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: larsbo@rh.dk

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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