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Research Article Cell Biology International
10.1002/cbin.10601
Tube formation in the first trimester placental trophoblast cells:
Differential effects of angiogenic growth factors and fatty acids
Abhilash D. Pandyaa, Mrinal K. Das
a, Arnab Sarkar
a, Srinivas
Vilasagaramb, Sanjay Basak
band Asim K. Duttaroy
a*
aDepartment of Nutrition
Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo,
Norway
bNational Institute of Nutrition, Hyderabad, India
*Corresponding Author:
Professor Asim K. Duttaroy
Department of Nutrition,
Institute of Basic Medical Sciences,
Faculty of Medicine,University of Oslo,
Oslo, Norway
Email: [email protected]
Tel: +47 22 82 15 47
Fax: +47 22 85 13 41Abbreviations used: FABP4; cytosolic fatty acid binding protein-4, VEGF; vascular endothelial
growth factor, ANGPTL4;angiopoietin4likeprotein,DHA; docosahexaenoic acid, 22:6n-3; OA;
Oleic acid, MTT, 4,-dimethylthiazol-2-yl)-2,-diphenyl tetrazoliumbromide
This article has been accepted for publication and undergone full peer review but has not been throughthe copyediting, typesetting, pagination and proofreading process, which may lead to differences betweenthis version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10601]
This article is protected by copyright. All rights reserved
Received 16 November 2015; Revised 9 March 2016; Accepted 14 March 2016
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Abstract
The study aims to investigate whether cytosolic fatty acid binding protein-4 (FABP4) is involved
in angiogenic growth factors- and fatty acid-induced tube formation in first trimester placental
trophoblast cells, HTR8/SVneo. We determined the tube formation both at basal as well as
stimulated levels in the absence and presence of inhibitors of FABP4 and VEGF signaling
pathways. Basal level of tube formation was maximally reduced in the presence of 50M of
FABP4 inhibitor compared with those by VEGF signaling pathway inhibitors (rapamycin, L-
NAME, and p38 MAP kinase inhibitor). Whereas docosahexaenoic acid, 22:6n-3 (DHA)- and
VEGF- induced tube formation was maximally inhibited by p38 MAP kinase inhibitor (63.7%
and 34.5%, respectively), however leptin-induced tube formation was inhibited maximally by
FABP4 inhibitor (50.7%). ANGPTL4 and oleic acid (OA)-induced tube formation was not
blocked by any of these inhibitors. The FABP4 inhibitor inhibited cell growth stimulated by
DHA, leptin, VEGF, and OA (p
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1. Introduction
Angiogenesis is defined as a biological mechanism of new blood vessel formation from
preexisting ones and plays important roles in many processes including placentation (Khong and
Brosens, 2011). Angiogenesis is critical to successful fetal outcomes, as the placental blood flow
is dependent on placental vascularization. Lack of placental vascular development may
contribute to inadequate cytotrophoblast invasion as observed in preeclampsia (Reynolds and
Redmer, 2001). We have shown earlier that dietary fatty acids, vascular endothelial growth
factor (VEGF), leptin, and insulin stimulate angiogenesis in the first trimester placental
trophoblasts possibly via different mechanisms (Basak et al., 2013, Basak and Duttaroy, 2012,
Basak and Duttaroy, 2013a, Basak and Duttaroy, 2013b, Johnsen et al., 2011, Basak et al., 2015).
Fatty acid-binding protein-4 (FABP4) also known as adipocyte FABP (A-FABP) or aP2
(Duttaroy, 2009) was shown to be involved in VEGF-mediated angiogenesis in endothelial cells
(Ghelfi et al., 2013). Recent studies demonstrated that FABP4 as a novel target of VEGF and its
receptors (VEGF/VEGFR2) pathway, and a positive regulator of cell proliferation and
angiogenesis in endothelial cells (Elmasri et al., 2012, Elmasri et al., 2009, Ghelfi et al., 2013).
In fact, FABP4 plays a pro-angiogenic role in endothelial cells by promoting cell proliferation,
migration, survival, lipid accumulation, and morphogenesis. FABP4 has a role in activation of
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several mitogenic pathways and expression of several key mediators of angiogenesis (Elmasri et
al., 2009). In endothelial cells, FABP4 expression is induced by pro-angiogenic stimuli, such as
VEGF and basic fibroblast growth factor(Elmasri et al., 2009). The VEGF-mediated expression
of FABP4 was inhibited by siRNA-mediated knockdown of VEGFR2, whereas the VEGFR1
agonists, placental growth factors (PIGFs) had no such effect. FABP4 is primarily involved in
most of the VEGF mediated angiogenesis in endothelial cells (Harjes et al., 2014, Elmasri et al.,
2012). The disruption of stem cell factor (SCF)/c-kit signalling pathway played a critical role in
diminished VEGF mediated angiogenic responses in FABP4/ endothelial cells, indicating
FABP4 involvement in this process(Elmasri et al., 2012). It has been shown that the delta-like
ligand (DLL) 4-NOTCH directly regulates FABP4 gene expression by binding of the FABP4
promoter in endothelial cells(Guba et al., 2002). The FABP4 response to VEGF is dependent on
the NOTCH pathway, as inhibition of DLL4 binding to NOTCH and inhibition of NOTCH
cleavage leads to FABP4 reduction in response to VEGF (Harjes et al., 2014). Furthermore,
DLL4-NOTCH induced FABP4 is dependent on the insulin-responsive FOXO1 transcription
factor, providing a nodal point for the integration of angiogenic and metabolic signaling in
endothelial cells. One of the metabolic changes often found during angiogenesis is their
increased fatty acid synthesis and transport, lipid droplet formation, indicating possible
involvement of fatty acid transport system. It is also well known that cells alter their metabolism
to suit their needs for angiogenesis such as cell proliferation, invasion, and gene expression. Our
previous data showed that long chain fatty acids favored energy-intensive tube formation process
in the first trimester trophoblast cells (Johnsen et al., 2011, Basak and Duttaroy, 2013b, Basak
and Duttaroy, 2013a). FABP4 expression is induced by hypoxia that is essential for lipid
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accumulation in placental last trimester under increased lipid loads (Biron-Shental et al., 2008,
Scifres et al., 2011). Recent data demonstrated that maternal serum FABP4 is independently
associated with the subsequent development of preeclampsia. Elevated maternal serum FABP4
levels may also play a role in the pathogenesis of preeclampsia through pathways related to
insulin resistance, inflammation, and abnormal lipid metabolism (Scifres et al., 2011). All these
observations further warrant studies in order to understand the mechanisms as to how FABP4
regulates angiogenesis in the first trimester placenta. We demonstrated that leptin,
docosahexaenoic acid, 22:6n-3 (DHA) and c9, t11-conjugated linoleic acid (c9, t11-CLA)
stimulated FABP4 mRNA synthesis with concomitant enhanced tube formation in HTR8/SVneo
cells (Johnsen et al., 2011, Basak et al., 2013, Basak and Duttaroy, 2012, Basak and Duttaroy,
2013a). However, further study is required to ascertain the relationships between VEGF,
angiopoietin 4-like protein (ANGPTL4), dietary fatty acids and the roles of FABP4 in tube
formation of the placental first trimester trophoblasts.
In this paper we report for the first time about the differential effects of VEGF, leptin,
ANGPTL4 and dietary fatty acids (OA, and DHA) on FABP4 expression and its impact on tube
formation in placental first trimester trophoblasts. Expression of FABP4 protein was associated
with leptin, VEGF, and DHA induced-angiogenesis but not in ANGPTL4- and oleic acid (OA)-
mediated tube formation of these cells. In addition, FABP4 may not be involved as the key
regulator in these cells as observed in endothelial cells.
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2. Materials and Methods
2.1 Materials
The HTR8/SVneo trophoblast cell line was gifted by Dr. C.H. Graham, Queens University,
Canada. All the radiolabeled and unlabeled fatty acids were obtained as described previously
(Johnsen et al., 2011, Basak and Duttaroy, 2013a). Recombinant human VEGFA and ANGPTL4
were purchased from R&D and Abnova (USA) respectively. Lactate dehydrogenase (LDH)
assay kit was obtained from Roche Molecular Biochemical, Mannheim, Germany. Matrigel was
from BD Biosciences, USA. Rapamycin (mTOR inhibitor) and FABP4 inhibitor (BMS309403)
were obtained Calbiochem, UK. p38 MAP kinase inhibitor (SB203580) and NOS inhibitor, L-
Ng-nitro-L-arginine methyl ester (L-NAME) were obtained from Cell signaling Technology,
Inc., USA. TrypsinEDTA, penicillinstreptomycin solution, 3-(4, 5-dimethyl thiazol-2-yl)-2,5
diphenyl tetrazoliumbromide (MTT) and RPMI-1640 medium and all other chemicals were
obtained from Sigma Aldrich AS Norway.
2.2 Methods
2.2.1 Cell culture
The HTR8/SVneo cells were maintained in RPMI-1640 medium supplemented with 10% fetal
calf serum (Integro, Dieren, Holland), 2 mM L-glutamine, penicillin (50 units/ml), and
streptomycin (50g/ml) at 37 C in 5% CO2as described before (Johnsen et al., 2011).
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2.2.2 Cellular viability and proliferation assay
Cell viability and proliferation was performed as a measure of cellular growth and differentiation
as described before (Basak and Duttaroy, 2013a). Cells were incubated with different
angiogenic modulators including VEGF (10ng/ml), leptin (25ng/ml), angiopoietin-4 like protein
(ANGPTL4) (40ng/ml), OA (50M) and DHA (50M) in the absence and presence of different
inhibitors such as rapamycin (20nM), p38 MAP kinase inhibitor (5M), L-NAME (2mM) and
FABP4 inhibitor (50M). 3-(4,-dimethylthiazol-2-yl)-2,-diphenyl tetrazoliumbromide(MTT)
was used to detect viable proliferating cells. The absorbance was read at 562nm.
2.2.3 Uptake of radiolabeled fatty acids by HTR8/SVneo cells: Effect of FABP4 inhibitor
Typically, radiolabeled fatty acid was dissolved in serum-free RPMI containing fat-free BSA to
which appropriate quantities of the corresponding unlabeled fatty acid were added in order to
achieve the desired final concentrations, as described (Basak and Duttaroy, 2013a). The fatty
acid uptake was carried out as described before (Basak and Duttaroy, 2013a). The cells were pre-
incubated with FABP4 inhibitor (50M) for 1h, followed by 3h incubation with14
C fatty acids
with 100M of radiolabeled fatty acids of ([14
C]Oleic acid, [14
C]Linolenic acid,
[14
C]Arachidonic acid, [14
C]Eicosapentaenoic acid and [14
C]Docosahexaenoic acid (specific
activity 10002000 cpm/nmol). Fatty acid uptake was stopped by the addition of an ice-cold
solution of 0.5% fatty acid-free BSA and the cells were washed twice with 0.5% fatty acid-free
BSA and twice with PBS to remove any surface-bound fatty acid. The cells were dissolved by
the addition of 1 ml of 0.1M NaOH and left overnight at 4C. Cells were then scraped and 300l
aliquots of cell homogenate were transferred into scintillation vials containing 2ml of
scintillation cocktail. The radioactivity was determined using a scintillation counter. Data were
expressed as picomol of fatty acid taken up/g of cellular protein.
2.2.4 Tube formation assay
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Cellular angiogenesis was measured in vitro based on tube formation on an extracellular
matrigel, as described before (Johnsen et al., 2011). The cells were seeded (5x104cells/well/24
well plate) on matrigel (growth factor reduced) and FABP4 inhibitor (50M), rapamycin
(20nM), p38 MAP kinase inhibitor (5M), L-NAME (2mM) or all the inhibitors were added to
the cells in designated wells. In other experiments, angiogenic factors such as DHA (50M),
VEGF (10ng/ml), leptin (25ng/ml), ANGPTL4 (40ng/ml) or OA (50M) were added in separate
wells with or without mentioned inhibitors (same concentrations) to observe relative effects on
tube formation. The wells were captured after 16h by an inverted microscope at 40X
magnification (Nikon TS100F, Japan). Capillary tube length was quantified and expressed in
pixel [7]. Images were captured from the central view of at least five different fields per well and
extreme edges were excluded due to gel meniscus formation. Adobe Photoshop (version CS4)
was used to quantify tubule length of the capillary network formation. The results were
expressed pixel or as % over control using the formula: % over control = the mean length of total
tubes (assay groups) 100 / mean length of tubes (control groups).
2.2.5 Western blot analysis of FABP4 expression
HTR8/SVneo cells were pre-incubated in the absence and presence of VEGF (10ng/ml) and
leptin (25ng/ml) and fatty acids (50-100M) for 24h. Cells were lysed with 200l of
radioimmuno precipitation assay (RIPA) buffer followed by sonication and centrifugation as
described previously [8]. The supernatants were estimated for protein levels with BCA protein
assay kit (Pierce, USA) and 10g of protein/lane was resolved by SDS-PAGE (12%) prior to
their transfer to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp.).
Immediately after blocking, membranes were immunoblotted with antibodies against anti
FABP4 (1:5000, PA-530591, Thermo Scientific Pierce, USA), anti -actin (1:5000; ab-8227,
Abcam) and incubated with peroxidase conjugated goat anti-rabbit IgG (1:10,000, 31460
Thermo Scientific Pierce, USA). The blots were detected by using enhanced chemiluminescence
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substrate (Cat-32132, Pierce, USA). Immunoblot signals were captured by Storm860 phosphor
imager and quantified by Image Quant software (GE healthcare).
2.2.6 Quantitative estimation of gene expression by real-time PCR
Total RNA was isolated from the HTR8/SVneo cells using TRI reagent (Sigma T9424) as per
the instruction of the supplier. Total RNA was purified with DNase I (Sigma AMPD1) and
cDNAs were synthesized using iScript cDNA synthesis kit (Biorad #1708891). Reverse
transcription of cDNA was performed by power SYBR green PCR master mix (Life technologies
Part no.4367659) along with predesigned primers, KiCqStart SYBR green (Sigma) (Table
1). Real time PCR was carried out in ABI 7500 (Life Technology, USA). The Ct value of an
endogenous control gene TBP (TATA binding protein) was subtracted from the corresponding
Ct value for the target gene resulting in the delta Ct value which was used for relative
quantification of gene expression by the comparative Ct method (2Ct
).
3. Statistics and data analysis
All the values are presented as mean and standard errors of mean (SEM). Level of significance
was calculated by using Students t-test. A p-value of
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4. Results
4.1 Basal tube formation in HTR8/SVneo cells: Effect of inhibitors of FABP4 and VEGF
mediated angiogenic signaling pathways
The basal tube formation (as a measure of in vitroangiogenesis) was performed on matrigel in
the presence and absence of various inhibitors of VEGF signaling pathways and FABP4 in order
to evaluate the effect of these inhibitors on angiogenesis in HTR8/SVneo cells. Inhibitors used
were rapamycin (mTOR inhibitor, 2nM), SB203580 (p38 MAP kinase inhibitor, 5 M), L-
NAME (eNOS inhibitor, 2mM), FABP4 inhibitor (BMS309403, 50M). Basal tube formation
and the effects of inhibitors were measured by tube length. Total length of tubular network as
well as number of branches and connection points were significantly inhibited upon the
treatment compared with the basal tube formation (control, p< 0.05). Fig. 1shows the effect of
different inhibitors on basal tube formation capacity of the HTR8/SVneo cells. All these
inhibitors blocked tube formation significantly but FABP4 inhibitor mediated its inhibitory effect
on the tube formation to the greatest extent (p
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4.2 Inducer mediated tube formation in the first trimester trophoblast cells, HTR8/SVneo
: Effects of angiogenesis signaling pathway inhibitors
Effects of inhibitors (rapamycin, L-NAME, p38 MAP kinase inhibitor, and FABP4 inhibitor) on
stimulated tube formation in the presence of DHA, leptin, VEGF, OA, or ANGPTL4 in these
cells is as shown in Fig 2. DHA-induced tube formation was inhibited in the order: p38 MAP
kinase inhibitor (63.7%; 2025 38.19, n=3) > rapamycin (60.1%; 2225 58.38, n=3) > FABP4
inhibitor (28.1%; 4015 67.14, n=3) (Fig 2A). However, L-NAME inhibited the least (10.2%;
5015 110.6, n=3), p< 0.05. Fig 2Bshows the inhibition of leptin-induced tube formation by
these inhibitors. Unlike DHA and VEGF, FABP4 inhibitor blocked leptin stimulated tube
formation to the largest extent (50.7%; 2590 16.07 n=3) followed by L-NAME (35.2%; 3400
117.3, n=3) whereas p38 MAP kinase inhibitor had no effect. VEGF-induced tube formation was
inhibited in the order of p38 MAP kinase inhibitor (34.5%; 2725 38.19, n=3) > rapamycin
(27.8%; 3000 28.87, n=3) > FABP4 inhibitor (14.2%; 3567 22.05, n=3), p< 0.005 (Fig 2C).
WiththeexceptionofFABP4andLNAMEinhibitor,ANGPTL4andOA inducedtubeformation
wasnotinhibitedbymajorityoftheseinhibitorsofVEGFsignallingmediators(Fig 2D and E).
FABP4 inhibitor significantly blocked the tube formation stimulated by DHA (p
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inhibited by BMS309403 in the range of ~15-25% when treated with DHA, OA, VEGF and
leptin as compared with their respective controls (p
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In order to investigate the expression of FABP4 at protein level, HTR8/SVneo cells were pre-
incubated with VEGF (10ng/ml), leptin (25ng/ml), DHA and OA (50 M) for 24h and harvested
whole cell lysate for Western blotting. Fig. 5 showed increased expression of FABP4 protein
level in these cells in the presence of VEGF, leptin and OA compared with control. DHA
however had no effect on FABP4 protein expression in these cells. Relative expression of
FABP4 was increased significantly by VEGF (34%; p
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5. Discussion
Cell tube network formation on matrigel occurs as a consequence of a number of necessary
biological activities, including cell migration, proliferation, cellcell junction formation and cell
elongation. However, these processes do not mimic the whole process of in vivoangiogenesis. We
investigated the tube formation as a measure of angiogenesis as evident in early placentation
process. The mechanisms that determine the angiogenic capacity of VEGF, leptin, and fatty acids
in first trimester placental trophoblast cells may underlie important differences in the mechanism
of actions between them. We previously demonstrated that DHA stimulated the expression of
VEGF with concomitant increase in the cellular proliferation and tube formation (as a measure of
angiogenesis) in the first trimester trophoblast cells, HTR8/SVneo (Johnsen et al., 2011, Basak
and Duttaroy, 2013b). In contrast to DHA, other long chain fatty acids such as EPA, AA, OA and
CLA promote synthesis of ANGPTL4 and tube formation without affecting VEGF synthesis in
these trophoblast cells (Johnsen et al., 2011, Basak and Duttaroy, 2013b). Based on these data, we
proposed that different mechanisms of action of DHA and other long chain fatty acids on tube
formation may operate in the tube formation of the first trimester trophoblast cells.
Recent studies have highlighted FABP4 as a novel target of VEGF and its mediators of the
VEGF signalling pathway in endothelial cells (Ghelfi et al., 2013, Elmasri et al., 2012, Cataltepe
et al., 2012). In addition, FABP4 has been reported as a positive regulator of cell proliferation
and angiogenesis in endothelial cells (Ghelfi et al., 2013). We previously reported that fatty acids
such as, EPA, DHA, c9t11-CLA and leptin stimulate mRNA expression of FABP4 in the first
trimester trophoblast cells, HTR8/SVneo(Johnsen et al., 2011, Basak et al., 2013, Basak and
Duttaroy, 2012, Basak and Duttaroy, 2013a). However, the role of FABP4 on the tube formation
mediated by VEGF, ANGPTL4 and fatty acids in these cells is not known.
In order to ascertain the role of FABP4, we investigated various aspects of angiogenesis
processes such as cellular growth and proliferation, tube formation, and fatty acid uptake in the
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presence of different angiogenic factors, and FABP4 inhibitor in placental first trimester cells,
HTR8/SVneo. In order to understand VEGF-FABP4 cross talk, we used several inhibitors of
VEGF signaling pathway mediators such as rapamycin (mTOR inhibitor), P38 kinase inhibitor
and L-NAME (eNOS inhibitor) to further elucidate the mechanism of DHA, VEGF and
ANGPTL4 and fatty acid-mediated angiogenesis in these cells.
Angiogenesis is regulated by a complex interplay between pro-angiogenic and anti-angiogenic
factors. In order to explore the involvement of VEGF signaling pathways that are activated
downstream during angiogenesis, we used several inhibitors of VEGF signaling pathways
mediators. A major signaling event downstream of VEGF is the activation of AKT which is
regulated by phosphoinositide-dependent kinase 1 and mammalian target of rapamycin (mTOR)
complex. mTOR is a serine/threonine kinase that regulates a diverse array of cellular processes,
including cell growth, survival, metabolism, and cytoskeleton dynamics. Angiogenesis depends
on Akt/mTOR and VEGF signaling cascade. Rapamycin, is an inhibitor of mammalian target of
mTOR. Inhibition of mTOR has been shown to block the actions of VEGF through both
inhibition of VEGF synthesis and signal transduction (Del Bufalo et al., 2006, Guba et al., 2002).
Rapamycin has been shown to block tube formation in endothelial cells (Luo et al., 2012).
Placenta expresses high level of p38 and p38 but not p38 and p38 (Wang et al.,
1997)whereas vascular endothelial cells co-express p38 and p38 (Hale et al., 1999). VEGF
activate p38 (Rousseau et al., 1997). p38 MAP kinase inhibitor (SB203580) has been shown to
inhibit p38 and p38(Lee et al., 1999) and VEGF-induced tube formation in different cell
systems (Lin et al., 2015, Wu et al., 2006). Whereas, eNOS inhibitor (L-NAME) has been used
in inhibition of angiogenesis in cells (Lin et al., 2015).
We reported previously that tube formation is spontaneous at the basal level in HTR8/SVneo cell
(Johnsen et al., 2011). Secretion of VEGF in the basal condition media of HTR8/SVneo cells in
the matrigel indicates that VEGF predominantly drives tube formation at the basal level. At the
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basal level, inhibition of the tube formation in the HTR8/SVneo cells was observed in the
following order: FABP4 inhibitor> P38 inhibitor kinase> rapamycin >L-NAME. Since effect of
FABP4 inhibitor was more potent at basal level (or non-induced state) of HTR8/SVneo cells, it is
reasonable to argue that FABP4 may be more involved in VEGF mediated tube formation
compared with other VEGF signaling mediators in these cells. However, inhibition of tube
formation at the stimulated levels by these compounds such as p38 MAP kinase inhibitor,
rapamycin, and L-NAME demonstrated differential effects on tube formation in first trimester
trophoblast cells, HTR8/SVneo. Rapamycin and p38 MAPK inhibitor blocked both VEGF- and
DHA- mediated tube formation in these cells. It was demonstrated that DHA stimulated tube
formation via VEGF (Johnsen et al., 2011). Therefore, as expected, the inhibitors of VEGF
signaling pathways blocked VEGF-, DHA- and leptin- stimulated tube formation to a different
degree without affecting ANGPTL4- and OA-induced tube formation. These data and others
indicate that FABP4 is involved in VEGF-mediated tube formation of endothelial cells (Elmasri
et al., 2012, Ghelfi et al., 2013, Elmasri et al., 2009). Our data showed that FABP4 was involved
in cellular growth and proliferation, and its inhibitor blocked DHA-, VEGF- and leptin-mediated
tube formation in vitro but with different degrees. We showed that leptin stimulated tube
formation was not inhibited by the selective inhibitor of VEGF, indicating that its action was
independent of VEGF and ANGPTL4 (Basak and Duttaroy, 2012). Leptin, however,
significantly increased the expression of FABP4 and genes those are involved in angiogenesis
pathways(Basak and Duttaroy, 2012).
This paper also reports for the first time that FABP4 protein expression is increased in the
presence of VEGF in the first trimester trophoblast cells, HTR8/SVneo. Unlike protein level, the
basal levels of VEGF and FABP4 mRNA expression were lower in HTR8/SVneo cells as
compared to Ea.Hy926 (an endothelial cell line) as evidenced by the differential Ct values in
these conditions (unpublished data). It is possible that VEGF-induced FABP4 expression at
protein level contributed in the augmented tube formation of the first trimester trophoblast cells.
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FABP4 has been shown as essential for trophoblast lipid accumulation (Scifres et al., 2011).
FABP4 delivers fatty acids to different intracellular compartments and thus effect fatty acid
metabolism, and also gene expression by delivering fatty acid ligands to the peroxisome
proliferator-activated receptors(Duttaroy, 2009). VEGF down regulated mRNA expression of
ADRP in the HTR8/SVneo cells possibly sequestered fatty acids for metabolic activities instead
of storage as a cellular energy. However, further work of ADRP expression at protein level and
lipid droplets would be required in the first trimester cells for definitive conclusions.
FABP4 had less effect on ANGPTL4- and OA- induced tube formation compared with VEGF-
mediated tube formation in these cells despite the facts that ANGPTL4 played a crucial role in
angiogenesis, metabolism and uptake of fatty acids particularly as an inhibitor of lipoprotein
lipase activity (Georgiadi et al., 2010, Chi et al., 2015). FABP4 which is the principal and main
target of VEGF induced angiogenesis in endothelial cells(Elmasri et al., 2012, Elmasri et al.,
2009, Harjes et al., 2014), may not be solely responsible for angiogenesis processes in these
cells, as suggested by different experimental observations obtained from tube formation, cellular
growth and its mRNA and protein expression.
In conclusion, we demonstrate that the differential effects of VEGF, leptin, ANGPTL4 and fatty
acids on FABP4 expression and its impact on angiogenesis (as measured as tube formation) in
placental first trimester trophoblasts. Expression of FABP4 protein was associated with leptin-,
VEGF-, and DHA- induced-tube formation but not in ANGPTL4- and OA mediated tube
formation in these cells. In addition, at basal level of tube formation in HTR8/SVneo cells,
FABP4-VEGF axis may be more involved in tube formation. However, FABP4 is not the key
regulator in stimulated tube formation by DHA, VEGF, and leptin in these cells and has no or
little involvement in ANGPTL4-, OA- mediated tube formation.
Acknowledgements
This study was supported by the grant from the Thune Holst Foundation.
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Table 1: List of predesigned SYBR Green I primers for gene expression analysis
SL.
No.
PrimerID Gene
symbol
GeneID Genename Nucleotidesequences(53) Ref_seqID
1 H_PLIN2_1 ADRP 123 Adiposedifferentiation
relatedprotein
F5 GTTCACCTGATTGAATTTGC3
R5
GAGGTAGAGCTTATCCTGAG
3
NM_001122
2 H_ACSL3_3 ACSL3 2181 AcylCoAsynthetaselong
chainfamilymember3
F5 GAGAGGAAGATGTCTACATTG3
R5 CTGATCTGCTAAAGTCTGTG3
NM_004457
3 H_ACSL5_3 ACSL5 51703 AcylCoAsynthetaselong
chainfamilymember5
F5 CATCCTTAGTAGGAGTGGTG3
R5 TTTAAGGCCACTTTCTTTCC3
NM_016234
4 H_FABP4_1 FABP4 2167 Fattyacidbindingprotein4 F5 CAAGAGCACCATAACCTTAG3
R5 CTCGTTTTCTCTTTATGGTGG3
NM_001442
5 H_LPL_1
LPL
4023
Lipoproteinlipase
F5 ACACAGAGGTAGATATTGGAG3
R5 CTTTTTCTGAGTCTCTCCTG3
NM_000237
6 H_TBP_1 TBP 6908 TATAbindingprotein F5 GCCAAGAGTGAAGAACAG3
R5 GAAGTCCAAGAACTTAGCTG3
NM_003194
7 H_HIF1A_2
HIF1A
3091
Hypoxiainduciblefactor1F5
GAAACTACTAGTGCCACATC3
R5GGAACTGTAGTTCTTTGACTC
3
NM_001243084
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Figure Legends
Figure 1. Effects of angiogenesis signaling pathway inhibitors on basal tube formation of
the HTR8/SVneo cells
Serum-starved cells (5104/ well) were cultured on pre-solidified matrigel in the presence of and
absence of inhibitors (P38 kinase inhibitor, Rapamycin, L-NAME, and FABP4 inhibitor), as
described in the Methods section. The micrographs show basal level tube formation in vitro in
HTR8/SVneo trophoblast cells in the absence and presence of inhibitors. (A) Control without the
presence of any angiogenic inhibitors, (B) in the presence of 5M P38 MAP kinase inhibitor, (C)
20nM rapamycin, (D)2mM L-NAME , (E) 50M FABP4 inhibitor and (F)all inhibitors. (G)
shows graphical presentation of the relative tube length (pixel) of HTR8/SVneo trophoblast cells
in the absence or presence of angiogenic inhibitors. The data are a representative experiment of
three independent experiments performed in triplicate (n = 3) SEM. Statistical significance was
determined using the unpaired t-test. Asterisks indicate level of significance of each data set.
**** p< 0.0001
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Figure 2. Effects of angiogenesis signaling pathway inhibitors on tube formation induced
by VEGF, ANGPTL4, leptin, DHA and OA in the first trimester trophoblast cells,
HTR8/SVneo
The graphs indicate relative tube formation profile in the presence of (A) 50M DHA, (B)
25ng/ml leptin, (C)VEGF 10ng/ml, (D)50M OA (E)40ng/ml ANGPTL4 in HTR8/SVneo
trophoblast cells in the absence or presence of angiogenic inhibitors (p38 MAP kinase inhibitor,
rapamycin, L-NAME, and FABP4 inhibitor). Statistical significance was determined using the
unpaired t-test. The data are a representative experiment of three independent experiments
performed in triplicate (n = 3) SEM. Statistical significance was determined using the unpaired
t-test. Asterisks indicate significance of each data set. Strength of significant data was
categorized by number of asterisks ranging from 1 to 4 *p
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Figure 4 Effects of VEGF on mRNA expression of lipid metabolic genes in the first
trimester placental trophoblast HTR8/SVneo cells.
Expression of mRNA was measured after the cells were incubated with VEGF (0 and 10 ng/ml)
for 24 h. The mRNA expression was analyzed using quantitative real-time RT-PCR normalized
to the endogenous control TBP. Fold change of gene expression was calculated according to the
Ct method. Data represent means SEM obtained from two separate experiments in
triplicates. *p
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