MECHANISMS OF PLACENTAL DYSFUNCTION IN PREGNANCY MALARIA
By
Jared Lybbert
A Thesis Submitted to the Faculty of
The Charles E. Schmidt College of Medicine
In Partial Fulfillment of the Requirements for the Degree of
Master of Science
Florida Atlantic University
Boca Raton, Florida
December 2015
Copyright by Jared Lybbert 2015
ii
ACKNOWLEDGEMENTS
Most importantly I would like to thank and applaud the patience and direction
given to me by Dr. Andrew Oleinikov, whose help and support have been invaluable. I
would also like to thank the members of the Oleinikov laboratory: Olga Chesnokova,
Jordan Merritt, and Irina Oleinikov, for their guidance and their friendship. Lastly, I
would like to thank my family and friends for their unwavering support, specifically
Stephanie Hamrick and Cristine Lybbert for their relentless encouragement.
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ABSTRACT
Author:
Jared Lybbert
Title:
Mechanisms of Placental Dysfunction in Pregnancy Malaria
Institution:
Florida Atlantic University
Thesis Advisor: Dr. Andrew Oleinikov
Degree:
Master of Science
Year:
2015
The molecular mechanisms by which pregnancy malaria affects the outcome of
fetal development are unknown. Megalin, which has been well studied in kidney, has
high expression in the placenta from early stages to term, and is proposed to be an
important factor in extensive maternofetal exchange during development of the fetus.
Pregnancy malaria (PM) is characterized by inflammation in placenta and is associated
with low birthweight (LBW), stillborn birth, and other pathologies. It is hypothesized that
PM disturbs megalin function/expression/distribution in the brush boarder of
syncytiotrophoblast which, in turn, may contribute significantly to pathology of LBW.
Our studies show that the presence of infected erythrocytes in placenta at the time
of delivery negatively affects protein abundance for megalin and Dab2. This is the first
report associating the abundance of placental megalin system proteins with the birth
weight of newborn babies, and associating PM with changes in megalin system protein
abundance.
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MECHANISMS OF PLACENTAL DYSFUNCTION IN PREGNANCY MALARIA
LIST OF FIGURES ......................................................................................................... viii
LIST OF TABLES .............................................................................................................. x
INTRODUCTION .............................................................................................................. 1
Plasmodium’s blood stage causes pathology in humans ................................................. 1
Mother-fetus interactions and placental malaria ............................................................. 2
Megalin function ............................................................................................................. 3
Cubilin function............................................................................................................... 4
Disabled 2 (Dab2) function ............................................................................................. 4
Actin function .................................................................................................................. 4
Molecular and cellular events in PM that may affect megalin functions ........................ 5
Hypothesis ....................................................................................................................... 6
METHODS AND MATERIALS ........................................................................................ 7
Antibodies ....................................................................................................................... 7
Measurement of megalin and Dab2 abundance by fluorescence microscopy ................ 8
Statistical analysis ........................................................................................................... 9
RESULTS ......................................................................................................................... 12
Megalin and Dab2 protein expression within syncytiotrophoblast ............................... 12
Placental malaria is associated with reduced abundance of megalin and Dab2 ............ 13
vi
Megalin and Dab2 protein abundance are positively correlated in placental brush
boarder ........................................................................................................................... 14
Abundance of megalin is positively correlated with birth weight and is
significantly reduced in low birth weight placentas infected with malaria parasites .... 14
Actin abundance within the syncytiotrophoblast is not correlated to birth weight or
with pregnancy malaria ................................................................................................. 14
Abundance of cubilin is not correlated to birth weight or with pregnancy malaria
status .............................................................................................................................. 15
DISCUSSION ................................................................................................................... 21
Outcomes from megalin signaling/function disturbance .............................................. 21
CONCLUSION ................................................................................................................. 24
REFERENCES ................................................................................................................. 25
vii
LIST OF FIGURES
Figure 1 Immunofluorescence image of human placenta stained with megalin
(green) and DAPI (blue). .................................................................................. 10
Figure 2 Strong correlation of protein abundance measurements in sections of
placental samples obtained in independent blind experiments using two
different antibody preparations. ....................................................................... 15
Figure 3 Megalin and Dab2 abundance in the brush boarder of syncytiotrophoblast
in placentas with malarial infection at delivery is reduced (PE+). ................... 16
Figure 4 Parity is not a confounding factor in association of placental infection with
reduced abundance of megalin and Dab2. ........................................................ 16
Figure 5 Megalin and Dab2 abundance is reduced in brush boarder of
syncytiotrophoblast in placentas with malarial associated inflammation
(PE(+) and INFL(+)) compared to control. ...................................................... 17
Figure 6 Megalin and Dab2 abundance in brush boarder of syncytiotrophoblast are
highly correlated. .............................................................................................. 17
Figure 7 Megalin and Dab2 abundance in brush boarder of syncytiotrophoblast
correlated to birth weight. ................................................................................ 18
Figure 8 Megalin and Dab2 abundance in brush boarder of syncytiotrophoblast is
reduced in samples with low birth weight and pregnancy malaria at
delivery. ............................................................................................................ 18
viii
Figure 9 Representative images of expression of megalin and Dab2 staining in
placental sections (displayed as green). ........................................................... 19
Figure 10 Representative image of actin staining (green) on placental tissue. ................. 20
Figure 11 Actin and cubilin abundance in the brush boarder of syncytiotrophoblast
did not differ between PE+ samples and PE- samples. .................................... 20
ix
LIST OF TABLES
Table 1
Clinical information of on placental sections and corresponding babies and
women. ............................................................................................................. 11
x
INTRODUCTION
Approximately 3.2 billion people, about half the world’s population is at risk for
malaria. About 200 million malaria cases a year lead to an estimated 584,000 deaths from
infection of the parasite. 90% of the world’s death occur in Africa and over 430,000
children die before reaching the age of 5. During 2013, it was estimated that 128 million
people were infected with Plasmodium falciparum [1]. It is estimated that each year
10,000 women and 200,000 infants die as a result of infection by P. falciparum during
pregnancy. Infant mortality from pregnancy malaria is largely from low birth weight
(<2.5kg), but can also be caused by severe anemia, stillbirth and premature abortion [2].
Plasmodium’s blood stage causes pathology in humans
The plasmodium parasite has a complex life cycle involving both an insect vector
(Anopheles mosquito) and human host. Of the five Plasmodium species which infect
humans, P. falciparum is the most dangerous. All pathology resulting from infection is
caused by the asexual blood stage in which parasites infect red blood cells. Once red
blood cells are infected, the parasite consumes hemoglobin and creates hemozoin, a
metabolically crystalized byproduct [3] which may be visualized under a microscope, and
indicates ongoing (within erythrocytes) or past (in extracellular fibrin deposits) infection
in the tissue. Pathology for uncomplicated malaria is linked to fevers and chills which
coincide with intraerythrocytic cycles of growth of the parasite (~48 hours). Severe
malaria to the host includes lactic acidosis, cerebral malaria (resulting of adhesion of
parasites to brain endothelial cells), severe anemia, respiratory distress, and placental
1
malaria [4]. To avoid clearance by the spleen and ensure parasite survival, the parasite
sequesters along the vasculature of various tissues [5]. Sequestration occurs as the
parasite expresses variant proteins, known as PfEMP1 proteins, produced by the var gene
family (~60 variants), onto the surface of infected red blood cells to allow binding to
endothelial surface proteins such as ICAM-1, CD36, and placental surface molecule
chondroitin sulfate A (CSA) [6, 7]. One of the PfEMP1 proteins called VAR2CSA has
been implicated in CSA binding and is responsible for parasite sequestration within the
placenta [7].
Mother-fetus interactions and placental malaria
The essential interface which mediates nutrient transfer between mother and fetus
is the syncytiotrophoblast. This tissue initially is comprised of many cells which
eventually fuse into a single multi-nuclear cell and persists from early conception through
to term indicating its essential function in pregnancy. The syncytiotrophoblast forms
microvilli that expand the total surface area to almost 100m² allowing for efficient
transfer of nutrients [8]. The syncytiotrophoblast is also the surface where P. falciparum
infected erythrocytes sequester in the placenta and is consequently exposed to host
macrophages during host-mediated immune responses [9]. The inflammatory responses
initiated by host macrophages due to P. falciparum infection can cause local placental
necrosis, and is implicated in low birth weight and poor pregnancy outcomes [10].
Incidence of PM in malarial endemic regions decreases with parity [11] as specific antiPM immune response develops. This response is associated with the appearance of antiadhesion antibodies that prevent malaria-parasitized erythrocyte (PE) sequestration
through chondroitin sulfate A (CSA) in the placental villi [12]. The molecular
2
mechanisms caused by parasitized erythrocyte sequestration in placenta were studied here
by quantification of the abundance of proteins involved in the megalin
signaling/transporting system, including: megalin, a transmembrane protein; Dab2, its
intracellular ligand; and cubilin, an extracellular membrane protein which interacts with
megalin. The details of these proteins functions are discussed in greater detail below.
Megalin function
Megalin is a giant (~600kDa) multi-ligand ancient low density lipoprotein
receptor (also called LRP-2, gp330, gp600) with significant physiological functions [13].
This protein is part of the LDL receptor family, including LDL receptor-related protein
(LRP-1). Megalin knockout in mice leads to perinatal death (98%) as a result of
complications with megalin protein expression in organs such as brain, kidney and lung
[14, 15]. Megalin is highly expressed in the syncytiotrophoblast implicating importance
in placental function. Many functions of megalin have been studied in kidney and in early
embryonic development [16, 17]. Its role in PM is not yet well characterized. It has been
shown that megalin expresses in the placenta and is localized on the surface of the
syncytiotrophoblast and within placental cytotrophoblast [18, 19]. Megalin is expressed
on the placental micro-villi (brush boarder) apical surface of the syncytiotrophoblast
which is exposed to maternal blood [18, 19]. Megalin’s ligands (n~50) include insulin,
insulin-like growth factor I (IGF-I), apolipoproteins, morphogens, albumin, extracellular
matrix proteins, and other nutrients, hormones, vitamin-binding and signaling molecules,
[17]. Taking into account the broad spectrum of ligands which involve megalin it is likely
that the impaired functions of this protein in the syncytiotrophoblast may result in poor
3
outcomes, due to restricted availability of nutrients, for fetal development. The role of
megalin in placental malaria was not studied previously.
Cubilin function
A multi-ligand endocytic receptor that forms a complex with megalin and
expands the number of ligands that can be internalized through megalin-mediated
endocytosis [16]. These ligands comprise some of those which also bind to megalin and
include albumin, Intrinsic Factor-Vitamin B 12, Vitamin D binding protein, hemoglobin
and Apolipoproteins A-I, as well as immunoglobulin light chains [Reviewed in 17].
Cubilin function is well studied in the kidney but its role in the placenta is unknown.
Cubilin has been reported to express within the placenta, with increasing mRNA levels
across gestation [20].
Disabled 2 (Dab2) function
Cytosolic protein Dab2 has been identified as the adaptor protein by which
megalin internalization occurs, and interacts with megalin through a conserved
phosphotyrosine interaction domain (PID) [21]. Dab2 is a tumor suppressor protein [22].
It inhibits Wnt/ B-cantenin signaling and binds a variety of other intracellular proteins
[23]. Dab2 was shown to be critical in the endocytosis function of megalin within the
endoderm during fetal development [24]. Early work by this thesis advisor on Dab2
hypothesized that megalin has dual function in signal transduction and in endocytosis
through Dab2 interaction [21]. This view is now widely accepted [17].
Actin function
Actin contributes to the structure of syncytiotrophoblast tissue which serves as a
physical barrier between mother and fetal tissue. Actin is expressed in
4
syncytiotrophoblast tissue and is also localized in underlying mesenchyme [25]. We
observed staining which was consistent with previous studies of actin on placental tissue
(see Results section below). In our experiments, actin could serve as a useful control
protein which most likely is unaffected by PE adhesion to syncytiotrophoblast tissue.
Molecular and cellular events in PM that may affect megalin functions
Multiple factors acting during placental malaria may affect placental development
and functions. In relevance to the megalin system, the sequestration of PE through
surface-expressed PfEMP1 parasite adhesions may cross-link corresponding host
adhesion receptor, CSA, on the syncytiotrophoblast surface. This cross-linking over a
large part of the placental surface may directly affect megalin-mediated endocytic and/or
signaling pathways similar to other studies on the effect of cross-linking of cell surface
receptors [26, 27]. Infiltrating macrophages interacting with sequestered PE likely further
exacerbate negative effects to the syncytiotrophoblast tissue.
TGF-beta is a cytokine released by host macrophages which can have varying
effects dependent upon other co-signals. During pregnancy malaria TGF-β concentration
within the placenta is significantly increased [28]. TGF-β negatively regulates
endocytosis of albumin through megalin and cubilin in kidney [29]. Analogously, during
pregnancy malaria, macrophage infiltration and release of TGF-β may cause systemic
regulation of megalin expression in the syncytiotrophoblast reducing endocytosis and
signaling processes.
Due to the large number of ligands, and abundance of megalin found within the
placenta, it is likely that it has high importance in proper placental function and proper
5
fetal development. Cubilin and Dab2, being accessory proteins, will need to be studied as
well to better understand molecular mechanisms of placental pathology during malaria.
Hypothesis
We hypothesize that abundance levels of receptor megalin and its ancillary
proteins that are expressed along the syncytiotrophoblast brush border are affected by the
presence of infected erythrocytes in the placenta and this may correlate to low birth
weight outcomes.
Aim 1: To quantify the abundance of proteins megalin, Dab2, and cubilin in the
syncytiotrophoblast brush border of placental sections obtained from Ugandan women at
term using fluorescent microscopy.
Aim 2: To statistically analyze changes in protein abundance and associate protein
abundance measurements with various clinical parameters (placental infection, low birth
weight) using categorical analysis.
6
METHODS AND MATERIALS
Antibodies
Megalin – rabbit polyclonal antibody raised against 17mer peptide of cytoplasmic
tail of megalin of human origin [21]. Antibodies were tested on mouse kidney tissue and
showed strong staining to proximal tubules and significantly less staining in glomeruli
which is consistent with previous studies of megalin staining [30]. Megalin antibodies
were used at concentrations of 1:50 for two independent preparations purified on 17-mer
peptide column [21].
Dab2 - rabbit polyclonal antibody raised against amino acids 661-770 of Dab2 of
human origin (H-110, Santa Cruz). We also have antibodies raised against a GST- Dab2
phosphotyrosine interaction domain (PID) fusion protein, which binds to megalin [21].
Antibodies were tested on mouse kidney tissue and showed strong staining to proximal
tubules and significantly less staining in glomeruli which is consistent with previous
studies of Dab2 staining. Anti-dab2 primary antibodies were used at concentrations of
1:50 for both.
Cubilin – sheep polyclonal antibody raised against Escherichia coli derived
recombinant human cubilin (10ug/mL, R&D Systems, AF370). Antibodies were tested
on mouse kidney tissue and showed strong staining to proximal tubules and significantly
less staining in glomeruli, which is consistent with previous studies of cubilin staining
[31].
7
Actin – sheep polyclonal antibody raised against human platelet derived actin
(10ug/mL, R&D Systems, AF4000). Antibodies were tested on mouse kidney tissue and
showed strong staining to proximal tubules and significantly less staining in glomeruli,
which is consistent with previous studies of actin staining [32]. Antibodies were tested on
human placental tissue and showed low staining in syncytiotrophoblast which is
consistent with previous studies of actin staining of placental tissue [25].
Secondary antibodies – Fluorescent proteins used were polyclonal Donkey antisheep (Alexa Fluor 488) or anti-rabbit (Alexa Fluor 598) IgG antibodies (Jackson
Immunoresearch).
Measurement of megalin and Dab2 abundance by fluorescence microscopy
The paraffin embedded tissue samples (n=28) (Table 1) [33] were rehydrated by a
series of graded xylene/ethanol washes(100% xylene, 50/50%, xylenes and ethanol,
100% ethanol, 95% ethanol, 75%, ethanol, 50% ethanol for 3 minutes each) and washed
in phosphate buffer solution (PBS). The rehydrated slides were then heated in Tris-EDTA
buffer (10 mM Tris Base, 1mM EDTA solution, 0.05% Tween-20, pH 9.0) and
microwaved to boiling at full power and then maintained at 50% power for 8 minutes and
cooled in PBS [34]. Sections were then pre-incubated with PBS, 1% BSA and 0.25%
Triton-X for 30 minutes, incubated with primary antibody in the presence of 2.5% nonimmune donkey serum (Jackson Immunoresearch, 017-000-121) either for one hour at
room temperature or overnight at 4°C in a humidified chamber, washed with PBS for 5
minutes 3 times. Slides were then incubated with secondary antibody in the presence of
non-immune donkey IgG (1:100, Jackson Immunoresearch, 017-000-002) for one hour at
room temperature and counterstained with DAPI (5μg/mL), (Sigma, D9542) for 3
8
minutes. After three washes with PBS for 5 minutes, slides were processed with
Vectashield mounting medium (Vector Laboratories, H-1000) and coverslip applied.
Negative controls were processed without primary antibody. For the staining, to provide
maximally identical conditions, each placental section was divided in sub-sections,
processed with all primary antibodies, and then incubated with secondary antibody
simultaneously.
Slides were then imaged using a confocal microscope (Zeiss LSM 700 equipped
with Plan-Apochromat 63x/1.40 Oil DIC M27 objective) and processed using software
Zen 2012 (Blue). Images were captured using identical conditions to allow for
quantification of signal strength. Collected Images of syncytiotrophoblast were selected
for the brush boarder only or the entire syncytiotrophoblast. The process of selection is
illustrated in Figure 1, selected region A indicates brush border area, and region A+B
indicates entire syncytiotrophoblast. Average intensity was recorded. Each antibody stain
and control stain was imaged in triplicate, and averaged yielding a single value for each
stain per slide. Control values were subtracted from test values to obtain final value for
each sample. Final numbers were analyzed. Slides were processed blindly.
Statistical analysis
Statistical analyses were performed using Prizm statistical software using nonparametric statistics: Mann-Whitney tests for group differences and Spearman tests for
analyses of correlations.
9
Figure 1 Immunofluorescence image of human placenta stained with megalin
(green) and DAPI (blue). Highlighted regions display only the brush boarder of the
syncytiotrophoblast (selection region A) or the entire syncytiotrophoblast
(selection region A and B). This image illustrates the two different methods for
determining protein concentration within the syncytiotrophoblast.
10
Table 1 Clinical information of on placental sections and corresponding babies and
women. Parasite – parasites found in placenta at delivery; hemozoin – extracellular
hemozoin in fibrin found in placenta indicating previous (resolved) placental infection.
11
RESULTS
Megalin and Dab2 protein expression within syncytiotrophoblast
Our immunofluorescence staining studies confirmed expression of megalin and
Dab2 proteins in syncytiotrophoblast tissue of human term placenta as previously
reported [21, 35]. The distribution of these proteins is not uniform throughout the
syncytiotrophoblast and may show little to no presence at all, thus complicating reliable
quantification. Therefore we 1) measured the most abundant parts of the
syncytiotrophoblast as reflecting the ability of the tissue to express these proteins; 2)
perform repeated blind experiments using different antibody preparations. In each
placental section three areas were selected encompassing syncytiotrophoblast tissue;
regions for analysis were selected based on highest density of signal. If staining was
similar throughout the section three random areas along the syncytiotrophoblast were
selected.
Both proteins were measured for abundance using two different antibodies against
each protein in independent blind experiments. The abundance measurements of megalin
using two different antibody preparations highly correlated, as well as the abundance
measurements using two different antibodies against Dab2. (Figure 2). This data
illustrates that our measurements reflect abundance of proteins in the tissue and are
usable for making quantitative comparisons between samples.
12
Placental malaria is associated with reduced abundance of megalin and Dab2
Figure 3 displays the results of quantification of megalin (A) and Dab2 (B)
abundance by indirect IFM, in the brush boarder of syncytiotrophoblast and stratified by
malarial infection in placenta present at time of delivery. The abundance of proteins,
megalin and Dab2, are substantially reduced in placental malaria sections with statistical
significance. The presence of insoluble hemozoin detected in the extracellular fibrin
deposits (Hz+) is indicative of past placental malaria infection. If we combine samples
with placental PE(+) and Hz(+) into one group, stratified against control samples,
megalin and Dab2 continue to demonstrate statistically significant reduction of
abundance in these samples against control (p=0.04 and 0.005 for megalin and Dab2
respectively, blue triangles indicate Hz(+) PE(-) samples). Similar results (as in figure 3)
were obtained when protein abundance was measured in the entire syncytiotrophoblast
rather than exclusively along the brush boarder (data not shown).Parity is not a
confounding factor. Comparing samples for which parity data is known, we see no
difference in median parity, and the same decrease in megalin and Dab2 abundance for
PE+ vs PE- (Figure 4).
The host response to malarial infection within the placenta involves monocyte
recruitment causing inflammation [36]. Comparison of data involving samples which
show placental infection and inflammation against samples which are negative for both
displays a similar decrease in abundance of megalin and Dab2 with significance for Dab2
and close to significance for megalin (Figure 5).
13
Megalin and Dab2 protein abundance are positively correlated in placental brush
boarder
Dab2 is an intracellular ligand of megalin and has been shown to co-localize with
megalin in the proximal kidney tubule [21, 37]. Their expression is mutually dependent
as knockout of megalin abolishes Dab2 expression, and knockout of Dab2 inhibits
megalin function [37]. Megalin and Dab2 are two proteins which interact with each other
for signaling and endocytosis [21]. Our data shows that megalin and Dab2 abundance
directly correlated in the brush boarder of the syncytiotrophoblast (Figure 6) further
indirectly confirming their interdependence.
Abundance of megalin is positively correlated with birth weight and is significantly
reduced in low birth weight placentas infected with malaria parasites
Birthweight is positively correlated with abundance of megalin and Dab2 in the
brush boarder of syncytiotrophoblast although not found to be statistically significant
(Figure 7). However comparing samples with placental infection at delivery with LBW
against those with normal birthweight and no present or previous infection demonstrates
that PE increases the difference in abundance for both megalin and Dab2, with statistical
significance for Dab2 (Figure 8). Figure 9 illustrates comparative differences observed
between these samples.
Actin abundance within the syncytiotrophoblast is not correlated to birth weight or with
pregnancy malaria
A representative image of actin staining is shown in Figure 10. Actin abundance
in the brush boarder of syncytiotrophoblast did not differ between PE+ samples and PE-
14
samples (Figure 11). There also no reportable difference of actin abundance in PE+ LBW
samples stratified against PE- NBW samples (data not shown).
Abundance of cubilin is not correlated to birth weight or with pregnancy malaria status
It has been reported that cubilin expresses in the yolk sac and placenta [20].
Median values for cubilin reflect that the presence of PEs had no discernable effect on its
expression in the samples tested (Figure 11).
Figure 2 Strong correlation of protein abundance measurements in sections of placental
samples obtained in independent blind experiments using two different antibody
preparations. A, megalin measurements. B, Dab2 measurements.
15
Figure 3 Megalin and Dab2 abundance in the brush boarder of syncytiotrophoblast in
placentas with malarial infection at delivery is reduced (PE+). AFM, arbitrary fluorescent
units. Red line indicates median value.
Figure 4 Parity is not a confounding factor in association of placental infection with
reduced abundance of megalin and Dab2. Samples with data on parity (n=22, Table 1)
were used for this analysis. Red bars, medians.
16
Figure 5 Megalin and Dab2 abundance is reduced in brush boarder of syncytiotrophoblast
in placentas with malarial associated inflammation (PE(+) and INFL(+)) compared to
control. AFU, Arbitrary fluorescent units. Red line indicates median value.
Figure 6 Megalin and Dab2 abundance in brush boarder of syncytiotrophoblast are
highly correlated. AFU, arbitrary fluorescent units. Line indicates linear regression
p=0.002.
17
Figure 7 Megalin and Dab2 abundance in brush boarder of syncytiotrophoblast
correlated to birth weight. AFU, arbitrary fluorescent units.
Figure 8 Megalin and Dab2 abundance in brush boarder of syncytiotrophoblast is
reduced in samples with low birth weight and pregnancy malaria at delivery. AFU,
arbitrary fluorescent units. Red line indicates median.
18
Figure 9 Representative images of expression of megalin and Dab2 staining in placental
sections (displayed as green). Staining along the syncytiotrophoblast showing expression
of Dab2 in NBW PE- placenta (C) against LBW PE+ (D). Blue is DAPI staining, Bright
cells inside of fetal vessels are self-fluorescing erythrocytes (e). (Magnification: 630x).
19
Figure 10 Representative image of actin staining (green) on placental tissue. Low
abundance of protein actin is seen on syncytiotrophoblast (S, between red and white
dotted lines), with high abundance in mesenchyme (M, inside red dotted line), fetal
endothelial tissue (F). Blue DAPI staining indicates nuclei.
Figure 11 Actin and cubilin abundance in the brush boarder of syncytiotrophoblast did
not differ between PE+ samples and PE- samples. Numbers indicate p values.
20
DISCUSSION
Our results clearly indicate that placental malaria reduces the abundance of
megalin and Dab2 proteins in the brush boarder of syncytiotrophoblast. This may
negatively affect the function/signaling pathways within the placenta, which may lead to
fetal growth restriction and low birth weight pathology.
We did not see statistical significant changes in cubilin expression depending on
placental infection status. This may indicate that interaction between megalin and cubilin,
two co-receptors in kidney, may be different in placenta and requires more studies.
Nevertheless cubilin only expands the extensive list of megalin’s ligands and may have a
smaller or different role in placenta compared to kidney.
Outcomes from megalin signaling/function disturbance
Megalin interacts with a broad range of ligands including lipoproteins, vitamins,
and hormones, which are important for proper fetal development. Disruption of megalin
signaling/function may play an important role of low birth weight pathology.
Lipid synthesis is can be accomplished by the fetus, however supplementation
from the mother is essential for proper growth. Early in pregnancy fetal demands of
maternal lipids are high, as gestation increases, however, fetal capacity for maternal
lipoproteins decreases [38]. Early maternal cholesterol supplementation is needed to
activate sonic hedgehog signaling, an important pathway for neural pathway and proper
forebrain development [39]. Cholesterol is also the origin of a number of signaling
21
cascades, and is a part of every lipid membrane. Several lipoprotein receptors express in
the placenta, which includes megalin. Disruption of megalin’s function in transporting
lipids across the membrane may contribute to low birth weight outcomes.
Vitamins utilized by the fetus can only be provided by maternal blood. Vitamin-D
Binding Protein (DBP), the carrier for 25-OH Vitamin-D3, interacts with megalin which
is important for calcium absorption and bone mineralization in kidney tubules [40].
Inhibiting vitamin-D supplementation to a developing fetus may increase preterm
delivery risk and fetal growth restriction outcomes [41]. Retinol binding protein and
cobalamin (Vitamin B-12) have been shown to interact with megalin via their respective
binding proteins in kidney tubules [42] and may similarly be transported to fetus through
placenta.
Hormones play an essential role in regulation of fetal growth. As stated
previously insulin and insulin-like growth factor are ligands of megalin [16]. Megalin
also binds to transthyretin, parathyroid hormone, and sex hormone binding globulin
which all serve important roles in fetal development [43, 44].
Megalin interacts with a variety of ligands and serve many different roles in a
tissue dependent manner. Within the placenta its role most likely revolves around the
purpose and function of the placenta, which is to aid and assist in the growth of the fetus
through nutrient supplementation and hormone signaling. Disruption of megalin
transporting/signaling function may affect development of the placenta and/or fetus.
In vivo studies of malaria parasitized erythrocyte’s effect on placenta are
extremely difficult to perform. However, an in vitro placental model involving the
22
utilization of BeWo cells can be used. BeWo cells are an immortalized cytotrophoblast
cell line capable of syncytialization, which shares similarities with placental
syncytiotrophoblast. Exploring the effect on parasitized erythrocytes on BeWo cells as
well as studying the interaction of human macrophages in the presence of PEs may help
to understand the molecular mechanisms in vivo. This will be used in future studies to
associate our findings in human placenta with molecular mechanisms of megalin
functions.
23
CONCLUSION
In conclusion, our findings suggest that placental malaria reduces abundance of
megalin-associated transport/signaling systems in human syncytiotrophoblast, which
correlates with birthweight at delivery and may ultimately contribute toward low birth
weight outcomes. To our knowledge, this is the first report associating the abundance of
placental megalin system proteins with the birth weight of newborn babies and
associating placental malaria with changes in abundance of megalin system proteins.
The results of this work were submitted for publication and are currently under
review. The thesis candidate is the first author in the submitted manuscript.
24
REFERENCES
1. World malaria report 2014, World Health Organization, Geneva
2. McGregor, I.A. (1987) Thoughts on malaria in pregnancy with consideration of some
factors which influence remedial strategies. Parassitologia 29: 153-163.
3. Coronado, L.M., Nadovich, C.T., Spadafora, C., (2014) Malarial Hemozoin: From
target to tool. Biochim Biophys Acta. 1840(6):2032-2041.
4. Weatherall, D.J., et al. (2002) Malaria and the Red Cell. Hematology. Am Soc
Hematol Educ Program. 2002:35-57.
5. Sherman, I.W., Eda, S., Winograd, E., (2003) Cytoadherence and sequestration in
Plasmodium falciparum: defining the ties that bind. Microbes and Infection,
5(10):897-909.
6. Baruch, D.I., et al. (1995) Cloning the P. falciparum Gene Encoding PfEMPl a
Malarial Variant Antigen and Adherence Receptoron the Surface of Parasitized
Human Erythrocytes. Cell 82:77-87.
7. Srivastava, A., et al. (2010) Full-length extracellular region of the var2CSA variant of
PfEMP1 is required for specific, high-affinity binding to CSA. Proc Natl Acad Sci U
S A. 107(11):4884-9.
8. Benirschle, K., Kauffmann, P. (2000) Pathology of the human placenta. Springer.
9. Walter, P.R., Garin, Y., Blot P. (1982) Placental pathologic changes in malaria. A
histologic and ultrastructural study. Am J Pathol 109:330-342.
10. Fried, M., Duffy, P. (1998) Maternal malaria and parasite adhesion. J Mol Med (Berl)
76: 162-171.
25
11. McGregor, I.A. (1987) Thoughts on malaria in pregnancy with consideration of some
factors which influence remedial strategies. Parassitologia. 29(2-3): p. 153-63.
12. Duffy, P.E., Fried, M. (2003) Antibodies that inhibit Plasmodium falciparum
adhesion to chondroitin sulfate A are associated with increased birth weight and the
gestational age of newborns. Infect Immun.71:6620-6623.
13. Yochem, J., Greenwaled, I. (1993) A gene for a low density lipoprotein receptorrelated protein in the nematode Caenorhabditis elegans. Proc Natl Acad Sci USA
90:4572-4576.
14. Willnow, T.E., Hilpert, J., Armstrong, S.A., Rohlmann, A., Hammer, R.E., et al.
(1996) Defective forebrain development in mice lacking gp330/megalin. Proc Natl
Acad Sci USA 93:8460-8464.
15. Leheste, J.R., et al. (1999) Megalin knockout mice as an animal model of low
molecular weight proteinuria. Am J Pathol 155:1361-1370.
16. Christensen, E.I., Birn, H. (2002) Megalin and cubilin: multifunctional endocytic
receptors. Nat Rev Mol Cell Biol 3:256-266
17. Marzolo, M.P., Farfan, P. (2011) New insights into the roles of megalin/LRP2 and the
regulation of its functional expression. Biol Res 44:89-105.
18. Lundgren, S., et al. (1997) Tissue distribution of human gp330/megalin, a putative
Ca(2+)-sensing protein. J Histochem Cytochem. 43(3):383-92.
19. Lambot, N., et al. (2006) Evidence for a Clathrin-Mediated Recycling of Albumin in
Human Term Placenta. Biol Reprod. 75(1):90-97.
20. Burke, K.A., Jauniaux, E., Burton, G.J., Cindrova-Davies, T. (2013) Expression and
immunolocalisation of the endocytic receptors megalin and cubilin in the human yolk
sac and placenta across gestation. Placenta. 11:1105-9.
21. Oleinikov, A.V., Zhao, J., Makker, S.P. (2000) Cytosolic adaptor protein Dab2 is an
intracellular ligand of endocytic receptor gp600/megalin. Biochemical J 347:613-621.
26
22. Mok, S.C., et al. (1998) DOC-2, a candidate tumor suppressor gene in human
epithelial ovarian cancer. Oncogene. 16:2381-2387.
23. Prunier, C., Hacevar, B.A., Howe, P.H., (2004) Wnt signaling: physiology and
pathology. Growth Factors. 22:141-150.
24. Maurer, M., Cooper, J., (2005) Endocytosis of megalin by visceral endoderm cells
requires the Dab2 adaptor protein. J of Cell Sci. 118:5345-5355.
25. Zeldovich, V.B., et al (2013) Placental Syncytium Forms a Biophysical Barrier
against Pathogen Invasion. PLoS Pathog, 9(12): e1003821.
26. Nolan, C.M., Creek, K.E., Grubb, J.H., Sly WS (1987) Antibody to the
phosphomannosyl receptor inhibits recycling of receptor in fibroblasts. J Cell
Biochem 35:137-151.
27. Fanning, S.L., George, T.C., Feng, D., Feldman, S.B., Megjugorac, N.J., et al. (2006)
Receptor cross-linking on human plasmacytoid dendritic cells leads to the regulation
of IFN-alpha production. J Immunol 177:5829-5839.
28. Fried, M., Muga, R.O., Misore, A.O., Duffy, P.E. (1998) Malaria elicits type 1
cytokines in the human placenta: IFN-gamma and TNF-alpha associated with
pregnancy outcomes. J Immunol 160:2523-2530.
29. Gekle, M., Knaus, P., Nielsen, R., Mildenberger, S., Freudinger, R., et al. (2003)
Transforming growth factor-beta1 reduces megalin- and cubilin-mediated endocytosis
of albumin in proximal-tubule-derived opossum kidney cells. J Physiol 552:471-481.
30. Hou, J., Wen, Y.H., Feng, K.N., Ma, X.F., Yao, J.P., (2015) DACT1 is involved in
human placenta development by promoting Wnt Signaling. Arch Gynecol Obstet,
6:1289-96.
31. Abrams, E.T., et al. (2003) Host Response to Malaria During Pregnancy: Placental
Monocyte Recruitment Is Associated with Elevated β Chemokine Expression. J
Immunol. 170(1):2759-64.
32. Nagai, J., et al. (2005) Mutually dependent localization of megalin and Dab2 in the
renal proximal tubule. Am J Physiol Renal Physiol. 289(9):569-76.
27
33. Makker, S.P., Widstrom, R., Huang, J., (1995) Transcription and translation of gp600
and receptor-associated protein (RAP) in active Heymann nephritis. Am. J. Pathol.
146:1481-1487.
34. Christensen, E.I., Henrik, B., Storm, T., Weyer, K., Nielsen, R., (2012) Endocytic
Receptors in the Renal Proximal Tubule. Physiology 27(4):223-236.
35. Brzica, H., Brelijak, D., Vrhovac, I., Sabolić, I., (2011). Role of microwave heating in
antigen retrieval in cryosections of the formalin-fixed mammalian tissues, Microwave
Heating, Dr. Usha Chandra (Ed.), ISBN: 978-953-307-573-0, InTech, DOI:
10.5772/20319.
36. Muehlenbachs, A., et al. (2012) Artemether-lumefantrine to treat malaria in
pregnancy is associated with reduced placental haemozoin deposition compared to
quinine in a randomized controlled trial. Malaria Journal 11:150
37. Shahed, A., Young, K.A., (2013) Anti-Müllerian Hormone (AMH), Inhibin-α,
Growth Differentiation Factor 9 (GDF-9), and Bone Morphogenic Protein-15 (BMP15) mRNA and protein are influenced by photoperiod-induced ovarian regression and
recrudescence in Siberian hamster ovaries. Mol Reprod Dev. 80(11):895-907.
38. Herrera, E., (2002) Lipid metabolism in pregnancy and its consequences in the fetus
and newborn. Endocrine Oct;19(1):43-55
39. Chiang, C., et al (1996) Cyclopia and defective axial patterning in mice lacking sonic
hedgehog gene function. Nature, Oct 3;383(6599):407-13
40. Nykjaer, A., et al (1999) An Endocytic Pathway Essential for Renal Uptake and
Activation of the Steroid 25-(OH) Vitamin D3. Cell, 96(4):507-515
41. Ray, J.G., Laskin, C.A., (1999) Folic acid and homocyst(e)ine metabolic defects and
the risk of placental abruption, pre-eclampsia and spontaneous pregnancy loss: A
systematic review. Placenta, 1999. 20(7):519-29
42. Christensen, E.I., Willnow T.E., (1999) Essential Role of Megalin in Renal Proximal
Tubule for Vitamin Homeostasis. J Am Soc Nephrol. Oct; 10(10):2224-36
43. Verroust, P.J., Christensen, E.I., (2002) Megalin and cubilin—the story of two
multipurpose receptors unfolds, Nephrol Dial Transplant. 17:1867-1871
28
44. Hammes, A., et al (2005) Role of Endocytosis in Cellular Uptake of Sex Steroids.
Cell. 2005 122(5):751-62
29