Tube formation in the first trimester placental trophoblast cells: Differential effects of angiogenic growth factors and fatty acids

7/26/2019 Tube formation in the first trimester placental trophoblast cells: Differential effects of angiogenic growth factors an

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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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Documents

Tube formation in the first trimester placental trophoblast cells: Differential effects of angiogenic growth factors and fatty acids