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Department of Nutrition
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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, LNAME, 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<0.05) but did not affected by ANGPTL4. VEGF, leptin and OA
also increased FABP4 protein level in these cells though the uptake of fatty acids by these cells
was not affected by the presence of FABP4 inhibitor. Our data demonstrate that FABP4 may be
involved in part in the basal level, and stimulated tube formation by VEGF, DHA, and leptin
whereas it has little or no effect in ANGPTL4- and OA- induced tube formation in these cells.
Thus, FABP4 may play a differential role in fatty acids and angiogenic growth factors mediated
tube formation in the first trimester trophoblst cells in vitro.
Keywords: FABP4, VEGF, ANGPTL4, DHA, Tube formation, first trimester trophoblast cells,
HTR8/SVneo cells, FABP4 inhibitor.
1. Introduction
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
4
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.
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, LNg-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
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 preincubated with FABP4 inhibitor (50M) for 1h, followed by 3h incubation with 14C fatty acids
with 100M of radiolabeled fatty acids of ([14C]Oleic acid, [14C]Linolenic acid,
[14C]Arachidonic acid, [14C]Eicosapentaenoic acid and [14C]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
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 <0.05 was considered statistically
significant. Statistical significance for MTT data was determined using the Holm-Sidak method,
with alpha=5.000%.
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 vitro angiogenesis) 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), LNAME (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. 1 shows 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<0.0001, unpaired t-test). Tube formation was
maximally inhibited in the presence of FABP4 inhibitor compared with other inhibitors
(rapamycin, L-NAME, and p38 MAP kinase inhibitor) at their respective potent doses in these
cells. Comparison between the inhibitors on tube length showed differential inhibitory effects of
these compounds. Inhibitory potencies on basal tube formation was in the order of FABP4
inhibitor (57.3%; 1200 14.43, n=3), p38 MAP kinase inhibitor (36.7%; 1778 17.40, n=3),
rapamycin (32.2%; 1905 10.41, n=3), and L-NAME (28.5%; 2008 22.05, n=3). Presence of
all inhibitors could not however block the tube formation (42.5%; 1615 7,638, n=3) more than
that by FABP4 inhibitor alone (57.3%; 1200 14.43, n=3).
10
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 2B shows 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,ANGPTL4andOAinducedtubeformation
wasnotinhibitedbymajorityoftheseinhibitorsofVEGFsignallingmediators(Fig 2D and E).
FABP4 inhibitor significantly blocked the tube formation stimulated by DHA (p<0.005), leptin
(p< 0.0001), VEGF (p<0.005) and to some extent OA stimulated tube formation (p=0.0165),
whereas it did not considerably affect the tube formation induced by ANGPTL4 (p=0.90003).
4.3 Effects of inhibitors FABP4 and VEGF signaling pathways on cell growth and
proliferation in HTR8/SVneo cells
In order to elucidate further the roles of FABP4 on cellular growth and proliferation,
HTR8/SVneo cells were cultured in the presence and absence of FABP4 inhibitor (BMS309403)
along with VEGF, leptin, OA and DHA. The growth and proliferation of HTR8/SVneo cells was
11
inhibited by BMS309403 in the range of ~15-25% when treated with DHA, OA, VEGF and
leptin as compared with their respective controls (p<0.05). The cell proliferation inhibited by
BMS309403 at basal level (control) was 16% (p<0.05) without any treatment. FABP4 inhibitor
did not affect the cellular growth stimulated by ANGPTL4.
4.4 Effect of FABP4 inhibitor on fatty acid uptakes in HTR8/SVneo cells
In order to understand the role of FABP4 in the fatty acid uptake of the first trimester trophoblast
cells, we examined the uptake of ([14C]Oleic acid (OA), [14C]-Linolenic acid (LA),
[14C]Arachidonic acid (AA), [14C]Eicosapentaenoic acid (EPA) and [14C]Docosahexaenoic acid
(DHA) in the presence and absence of FABP4 inhibitor. The presence of FABP4 inhibitor did
not affect the uptake of these radiolabeled fatty acids compared with control (Data not shown).
4.5 Effects of VEGF on mRNA expression of lipid metabolic genes in HTR8/SVneo cells
In order to determine whether VEGF-induced tube formation was associated with lipid
metabolism of these cells, we measured the mRNA expression of FABP4 and other important
lipid metabolic genes such as LPL, ACSL3, and ACSL5, ADRP and HIF1 in the absence and
presence of this angiogenesis inducer. Pre-incubation of HTR8/SVneo cells with VEGF
(10ng/ml) did not alter the expression of genes associated with fatty acid uptake, binding, and
metabolic activities as compared with those in controls. However, there was a significant
decrease (2.7 fold), p<0.05) in the expression of ADRP, a gene encodes lipid droplet protein, in
the first trimester trophoblast cells as compared with the control (Fig. 4).
4.6 Effect of OA, DHA, VEGF, and leptin, on FABP4 protein expression in HTR8/SVneo
cells
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In order to investigate the expression of FABP4 at protein level, HTR8/SVneo cells were preincubated 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<0.0001), leptin and OA (15% ; p<0.002)
as compared with the control (Fig. 5 A-C).
13
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 vivo angiogenesis. 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.
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
15
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.
16
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 VEGFmediated 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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19
Table 1: List of predesigned SYBR Green I primers for gene expression analysis
SL.
PrimerID
Gene
symbol
GeneID
Genename
Nucleotidesequences(53)
Ref_seqID
H_PLIN2_1
ADRP
123
Adiposedifferentiation
relatedprotein
F5GTTCACCTGATTGAATTTGC3
NM_001122
No.
1
R5GAGGTAGAGCTTATCCTGAG3
2
H_ACSL3_3
ACSL3
2181
AcylCoAsynthetaselong
chainfamilymember3
F5GAGAGGAAGATGTCTACATTG3
NM_004457
R5CTGATCTGCTAAAGTCTGTG3
3
H_ACSL5_3
ACSL5
51703
AcylCoAsynthetaselong
chainfamilymember5
F5CATCCTTAGTAGGAGTGGTG3
NM_016234
R5TTTAAGGCCACTTTCTTTCC3
4
H_FABP4_1
FABP4
2167
Fattyacidbindingprotein4
H_LPL_1
H_TBP_1
H_HIF1A_2
NM_001442
R5CTCGTTTTCTCTTTATGGTGG3
LPL
4023
TBP
6908
Lipoproteinlipase
TATAbindingprotein
F5CAAGAGCACCATAACCTTAG3
F5ACACAGAGGTAGATATTGGAG3
NM_000237
R5CTTTTTCTGAGTCTCTCCTG3
F5GCCAAGAGTGAAGAACAG3
NM_003194
R5GAAGTCCAAGAACTTAGCTG3
HIF1A
3091
Hypoxiainduciblefactor1
F5
GAAACTACTAGTGCCACATC
R5
GGAACTGTAGTTCTTTGACTC
NM_001243084
20
Figure Legends
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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<0.05 (least significant), **p<0.005,
*** p<0.0005, **** p<0.0001 (most significant).
Figure 3. Effects of FABP4 inhibitor on cell growth and proliferation of HTR8/SVneo cells.
Cells (2-9 103/well/96 plate) viability was measured by MTT assay as described in the Method
section. The absorbance was read at 562 nm. Data represent mean SEM (n = 3). Percentage of
changes in proliferation was calculated by considering control as 100 % after normalized over
basal values. The graph shows relative growth effects of DHA (50M), OA (50M), VEGF
(10ng/ml), leptin (25ng/ml) and ANGPTL4 (40ng/ml) on HTR8/SVneo trophoblast cells in the
absence or presence of 50M FABP4 inhibitor in the cell proliferation assay. Statistical
significance was determined using the Holm-Sidak method, with alpha=5.000%. Statistical
significance was determined using the unpaired t-test. Asterisks indicate significance of each
data set.* p< 0.05
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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<0.05.
Figure 5. Effect of VEGF, leptin and fatty acids on FABP4 protein expression in
HTR8/SVneo cells.
Cell lysates were prepared after incubating these HTR8/SVneo cells in the absence and presence
of
(A) Fatty acids (50M, OA : Oleic acid ; DHA : Docosahexaenoic acid; Con: control (B)
VEGF 10ng/ml and (C) leptin 25ng/ml for 24 h. Western blot was performed for the expression
of FABP4 protein as described in the Methods section. Bar graphs showing quantification of the
expression level of each protein compared with the expression of -actin. Relative expression
was calculated after normalizing expression of the protein with -actin. Data are expressed as
arbitrary folds of changes over control. Data are expressed as Means SEM. Western blots are
representative of three independent experiments. **p<0.002; *** p<0.0001
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