Human placenta secretes apolipoprotein B-100-containing lipoproteins
Eva M. Madsen1, Marie L.S. Lindegaard1, Claus B. Andersen2, Peter Damm3, Lars B.
Nielsen1
Departments of 1Clinical Biochemistry and 2Pathology and 3Clinic of Obstetrics,
Rigshospitalet, University of Copenhagen, Denmark
Running title: Lipoprotein secretion by placenta
Correspondence:
Lars B. Nielsen, MD, PhD, DMSc
Department of Clinical Biochemistry KB3011
Rigshospitalet, University of Copenhagen
Blegdamsvej 9, DK-2100 Copenhagen, Denmark
Ph: +45 3545 3011 FAX: +45 3545 2524 E-mail: [email protected]
JBC Papers in Press. Published on October 25, 2004 as Manuscript M411404200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Supply of lipids from the mother is essential for fetal growth and
development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-
containing lipoproteins results in embryonic lethality. In humans, the yolk sac is
vestigial. Nutritional functions are instead established very early during pregnancy in
the placenta. To examine if the human placenta produces lipoproteins, we examined
apoB and microsomal triglyceride transfer protein (MTP) mRNA expression in
placental biopsies. ApoB and MTP are mandatory for assembly and secretion of apoB-
containing lipoproteins. Both genes were expressed in placenta and microsomal extracts
from human placenta contained triglyceride transfer activity, indicating expression of
bioactive MTP. In order to detect lipoprotein secretion, biopsies from term placentas
were placed in medium with [35S]-methionine and -cysteine for 3-24 hours. Upon
sucrose gradient ultracentrifugation of the labeled medium, fractions were analysed by
apoB-immunoprecipitation. [35S]-labeled apoB-100 was recovered in d ~1.02-1.04 g/mL
particles (i.e., similar to the density of plasma low density lipoproteins). Electron
microscopy of negatively stained lipoproteins secreted from placental tissue showed
spherical particles with a diameter of 47 ± 10 nm. These results demonstrate that
human placenta expresses both apoB and MTP and consequently synthesize and secrete
apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel
pathway of lipid transfer from the mother to the developing fetus.
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INTRODUCTION
Lipids from the maternal circulation are vital to sustain fetal growth and brain
development. In addition to essential fatty acids, fetal development is dependent on
cholesterol, glycolipids, and lipid soluble vitamins. Although fatty acids can passively diffuse
across the placental barrier (1), specific mechanisms for delivery of other lipids including
cholesterol remains to be determined (1). Elucidation of the molecular machinery involved in
lipid transport to the fetus will potentially help understand states of fetal overgrowth (e.g. in
maternal diabetes) or intrauterine growth restriction.
The most efficient system for delivery of lipids from a tissue to the blood is the
formation and secretion of apolipoprotein B (apoB)1-containing lipoproteins. The apoB-
containing lipoproteins can contain large amounts of cholesterol and triglycerides and also
serve as carrier of essential lipids such as lipophilic vitamins and glycolipids (2-4). The
importance of apoB-containing lipoprotein secretion is evident in the liver and intestine where
both apoB and microsomal triglyceride transfer protein (MTP) are needed to export large
amounts of lipids for usage in peripheral tissues (2;5;6).
In rodents, the yolk sac plays an essential role in lipid transport to the fetus
during the major part of pregnancy. The yolk sac of mice and rats express the apoB gene
(7;8) and synthesize apoB-containing lipoproteins (9;10). The importance of lipoprotein
secretion from the yolk sac in mice is emphasized by the observation that both apoB and MTP
knockout mice die in utero (11;12). In humans, apoB is also expressed the yolk sac (13).
However, the human yolk sac is a rudimentary organ and looses its nutritional functions early
in embryogenesis (14). Instead, the substrate transfer from the maternal to the fetal
circulation occurs in placenta. We speculated that even though rodent placentas only express
very low levels of apoB and MTP (7;8), the transfer of lipids from mother to fetus in humans 1 ApoB, apolipoproteinB; BSA, bovine serum albumin; FBS, fetal bovine serum; HDL, high density lipoprotein; LDL, low density lipoprotein; MTP, microsomal triglyceride transfer protein; PCR, polymerase chain reaction; VLDL, very low density lipoprotein.
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could be mediated, at least in part, by apoB-containing lipoprotein secretion from the
placenta. This hypothesis was addressed by characterizing human term placental biopsies
with respect to mRNA and protein expression of MTP and apoB and secretion of newly
formed apoB-containing lipoproteins.
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EXPERIMENTAL PROCEDURES
Tissues and cells
Biopsies from human term placenta (~0.3 g) were obtained after elective
cesarean section in healthy women with a non-pathological pregnancy. Biopsies were placed
in medium on ice (for metabolic labeling studies and electron microscopy) or frozen in liquid
N2 (for mRNA and MTP activity studies) within 7-15 minutes after childbirth. All mothers
gave informed, written consent and the study protocol was approved by the local ethics
committee (KF 01-048/01). Mouse hearts were taken from three 6-month old male C57Bl/6
mice (M&B, Ry, Denmark) that had been housed at the Panum Institute, University of
Copenhagen and fed standard laboratory chow (Altromin no. 1314, Rugaarden, Denmark).
HepG2-cells were grown in a humidified atmosphere of 90 % air and 10 % CO2
at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax-I, 4500 mg/L
glucose, and pyridoxine and without sodium pyruvate (61965-026, Gibco, Invitrogen)
supplemented with 10 % fetal bovine serum (FBS) (10106-169, Gibco, Invitrogen) and 1 %
penicillin/streptomycin (15140-122, Gibco, Invitrogen). The cells were split 1:4 or 1:5 twice
a week. Labeling experiments were carried out with subconfluent monolayered HepG2 cells
in 6-well Multi-dishes (9.6 cm2/well) (150229, Nunc, Roskilde, Denmark).
mRNA expression
Total RNA was isolated from human placental biopsies and HepG2-cells with
Trizol (15596-026, Gibco, Invitrogen) and used for cDNA synthesis and quantitative real-
time PCR analysis of apoB and MTP mRNA expression with a Lightcycler (Roche A/S,
Hvidovre, Denmark) (15;16). The primers used for MTP and apoB mRNA amplification have
already been described (17). For amplification of ß-actin the primers were: h-ß-actin-31 (5’-
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GAAGCATTTGCGGTGGACGAT-3’) and h-ß-actin-51 (5’-
TCCTGTGGCATCCACGAAACT-3’).
Placental microsomal triglyceride transfer activity
Extracts of microsomal proteins were prepared from biopsies of two human
placentas, pig heart, and pig liver by homogenization of approximately 100 mg of each tissue
separately in 1 mL buffer (50 mmol/L Tris-HCl, 50 mmol/L KCl, 5 mmol/L EDTA, and
protease inhibitor, Roche A/S) with a PT 1200 Polytron (Buch & Holm A/S, Herlev,
Denmark). The total protein concentration in each homogenate was determined with the
bicinchoninic acid protein assay (Pierce, Copenhagen, Denmark) using bovine serum albumin
(BSA) as standard. The protein concentration was adjusted to 1.75 mg/mL, and placental
homogenates were centrifuged for 60 minutes at 100,000 g in a Beckman Optima LE-80K
ultracentrifuge (Beckman Coulter Inc., Fullerton, CA). The supernatant (i.e., the microsomal
fraction) was added to 1/10 volume of 0.54 % sodium deoxycholate (pH 7.5) and incubated on
ice for 30 minutes, followed by overnight dialysis at 4 °C against 15 mmol/L Tris (pH 7.4), 40
mmol/L NaCl, 10 mmol/L EDTA, and 0.02 % NaN3. Triglyceride transfer activity in the
microsomal protein fraction was measured at 37 °C as the transfer of [14C]-triglycerides from
labeled donor vesicles to acceptor vesicles that contained unlabeled triglycerides (18). The
donor vesicles contained 40 nmol phosphatidylcholine, 0.08 nmol [14C]-triglyceride, 3.0 nmol
cardiolipin, and 100 cpm/nmol [3H]-phosphatidylcholine. The acceptor vesicles contained
240 nmol phosphatidylcholine, 0.48 nmol triolein, and 100 cpm/nmol [3H]-
phosphatidylcholine. We measured the triglyceride transfer activity in human placentas by
incubating the microsomal protein fraction corresponding to 100 µg of total placental or pig
heart protein or 10 µg of pig liver with donor and acceptor vesicles for 6 hours (19). The
triglyceride transfer activity in both placental extracts was corrected for the transfer activity in
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a heat-inactivated (incubation at 65 °C for 10 minutes) aliquot of the same extracts (18) and
for the spontaneous transfer between donor and acceptor vesicles in a test tube without
microsomal extract.
Metabolic labeling studies
Each human placental biopsy (~0.3 g) was placed in a 2.0 mL Eppendorf tube,
minced into approximately 1 mm3 pieces with scissors and washed three times with 1.0 mL
incubation medium [methionine- and cysteine-free Dulbecco’s Modified Eagle Medium (D-
0422, Sigma, Vallensbæk Strand, Denmark) with 7 % fetal bovine serum (FBS), 2.0 mM
Glutamax-I (35050-038, Gibco, Invitrogen), 2.0 mM sodium pyruvate and protease inhibitors
(Complete Mini, 1 836 153, Roche A/S)] before adding 1.4 mL incubation medium with 0.59-
0.86 mCi [35S]Promix (SJQ0079, Amersham Biosciences, Hoersholm, Denmark). In some
experiments the incubation media also contained 0.81 mM oleic acid complexed with bovine
serum albumin (BSA) (molar ratio 2:1) (O3008, Sigma) or 0.41 mM fatty acid-free BSA (A-
9205, Sigma). The tube was placed at 37 °C on a shaking table at 250 rpm. The time from
child delivery to incubation of the placental tissue with labeling medium was 45-65 minutes.
After 3-24 hours the tissue was pelleted by centrifugation (12,000 g for 1 minute at 4 °C) and
the medium was collected. HepG2 cells were pre-incubated for one hour with incubation
medium without Complete Mini followed by incubation for 22-24 hours with 1.5 mL
incubation medium, with or without 0.81 mM oleic acid, and with 0.23 mCi [35S]Promix in
each well. Cell debris was removed by brief centrifugation.
Labeled medium from placenta or HepG2 cells was subjected to discontinuous
sucrose gradient ultracentrifugation. The sucrose gradient (20;21) was formed by layering
from the bottom of 13.2 mL Ultra-Clear tubes (344059, Beckman Coulter Inc.): 2 mL 50 %
sucrose, 2 mL 25 % sucrose, 5 mL 12.5 % sucrose containing 1 mL of the sample and 3 mL
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of phosphate-buffered saline. Complete Mini (1 tablet/ 7 mL) was added to all solutions in
experiments with placental medium. After ultracentrifugation (35,000 rpm for 70-71 hours at
12 °C with a Beckman SW41 Ti rotor in a Beckman Optima LE-80K ultracentrifuge) the
gradients were unloaded from the top of the tube into 6 fractions: 1 mL (top fraction), 4 x 1.5
mL and 5 mL (bottom fraction) and the density of each fraction was determined by weight.
ApoB was recovered by immunoprecipitation from 500 µl of each fraction and
the unfractioned labeling medium. Initially, each sample was pre-cleared by incubation with
20 µl Protein A/G PLUS-Agarose (0.5 mL agarose/2.0 mL) (SC-2003, Santa-Cruz
Biotechnology, Inc., Santa Cruz, CA, USA) for 30 minutes followed by pelleting of the beads
by centrifugation (2 minutes at 13,000 g). A polyclonal human apoB-100 antibody (Q0497,
DAKO, Glostrup, Denmark) was added to the supernatant and the mixture was incubated for
12-16 hours, before Protein A/G PLUS Agarose (20 µl) was added. All incubations were
carried out at 4 °C on a rocking-type mixer. After two more hours the tubes were centrifuged
(2 minutes at 13,000 g), and the pellet was washed three times with TRIS buffered saline
before being subjected to sodium dodecyl sulfate polyacrylamid gel electrophoresis (SDS-
PAGE) with Novex 4-20 % tris-glycine gels (EC6025BOX, Gibco, Invitrogen). After drying
of the gels, [35S]-labeled proteins were visualized with a FUJIX BAS2000 PhosphorImager
(Fuji, Tokyo, Japan).
Electron microscopy
Medium containing placental lipoproteins was prepared by incubating placental
tissue with oleic acid for 24 hours as described for the metabolic labeling studies, however, no
FBS was added, and instead of Promix, non-radioactive methionine (100 µg/mL, M-5308,
Sigma) and cysteine (500 µg/mL,C-7477, Sigma) were used. The medium was adjusted to a
density of 1.100 g/mL with NaBr. Five mL of density adjusted medium was overlayered with
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a d = 1.063 g/mL NaBr solution in a 13.2 mL Ultra-Clear tube and ultracentrifuged in the
Beckman SW41 Ti rotor at 40,000 rpm and 20 °C for 20 hours. Subsequently, formvar-
coated copper grids (400 mesh, FCF400Cu(25), Ax-Lab, Copenhagen, Denmark) were placed
on the liquid surface of each tube for three minutes. Lipoproteins were visualized using
negative staining with 2 % phosphotungstate (pH 7,0) (22-24) and a Zeiss EM 900 electron
microscope with a Mega View camera system. To compare placental and plasma
lipoproteins, we isolated VLDL (d < 1.019 g/mL), LDL (1.019 < d < 1.063 g/mL) and HDL
(1.063 < d < 1.21 g/mL) from human plasma by sequential ultracentrifugation and visualized
them as described above.
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RESULTS
MTP and apoB expression in the human placenta
Real-time RT-PCR analyses were used to establish whether the genes necessary
for lipoprotein secretion, apoB and MTP, are expressed in human placenta. Both gene
products were present in biopsies from human placentas (Fig. 1A). Placental biopsies taken
adjacent to the umbilical cord, at the periphery of the placenta or in between the two showed
no systematic regional variation in apoB and MTP mRNA expression levels and the levels of
ApoB- and MTP mRNA expression in three placentas were similar to those in human hearts
(17) (data not shown).
MTP catalyzes triglyceride transfer during assembly of apoB-containing
lipoproteins in the endoplasmatic reticulum (5). To examine whether bioactive MTP is
present in human placenta, we measured the triglyceride transfer activity in microsomal
extracts of placental biopsies. Placental extracts catalysed the transfer of triglyceride between
vesicles more efficiently than extracts from pig heart, but less efficiently than extracts from
pig liver (Fig. 1B).
Lipoprotein secretion by placenta
To investigate whether placenta synthesizes and secretes lipoproteins we
performed metabolic labeling studies with placental biopsies. Initially, we incubated
placental biopsies with [35S]-labeled amino acids and immunoprecipitated ApoB directly from
the labeled medium. SDS-PAGE and PhosphorImager analyses of immunoprecipitates
revealed [35S]-apoB-100, but not [35S]-apoB-48 in the medium (data not shown). In order to
assess the density of the [35S]-apoB-100-containing lipoproteins, we subjected the labeled
medium to sucrose density gradient ultracentrifugation and immunoprecipitated [35S]-apoB in
six different density fractions. [35S]-apoB-100 appeared in fractions with densities of 1.02-
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1.04 g/mL (Fig. 2B), i.e. corresponding to particles with the same buoyancy as plasma LDL
(1.019 < d < 1.063 g/mL). The addition of oleic acid induces the secretion of lipoproteins
with a density similar to VLDL (d < 1.019 g/mL) in HepG2 cells and causes a reduction in the
amount of lipid and protein in the LDL and HDL density range (25). Whether oleic acid has
the same effect in placenta was investigated by adding oleic acid complexed to BSA to the
placental incubation medium. The density decreasing effect of oleic acid on apoB-100-
containing lipoproteins from HepG2 cells (Figs. 2C and D) was not seen in placenta (Figs. 2A
and B).
Placenta expresses lipase activity (26). We therefore considered the possibility
that lipoproteins from placenta might in fact be secreted as VLDL and subsequently converted
to LDL due to hydrolysis of the triglyceride moieties. To examine the extent of lipolytic
conversion of VLDL to LDL, medium containing [35S]-labeled VLDL was harvested from
HepG2 cells and incubated with human placenta or mouse heart tissue (which expresses high
levels of lipoprotein lipase (27)) for 24 hours. The incubation with mouse heart tissue lead to
disappearance of the [35S]-apoB-100 VLDL particles; only a faint band corresponding to
[35S]-apoB-100 LDL was seen (Fig. 3C). In contrast, incubation with placental tissue only
produced partial redistribution of [35S]-apoB-100 from VLDL to LDL (Fig. 3B). This
supports the conclusion that placenta, both with and without oleic acid supplementation,
mainly secretes apoB-100 in particles with LDL density. However, it also indicates that some
newly formed VLDL may have been converted to LDL upon secretion and therefore escaped
detection as [35S]-VLDL in our analysis of the medium.
To see placental lipoproteins, placental biopsies were incubated with serum free
medium for 24 hours before isolation of d < 1.063 g/mL lipoproteins. Electron microscopy of
negatively stained placental lipoproteins showed spherical lipoproteins of 47 ± 10 nm (mean ±
standard deviation, n = 65) (Fig. 4A). This result was seen in two independent experiments.
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Unexpectedly, the placental lipoproteins were larger than plasma LDL (mean diameter: 23 ± 7
nm) on similarly prepared grids (Fig. 4B). The diameter of the plasma d < 1.019 g/mL
lipoproteins varied considerably and was on average 78 ± 64 nm.
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DISCUSSION
This study shows that human term placenta produces and secretes apoB-100-
containing lipoproteins. The maternal lipoprotein profile changes with an increase of
triglyceride and cholesterol levels in all lipoprotein fractions during pregnancy (1;28). This
makes the mother the obvious donor of lipid moieties to placenta. The quadruplication of the
fetal weight from the 26th gestational week to delivery is mainly due to lipid accumulation
(29). We therefore suspect that the fetus is on the receiving end of placental lipoprotein
production. In support of this idea, Parker et al found that the LDL-cholesterol concentration
was ~10 % higher in the umbilical artery than in the umbilical vein (30).
The present results add placenta to a growing list of organs that produce apoB-
containing lipoproteins. In addition to liver and intestine, recent data suggest that cardiac
lipoprotein formation plays an integrated role in cardiac lipid metabolism (16;17;19;31) and
the kidney is a major lipoprotein producing organ in the chicken (32). We have also detected
apoB mRNA in the mouse kidney (unpublished) and would not be surprised if the kidney also
secretes apoB-containing lipoproteins in mammals. However, it is unlikely that lipoprotein
synthesis is important in all cells (e.g., as a generally expressed pathway for removal of
excess triglycerides) simply because the apoB expression levels are extremely low or
undetecable in other tissues than those discussed above, including skeletal muscle, adipose
tissue, lung, and spleen (7;33).
The present data suggest that placenta may produce apoB-containing
lipoproteins with an efficacy per gram tissue that is 2 % of that in the adult liver (a rough
estimate based on the relative MTP activity and MTP mRNA contents in placenta versus pig
liver and cultured human liver cells). Since the placenta weighs ~4 times more than the fetal
liver this result implies that ~8 % of the apoB-containing lipoproteins in fetal plasma might be
derived from the placenta. This estimate is of course rather speculative. Nevertheless, it
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illustrates that lipoproteins synthesized in placenta may contribute significantly to the plasma
pool of apoB-containing lipoproteins in the fetus.
As expected, oleic acid increased the buoyancy of newly secreted apoB-
containing lipoproteins from HepG2 cells (25). However, oleic acid failed to increase the
buoyancy of the apoB-containing lipoproteins from placental biopsies. Interestingly, a similar
lack of effect of oleic acid has been observed when studying the secretion of apoB-containing
lipoproteins from oleic acid-perfused mouse hearts (31). Moreover, the present data suggest
that the density of placental lipoproteins was only mildly affected by post-secretional lipolysis
(or preferential uptake of newly secreted VLDL) since the buoyancy of HepG2-cell derived
VLDL only changed partially after incubation with placental biopsies. Ultrastructural
analyses have shown that the size of nascent VLDL particles in mouse yolk sac cells diminish
in the late stages of pregnancy (from day embryonic day 13.5) (7). Since the present
experiments were carried out with term placentas, we cannot exclude that the placenta
produces larger and more buoyant lipoproteins at earlier time points during gestation. It is
puzzling that the size of placental lipoproteins was similar to that of plasma VLDL remnants
while the density resembled that of plasma LDL. Since we did not see any other [35S]-labeled
proteins than apoB-100 after immunoprecipitation of the apoB-containing lipoproteins, we
suspect that the high density might reflect differences in the lipid composition of placental
and plasma LDL. Although similarly sized placental lipoproteins were seen in two
independent experiments, the apparent discrepancy could also reflect an artifact of the
preparation of placental lipoproteins for electron microscopy (34).
What is the importance of placental lipoprotein in the fetus? Perhaps the
function as vehicle is not so much for triglycerides as it is for other lipid soluble molecules,
e.g., cholesterol, glycolipids, and lipid soluble vitamins. Comprehensive studies of knock-out
mouse models for the two genes involved in apoB-containing lipoprotein synthesis and
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secretion (MTP and apoB) have shown that homozygous embryos in both cases die in utero
and manifest severe neurodevelopmental abnormalities (11;12). At least two arguments
support that a defect α-tocopherol (vitamin E) delivery plays an important role in these lethal
phenotypes (7;11;12). First, plasma transport and tissue distribution of vitamin E relies to a
great extent on apoB-containing lipoproteins (35). Second, dietary or genetically induced
vitamin E deficiency in pregnant rats and mice (36-39) confers fetal resorption and embryonic
exencephalus similar to the phenotypes in MTP and apoB knock-out mice.
The principal role of α-tocopherol transfer protein (α-TTP) is to mediate the
incorporation of α-tocopherol into newly formed apoB-containing lipoproteins (4).
Interestingly, in humans, the placenta shows the second highest tissue expression of the α-
tocopherol transfer protein gene (only surpassed by the liver) (40). Despite this, human
individuals with mutations in the α-TTP gene (which cause severe vitamin E deficiency after
birth) or with apoB deficiency (i.e., abetalipoproteinemia or familial
hypobetalipoproteinemia) have only been reported to develop symptoms postnatally
(35;41;42). This implies that other pathways than LDL formation also can convey lipid and
vitamin E transport into the developing human fetus. Another mechanism could involve the
APT-binding cassette transporter 1 (ABC-AI), which is highly expressed in the placenta (43)
and mediates efflux of both cholesterol and vitamin E to HDL (44). Indeed, vitamin E in the
fetal circulation is found both in HDL and LDL lipoproteins (45).
Although the present findings cast light on a new aspect of transplacental lipid
transport it also undeniably highlights unanswered questions such as: what is the lipid
composition of placental lipoproteins and is the secretion regulated? Some of these questions
could be conveniently addressed in cell culture studies. RT-PCR studies of apoB and MTP
expression in isolated placental trophoblast cells suggested that the trophoblasts are capable of
making lipoproteins (data not shown). However, when we cultured two trophoblast derived
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cancer cell lines (BEWO and JAR) we only found very low expression levels of apoB and
MTP mRNA in those cells. Thus, future studies of human placental lipoprotein production
most likely will have to employ primary cultures of trophoblast cells (46) or ex-vivo dual
perfusion of isolated cotyledons (47).
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ACKNOWLEDGEMENTS
We thank Karen Rasmussen and Annemette Borch for technical assistance and
Nina Broholm, Lise-Lotte W. Niels-Christiansen and Gert H. Hansen for help with electron
microscopy. Professor G. Desoye, Graz, Austria generously provided isolated human
trophoblasts. The study was supported by the Danish Medical Research Council (22-03-
0087).
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FIGURE LEGENDS
FIG. 1. mRNA expression of MTP and apoB and triglyceride transfer activity in human
placenta.
A. RNA from term human placenta was used for cDNA synthesis. The apoB-
and MTP-transcripts were amplified with PCR and the products were analyzed on a 2 %-
agarose-gel. cDNA from HepG2 cells were used as positive controls. B. Mean triglyceride
transfer activity in microsomal extracts from two human term placentas. Porcine heart and
liver extracts were included as controls (48). The background activity in this assay was < 1
%.
FIG. 2. Secretion of apoB-100-containing LDL from human placenta.
Human placental biopsies (A and B) or cultured hepG2 cells (C and D) were
incubated with [35S]-methionine and -cysteine. The media were subsequently subjected to
sucrose density gradient ultracentrifugation. ApoB was isolated from each of six density
fractions by immunoprecipitation and analysed by SDS-PAGE and phosphor-imaging. The
relative intensity of the [35S]-apoB band is shown below each fraction. In some experiments,
oleic acid was added to the incubation medium (A and C). The polyclonal anti-apoB antibody
also precipitated several proteins of smaller size than apoB in both placental tissue and. The
nature of these proteins has not been investigated. The results are representative of at least
three independent experiments.
FIG. 3. Hydrolysis of VLDL by human placenta.
To investigate in vitro hydrolysis of VLDL by placenta, [35S]-apoB-100 VLDL
was harvested from HepG2 cells (A) and incubated with either 0.3 g minced human placental
(B) or mouse heart tissue (C) at 37 °C for 24 hours. The media were subsequently subjected
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to sucrose density gradient ultracentrifugation. ApoB was isolated from each of six density
fractions by immunoprecipitation and analysed by SDS-PAGE and phosphor-imaging. The
relative intensity of the [35S]-apoB band is shown below each fraction.
FIG. 4. Visualization of placental lipoproteins by electron microscopy.
After 3 initial washes of placental biopsies and an additional wash after 1 hour,
serum-free medium with oleic acids was added to placental tissue biopsies and the mixture
was left at 37 °C for 24 hours. Placental lipoproteins (d < 1.063 g/mL) in the medium and
lipoproteins in plasma of a healthy human subject were isolated by ultracentrifugation,
negatively stained, and inspected in an electron microscope. A. Placental lipoproteins (d <
1.063 g/mL). B. Human plasma LDL (1.019 < d < 1.063 g/mL). C. Human plasma VLDL (d
< 1.019 g/mL). D. Human plasma HDL (1.063 < d < 1.21 g/mL). The results are
representative of two independent experiments.
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NielsenEva M. Madsen, Marie L. S. Lindegaard, Claus B. Andersen, Peter Damm and Lars B.
Human placenta secretes apolipoprotein B-100-containing lipoproteins
published online October 25, 2004J. Biol. Chem.
10.1074/jbc.M411404200Access the most updated version of this article at doi:
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