase in the circulation of VLDL and LDL may have been
caused either by VLDL overproduction or by defective VLDL hydrolysis by
LPL, or both. Post-Triton VLDL secretion was similar in control and
transgenic mice, arguing against VLDL overproduction. Anyway, the
presence of chylomicron-like particles in transgenic mice was a strong
argument for defective VLDL-TG hydrolysis by LPL because large TG-rich
lipoproteins have a very short half-life in plasma, and chylomicronemia
results from congenital LPL deficiency in humans (33). The modest 30%
decrease in postheparin LPL activity in transgenic mice relative to
controls could not account for the apparent chylomicronemia. On the
contrary, the drastically decreased hydrolysis of VLDL-TG from
transgenic mice by exogenous LPL, strongly suggesting that this VLDL
was not a good substrate for LPL, may explain their accumulation in a
near native form.
Defective VLDL hydrolysis by LPL may be caused by the absence of
apoC-II, the obligatory cofactor of the enzyme (34). Because apoC-II is
less hydrophobic than apoA-II it may have been displaced from the
surface of VLDL by apoA-II (35). However, the presence of apoC-II in
VLDL from transgenic mice in greater amounts than in control VLDL
should have been sufficient for LPL activity because very little
apoC-II is needed for LPL activation (34).
We therefore tested the hypothesis of whether the presence of apoA-II
on the surface of VLDL was responsible for the defective VLDL
catabolism by LPL. We demonstrated for the first time, to our
knowledge, a dose-dependent inhibition of postheparin LPL activity by apoA-II. Whether apoA-II exerts a direct inhibitory effect
on LPL activity could not be established in this study because of the
presence of lipoproteins in the postheparin plasma used in our LPL
assay. Only an assay using purified components could address the
intrinsic inhibitory activities of apoA-II. Thus, the mechanism of LPL
inhibition by apoA-II remains at present unclear. By analogy to HL
(36), apoA-II carried on VLDL may saturate the surface of the particle
and impede binding of LPL to VLDL and thus TG hydrolysis. Such a
mechanism would be consistent with the decrease in
Vmax measured for LPL assayed with VLDL of transgenic mice.
The addition of apoA-II in postheparin plasma from control mice also
inhibited HL activity in a dose-dependent manner.
Inhibition of HL by apoA-II has been reported in several studies
in vitro (37, 38) and in vivo (8, 16), although
in vitro activation of HL by apoA-II has also been measured
(39). Two observations in the present study indicate that HL was
inhibited in our transgenic mice in vivo. First, the marked
TG enrichment of their HDL was proportional to human apoA-II
expression. Second, hypertriglyceridemia in fasted
mice persisted,
which may have been the result of partial HL inhibition because HL
hydrolyzes TG and phospholipid of chylomicron and VLDL remnants and
HDL.
Concerning remnant TG hydrolysis, the respective roles of HL and LPL
remain to be determined. Contrary to humans with functional HL
deficiencies, mice lacking HL through homologous recombination only
displayed increased HDL, whereas remnant TG clearance was affected only
after a massive oral fat load (40). On the other hand, adult mouse
liver has a significant amount of LPL activity, in contrast to humans
and rats (41), suggesting that mice under normal conditions may depend
more on LPL for facilitating clearance of TG-rich particles by the
liver than do humans and rats.
The following metabolic cascade is proposed to explain the lipoprotein
profile of fed transgenic mice overexpressing human apoA-II. Human
apoA-II produced at high levels in liver becomes associated in the
circulation with HDL and newly secreted VLDL. ApoA-II on the VLDL
surface hinders hydrolysis of VLDL-TG by LPL, the first obligatory step
of VLDL catabolism, thereby inducing accumulation of very large VLDL
with flotation characteristics of chylomicrons. At some point, apoA-II
progressively leaves the surface of VLDL, either to associate with HDL
or to be cleared (the site of apoA-II catabolism being unknown, to our
knowledge). This allows partial hydrolysis of VLDL-TG by LPL, and then
VLDL remnants can be further processed by HL, which cannot hydrolyse intact TG-rich lipoproteins. Furthermore, apoA-II may also reduce HL
activity, partly explaining the longer residence time of remnants in
the higher expressing
mice, the persistence of mild
hypertriglyceridemia after an overnight fast, and the TG enrichment of
HDL. The drastic decrease in HDL may result from diminished formation
in plasma caused by reduced availability of surface components after
the inhibition of VLDL catabolism by LPL and/or from increased
fractional catabolic rate after apoA-I displacement by human
apoA-II.
Most of the previous transgenic models of hypertriglyceridemia were
characterized by a decreased uptake of remnants of TG-rich lipoproteins, highlighting the role of the balance of apoCs to apoE in
the remnant particle for efficient clearance (42-45). To our
knowledge, the only mouse model that fits in an inverse fashion our
hypertriglyceridemic mice overexpressing human apoA-II is the apoA-II
knockout model. When these mice were crossed with apoE knockout mice,
which display dramatic accumulation of cholesterol-rich remnants, VLDL
clearance was increased greatly (9). Thus, when apoA-II was absent,
both LPL and HL apparently functioned at maximal activities.
Our model of hypertriglyceridemia caused by overexpression of human
apoA-II presents the potentially proatherogenic association of
postprandial hypertriglyceridemia and low plasma HDL. It has similarities with certain lipoprotein disorders in humans, such as
Tangier disease and type V hyperlipoproteinemia, where apoA-II was also
detected on VLDL subpopulations, and these VLDL were a bad substrate
for LPL (46, 47). Furthermore, a significant association between
elevated levels of apoA-II-containing VLDL and the progression of
coronary artery disease was shown in lovastatin-treated hypercholesterolemic subjects (48), whereas no correlation was found
with the severity of atherosclerotic lesions in another study (49). The
increased levels of apoA-II-transporting VLDL may explain the moderate
hypertriglyceridemia of carriers of the variant
apoA-IMilano, who present nevertheless no increased risk for atherosclerosis (50). At present, the inverse correlation between
risk for coronary heart disease and plasma HDL concentrations has been
clearly established by epidemiological studies, whereas the underlying
mechanism(s) remain(s) to be determined. It is debated whether low HDL
levels may act as markers for other atherogenic influence such as
hypertriglyceridemia (1). In this respect, HDL-cholesterol levels are
more highly correlated with postprandial than with fasting TG
concentrations. Further studies in our hypertriglyceridemic mice may
provide insight into the complex relationship between plasma TG and HDL concentrations.
 |
ACKNOWLEDGEMENTS |
We thank Nelly Poulain, Mai N'Guyen, and
Carole Lasnes for excellent technical assistance.
 |
FOOTNOTES |
*
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: Unité 505 INSERM,
Institut des Cordeliers, 15, rue de l'Ecole de Médecine, 75006 Paris, France. Tel.: 33-1-4234-6899; FAX: 33-1-4325-1615.
 |
ABBREVIATIONS |
The abbreviations used are:
HDL, high density
lipoprotein;
apo, apolipoprotein;
LDL, low density lipoprotein;
TG, triglyceride;
LPL, lipoprotein lipase;
hAIItg, human apolipoprotein
A-II transgenic;
IDL, intermediate density lipoprotein;
PAGE, polyacrylamide gel electrophoresis;
IEF, isoelectric focusing;
HL, hepatic lipase.
 |
REFERENCES |
-
Vega, G. L.,
and Grundy, S. M.
(1996)
Curr. Opin. Lipidol.
7,
209-216[Medline]
[Order article via Infotrieve]
-
Fielding, C. J.,
and Fielding, P. E.
(1995)
J. Lipid Res.
36,
211-228[Abstract]
-
Schultz, J. R.,
and Rubin, E. M.
(1994)
Curr. Opin. Lipidol.
5,
126-137[Medline]
[Order article via Infotrieve]
-
Rader, D. J.,
and Ikewaki, K.
(1996)
Curr. Opin. Lipidol.
7,
117-123[Medline]
[Order article via Infotrieve]
-
Banka, C. L.
(1996)
Curr. Opin. Lipidol.
7,
139-142[Medline]
[Order article via Infotrieve]
-
Castellani, L. W.,
Navab, M.,
Van Lenten, B. J.,
Hedrick, C. C.,
Hama, S. Y.,
Goto, A. M.,
Fogelman, A. M.,
and Lusis, A. J.
(1997)
J. Clin. Invest.
100,
464-474[Abstract/Free Full Text]
-
Williamson, R.,
Lee, D.,
Hagaman, J.,
and Maeda, N.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7134-7138[Abstract]
-
Plump, A. S.,
Azrolan, N.,
Odaka, H.,
Wu, L.,
Jiang, X.,
Tall, A.,
Eisenberg, S.,
and Breslow, J. L.
(1997)
J. Lipid Res.
38,
1033-1047[Abstract]
-
Weng, W.,
and Breslow, J. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14788-14794[Abstract/Free Full Text]
-
Schultz, J. R.,
Gong, E. L.,
McCall, M. R.,
Nichols, A. V.,
Clift, S. M.,
and Rubin, E. M.
(1992)
J. Biol. Chem.
267,
21630-21636[Abstract/Free Full Text]
-
Schultz, J. R.,
Verstuyft, J. G.,
Gong, E. L.,
Nichols, A. V.,
and Rubin, E. M.
(1993)
Nature
365,
762-764[CrossRef][Medline]
[Order article via Infotrieve]
-
Marzal-Casacuberta, A.,
Blanco-Vaca, F.,
Ishida, B. Y.,
Julve-Gil, J.,
Shen, J.,
Calvet-Marquez, S.,
Gonzalez-Sastre, F.,
and Chan, L.
(1996)
J. Biol. Chem.
271,
6720-6728[Abstract/Free Full Text]
-
Escolà-Gil, J. C.,
Marzal-Casacuberta, A.,
Julve-Gil, J.,
Ishida, B. Y.,
Ordonez-Llanos, J.,
Chan, L.,
Gonzalez-Sastre, F.,
and Blanco-Vaca, F.
(1998)
J. Lipid Res.
39,
457-462[Abstract/Free Full Text]
-
Warden, C. H.,
Hedrick, C. C.,
Qiao, J.-H.,
Castellani, L. W.,
and Lusis, A. J.
(1993)
Science
261,
469-472[Medline]
[Order article via Infotrieve]
-
Hedrick, C. C.,
Castellani, L. W.,
Warden, C. H.,
Puppione, D. L.,
and Lusis, A. J.
(1993)
J. Biol. Chem.
268,
20676-20682[Abstract/Free Full Text]
-
Zhong, S.,
Goldberg, I. J.,
Bruce, C.,
Rubin, E. M.,
Breslow, J. L.,
and Tall, A.
(1994)
J. Clin. Invest.
94,
2457-2467[Medline]
[Order article via Infotrieve]
-
Lopez, J.,
Roghani, A.,
Bertrand, J.,
Zanni, E.,
Kalopissis, A.,
Zannis, V. I.,
and Chambaz, J.
(1994)
Biochemistry
33,
4056-4064[Medline]
[Order article via Infotrieve]
-
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275[Free Full Text]
-
Nichols, A. V.,
Kraus, R. M.,
and Musliner, T. A.
(1986)
Methods Enzymol.
128,
417-431[Medline]
[Order article via Infotrieve]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Otway, S.,
and Robinson, D. S.
(1967)
J. Physiol.
190,
321-332[Medline]
[Order article via Infotrieve]
-
Francone, O. L.,
Kalopissis, A. D.,
and Griffaton, G.
(1989)
Biochim. Biophys. Acta
1002,
28-36[Medline]
[Order article via Infotrieve]
-
Folch, J.,
Lees, M.,
and Sloane-Stanley, G. H.
(1957)
J. Biol. Chem.
226,
497-509[Free Full Text]
-
Nilsson-Ehle, P.,
and Ekman, R.
(1977)
Artery
3,
194-209
-
Kalopissis, A. D.,
Griglio, S.,
and Le Liepvre, X.
(1982)
Biochim. Biophys. Acta
711,
33-39[Medline]
[Order article via Infotrieve]
-
Schmitz, G.,
Ilsemann, K.,
Melnik, B.,
and Assmann, G.
(1983)
J. Lipid Res.
24,
1021-1029[Abstract]
-
Plump, A. S.,
Smith, J. D.,
Hayek, T.,
Aalto-Setälä, K.,
Walsh, A.,
Verstuyft, J. G.,
Rubin, E. M.,
and Breslow, J. L.
(1992)
Cell
71,
343-353[Medline]
[Order article via Infotrieve]
-
Zhang, S. H.,
Reddick, R. L.,
Piedrahita, J. A.,
and Maeda, N.
(1992)
Science
258,
468-471[Medline]
[Order article via Infotrieve]
-
Ishibishi, 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]
-
Lagocki, P. A.,
and Scanu, A. M.
(1980)
J. Biol. Chem.
255,
3701-3706[Free Full Text]
-
Tall, A. R.,
and Small, D. M.
(1978)
N. Engl. J. Med.
299,
1232-1236[Medline]
[Order article via Infotrieve]
-
Wu, A.-L.,
and Windmueller, H. G.
(1981)
J. Biol. Chem.
256,
3615-3618[Abstract/Free Full Text]
-
Benlian, P.,
de Gennes, J. L.,
Foubert, L.,
Zhang, H.,
Gagné, S. E.,
and Hayden, M.
(1996)
N. Engl. J. Med.
335,
848-854[Abstract/Free Full Text]
-
Wang, C.-S.,
Hartsuck, J.,
and McConathy, W. J.
(1992)
Biochim. Biophys. Acta
1123,
1-17[Medline]
[Order article via Infotrieve]
-
Brasseur, R.,
Lins, L.,
Vanloo, B.,
Ruysschaert, J.-M.,
and Rosseneu, M.
(1992)
Proteins
13,
246-257[Medline]
[Order article via Infotrieve]
-
Kubo, M.,
Matsuzawa, Y.,
Yokoyama, S.,
Tajima, S.,
Ishikawa, K.,
Yamamoto, A.,
and Tarui, S.
(1982)
J. Biochem.
92,
865-870[Abstract]
-
Thuren, T.,
Wilcox, R. W.,
Sisson, P.,
and Waite, M.
(1991)
J. Biol. Chem.
266,
4853-4861[Abstract/Free Full Text]
-
Shinomiya, M.,
Sasaki, N.,
Barnhart, R. L.,
Shirai, K.,
and Jackson, R. L.
(1982)
Biochim. Biophys. Acta
713,
292-299[Medline]
[Order article via Infotrieve]
-
Mowri, H.-O.,
Patsch, J. R.,
Gotto, A. M., Jr.,
and Patsch, W.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
755-762[Abstract/Free Full Text]
-
Homanics, G. E.,
de Silva, H.,
Osada, J.,
Zhang, S. H.,
Wong, H.,
Borensztajn, J.,
and Maeda, N.
(1995)
J. Biol. Chem.
270,
2974-2980[Abstract/Free Full Text]
-
Peterson, J.,
Bengstsson-Olivecrona, G.,
and Olivecrona, T.
(1986)
Biochim. Biophys. Acta
878,
65-70[Medline]
[Order article via Infotrieve]
-
Shachter, N. S.,
Ebara, T.,
Ramakrishnan, R.,
Steiner, G.,
Breslow, J. L.,
Ginsberg, H. N.,
and Smith, J. D.
(1996)
J. Clin. Invest.
98,
846-855[Abstract/Free Full Text]
-
Shachter, N. S.,
Hayek, T.,
Leff, T.,
Smith, J. D.,
Rosenberg, D. W.,
Walsh, A.,
Ramakrishnan, R.,
Goldberg, I. J.,
Ginsberg, H. N.,
and Breslow, J. L.
(1994)
J. Clin. Invest.
93,
1683-1690[Medline]
[Order article via Infotrieve]
-
Aalto-Setälä, K.,
Fisher, E. A.,
Chen, X.,
Chajek-Shaul, T.,
Hayek, T.,
Zechner, R.,
Walsh, A.,
Ramakrishnan, R.,
Ginsberg, H. N.,
and Breslow, J. L.
(1992)
J. Clin. Invest.
90,
1889-1900[Medline]
[Order article via Infotrieve]
-
Ebara, T.,
Ramakrishnan, R.,
Steiner, G.,
and Shachter, N. S.
(1997)
J. Clin. Invest.
99,
2672-2681[Abstract/Free Full Text]
-
Wang, C.-S.,
Alaupovic, P.,
Gregg, R. E.,
and Brewer, H. B., Jr.
(1987)
Biochim. Biophys. Acta
920,
9-19[Medline]
[Order article via Infotrieve]
-
Alaupovic, P.,
Knight-Gibson, C.,
Wang, C.-S.,
Downs, D.,
Koren, E.,
Brewer, H. B., Jr.,
and Gregg, R. E.
(1991)
J. Lipid Res.
32,
9-19[Abstract]
-
Alaupovic, P.,
Mack, W. J.,
Knight-Gibson, C.,
and Hodis, H. N.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
715-722[Abstract/Free Full Text]
-
Koren, E.,
Corder, C.,
Mueller, G.,
Centurion, H.,
Hallum, G.,
Fesmire, J.,
McConathy, W. D.,
and Alaupovic, P.
(1996)
Atherosclerosis
122,
105-115[CrossRef][Medline]
[Order article via Infotrieve]
-
Bekaert, E. D.,
Alaupovic, P.,
Knight-Gibson, C. S.,
Franceschini, G.,
and Sirtori, C. R.
(1993)
J. Lipid Res.
34,
111-123[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.