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BOR - Papers in Press, published online ahead of print March 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.039479
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BIOLOGY OF REPRODUCTION 73, 172–179 (2005)
DOI: 10.1095/biolreprod.104.039479
© 2005 by the Society for the Study of Reproduction, Inc.

Tumor Necrosis Factor and Vascular Endothelial Growth Factor Induce Endothelial Integrin Repertories, Regulating Endovascular Differentiation and Apoptosis in a Human Extravillous Trophoblast Cell Line1

Kotaro Fukushima 2 3, Shingo Miyamoto 3, Kiyomi Tsukimori 3, Hiroaki Kobayashi 3, Hiroyuki Seki 5, Satoru Takeda 5, Egashira Kensuke 4, Kishio Ohtani 4, Masabumi Shibuya 6, and Hitoo Nakano 3

Departments of Obstetrics and Gynecology,3 Cardiovascular Medicine,4 Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, JapanDepartment of Obstetrics and Gynecology,5 Saitama Medical Center, Saitama Medical School, Saitama 350-8550, JapanDivision of Genetics,6 Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis is crucial in human development. Extravillous trophoblast (EVT) cells mimic endothelial cells in angiogenesis during endovascular differentiation, inducing a remodeling of spiral arteries that increases blood flow toward the intravillous space. We have previously shown that tumor necrosis factor (TNF) alpha regulates expression of ITGA6 and ITGA1, which are involved in cell survival, in the human EVT cell line TCL1. To further investigate endovascular differentiation, we examined the effects of vascular endothelial growth factor (VEGF), TNF, and extracellular matrix (ECM) on TCL1 cells. Seeded on Matrigel, TCL1 cells show tube-like formation that specifically recalls morphological changes in endothelial cells. Anti-ITGAV/ITGB3 antibodies significantly reduced the size of the capillary network (P < 0.05) on Matrigel and also suppressed TNF-induced apoptosis (P < 0.05) in TCL1 cells. VEGF induced expression of ITGAV/ITGB3 subunits and protein aggregation, as in the case of TNF, which in turn, induces synthesis of VEGF in TCL1 cells. Soluble FLT1 suppressed these activities in TCL1 cells, indicating that signals involving VEGF axis are essential for endovascular differentiation. These results suggest that TNF, VEGF, and ECM collaboratively regulate EVT behavior, including cell survival and endovascular differentiation, through integrin signaling during establishment and maintenance of successful human pregnancies.

cytokines, early development, placenta, pregnancy, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis plays a crucial role in physiological and pathological conditions, including human placental development. During early development, cytotrophoblast (CT) differentiation contributes to organization of the feto-maternal interface. One type of differentiated CT is extravillous cytotrophoblast (EVT), which invades the interstitium and promotes placental anchorage. Interstitial invasion is accompanied by endovascular differentiation wherein EVT finally replaces endothelial cells in uterine vessels. Moreover, EVT also intrudes into vascular muscles, replacing endothelial cells. This endovascular differentiation induces a remodeling of spiral arteries that increases blood flow toward the intravillous space, which is covered by syncitiotrophoblast, the other form of differentiated CT [1, 2]. It is reasonable to assume that EVT may mimic the behavior of endothelial cells in angiogenesis during endovascular differentiation.

Integrin subunits expressed in EVT are dramatically altered not only during endovascular differentiation, but also during interstitial invasion. The ITGA6 (integrin, alpha 6)/ ITGB4 (integrin, beta 4) is down-regulated in EVT, whereas ITGA5 (integrin, alpha 5)/ITGB1 (integrin, beta 1) and ITGA1 (integrin, alpha 1)/ITGB1 subunits are up-regulated during differentiation and invasion [3, 4]. The significance of this integrin subunit conversion is highlighted by the fact that in pre-eclampsia, in which both interstitial and endovascular invasion are abnormally shallow, EVT cells show significant defects in differentiation. Furthermore, EVT in pre-eclampsia increases expression of {alpha}5ß1, but fails both to up-regulate the ITGA1/ITGB1 subunit and to down-regulate the ITGA6/ITGB4 subunit [5, 6]. Thus, it is likely that expression of these integrin subunits is involved in regulation of normal EVT differentiation and is essential for normal pregnancy. The molecules involved in this process have been unclear. However, we have reported that tumor necrosis factor (TNF) alpha is able to switch integrin expression and induce apoptosis in a human EVT cell line. In addition, this apoptosis is suppressed by signals via ITGB1. These results suggest that TNF and extracellular matrix (ECM) collaboratively regulate EVT differentiation through integrin signaling [7] and contribute to normal placental development.

Integrins mediate cell adhesion and migration, orchestrate organization of the cytoskeleton, and activate signal transduction pathways [8]. It is believed that ITGAV (integrin, alpha V)/ITGB3 (integrin, beta 3) and ITGAV/ ITGB5 (integrin, beta 5) subunits play an important role in angiogenesis [9, 10]. Immunohistochemical analysis has shown that EVT cells start to express adhesion molecules typical of endothelial cells, including ITGAV/ITGB3 but not ITGAV/ITGB5 subunits, during invasion [11, 12]. In addition, it is well known that vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis [13]. VEGF family members and their receptors(VEGFRs) seem to be important early in angiogenesis [14]. Witmer et al. [15] reported that VEGF up-regulates the ITGAV/ITGB3 subunit. This suggests that on the bases of the similarity of this process to angiogenesis that these integrins and the VEGF-VEGFR system might be involved in endovascular differentiation in EVT.

In this context, we would expect that conversion of integrin subunit expression during endovascular invasion is controlled by soluble factors or ECM in placenta, as it is for interstitial invasion. To clarify the molecules involved in endovascular differentiation, we examined the effects of VEGF, TNF, and ECM on expression of the ITGAV/ITGB3 subunit and investigated the significance of these molecules using a human EVT cell line, TCL1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

ECM products were as follows: poly-L-lysine was purchased from Sigma Chemical Co. (St. Louis, MO) and growth factor-reduced Matrigel was purchased from Becton Dickinson Labware (Bedford, MA). TNF and mouse anti-human ITGAV/ITGB3 monoclonal antibody was purchased from Chemicon International Inc. (Temecula, CA). VEGF was purchased from Calbiochem (Darmstadt, Germany). Anti-VEGF antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) for immunoblotting, and from Upstate Biotechnology (Lake Placid, NY) for the neutralization assay. Fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG) (Molecular Probes Inc.) was used as the secondary antibody.

Cell Lines and Cell Culture

TCL1 cells were established from mixed primary cultures of cells isolated from chorionic membrane obtained from elective caesarean deliveries performed before term [16]. Primary cultures contained 8%–10% EVT. Isolated cells were immortalized by retroviral expression of SV40 large antigen; single-cell cloning techniques revealed that cells with an epithelial morphology were the only type present in the population after 6 mo of culture. Cells showed no tumorigenicity either in vitro or in vivo. The cloned population expressed human chorionic gonadotropin, alpha, beta, and colony stimulating factor 1, but they did not express markers for decidualized endometrial cells, macrophages, or natural killer cells. TCL1 cells were positive for cytokeratin, whereas none stained for vimentin. In addition, TCL1 cells constitutively expressed gelatinase-A, but gelatinase-B was expressed only when cells were cultured in the presence of ECM, which specifically restricted phenotype of the invading cytotrophoblast [7, 16–18].

A second cell line, JEG3 [19], was established from human choriocarcinoma. Cells were cultured in RPMI1640 (Nipro, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; Gibco Invitrogen Corporation, Carlsbad, CA) or conditioned serum in a humidified atmosphere containing 95% air-5% CO2 at 37°C.

DNA Transfection

The 2.3-kilobase (kb) human soluble FLT1 (fms-related tyrosine kinase 1) DNA, originally obtained from the human cDNA library, was cloned into the EcoRI sites of the eukaryotic expression vector plasmid cDNA3 (Invitrogen Corp., Carlsbad, CA) [20]. DNA transfection was performed using lipofectamin reagents as recommended by the supplier (Invitrogen).

Detection of Apoptotic Nuclei

Coverslips used for ECM coating (Matsunami, Kishiwada, Japan) were precleaned using aqua regia. Coverslips were coated with 10 µg/ml poly-L-lysine, 10 mg/ml IgG, or anti-ITGAV/ITGB3 antibody for 1 h at room temperature. After a rinse, 2 x 105 TCL1 cells were seeded on treated coverslips and incubated overnight at 37°C. After two PBS washes, TCL1 cells were incubated for TNF on complete media containing 10% FCS. Cells were fixed with 100% methanol and stained with Hoechst 33852 (Molecular Probes). After washing twice, cells were mounted onto glass slides with shielding buffer and analyzed with fluorescent microscopy (Olympus, Tokyo, Japan). Fragmented nuclei were counted (100x). More than 500 cells, including both apoptotic and normal cells, were examined.

Colony Survival Assay

Exponentially growing TCL1 cells were harvested and suspended in serum-free media for 1 h. Then, 100 cells were seeded in each well of a 24-well dish coated with 10 µg/ml antibodies or IgG, with serum-free media containing 100 pg/ml TNF. After 12 h of incubation, cells were rinsed twice with PBS and allowed to grow for 7 days before the number of colonies was counted microscopically.

Tube-like Formation Assay

Growth factor-reduced Matrigel (Becton Dickinson) was added (300 µl) to each well of a 24-well plate and allowed to polymerize for 1 h at 37°C. A total of 2 x 105 TCL1 cells, preincubated with serum-free media for 1 h, were seeded with or without 10 µg/ml antibodies. Cells were incubated at 37°C, viewed (magnification 40x–400x), and photographed using an Olympus IX71 microscope. At least five fields were examined per well; each experimental condition was tested in triplicate.

Reverse Transcriptase-Polymerase Chain Reaction

Total cellular RNA prepared with Trizol (Invitrogen) was reverse transcribed using the first-strand cDNA synthesis kit (Amersham Biosciences, Piscataway, NJ) as recommended. Transcribed cDNAs were amplified with KOD polymerase (Toyobo, Osaka, Japan). Polymerase chain reactions (PCRs) were carried out with the following primers used for ITGAV [21]: sense primer, 5'-TGA GGA TAT CAC CAA CTC CAC A-3' and antisense primer, 5'-GTT GCT AAT TCT AGT GGG TCA-3'; for ITGB3 [22]: sense primer, 5'-TGC TCA TTG GCC TTG CCG CCC TGC-3' and antisense primer, 5'-ACT ATT CGT CAG TAG GAG TCT AGT-3'. Primers for the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPD), were included in each reaction: sense primer, 5'-GAG TCA ACG GAT TTG GTC GT-3' and antisense primer, 5'-GTT GTC ATG GAT GAC CTT GG A-3'. PCR was carried out for 30 cycles, each at 94°C for 45 sec, an appropriate annealing temperature for 45 sec, and at 72°C for 1 min. The gel was visualized with ethidium bromide and photographed.

Immunofluorescence

TCL1 cells growing exponentially (2 x 105) were seeded on poly-L-lysine coverslips. After incubation overnight, the cells were incubated with TNF or VEGF. The cells were then fixed with 4% paraformaldehyde for 10 min and permeabilized with PBS containing 0.5% Triton-X. After blocking with 3% BSA for 30 min, cells were incubated with an anti-ITGAV/ITGB3 antibody overnight at 4°C followed by incubation with FITC-conjugated secondary antibody for 45 min at room temperature; nuclei were stained with Hoechst 33852. After washing twice, cells were mounted onto slide glasses with shielding buffer. The cells were analyzed using an Olympus BX50 confocal fluorescent microscope.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblotting

Cells were lysed in buffer containing 62.5 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% (w/v) SDS, and 10% glycerol. Cellular proteins were electrophoresed in a 12% SDS gel together with a prestained molecular weight marker (Bio-Rad Laboratories, Hercules, CA), transferred onto Immobilon-P (Millipore, Bedford, MA), and then analyzed by immunoblotting as described elsewhere [23].

Statistical Analysis

Statistical analysis was performed using the unpaired t-test program in Graphpad Prism (Graphpad Software, Inc. San Diego, CA). A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF Induction of ITGAV/ITGB3 Subunit mRNA Expression in TCL1 Cells

Because TCL1 cells were established from human preterm placenta that expressed a specifically restricted phenotype of invading cytotrophoblast [16] known to express ITGAV/ITGB3 subunit during endovascular differentiation [11, 12], we investigated the effect of VEGF on the ITGAV/ITGB3 subunit using reverse transcriptase-PCR analysis. As shown in Figure 1, the relative expression of ITGAV and ITGB3 in TCL1 cells incubated with 10 ng/ml VEGF was 3.29 and 2.06 at 4 h, and 3.08 and 3.53 at 8 h, a finding that is consistent with results reported with endothelial cells [15]. Then we investigated the effect of TNF, which induced integrin {alpha}6-{alpha}1 switching in TCL1 cells [7] and found that TNF induced ITGAV and ITGB3 expression in TCL1 cells in the same manner as VEGF.



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  		FIG. 1. Expression of integrin subunits in TCL1 cells. Reverse transcriptase-PCR was performed for integrin ITGAV and ITGB3 subunits and GAPD (internal control) after incubation with 10 ng/ml VEGF (left) or 100 pg/ml TNF (right)

VEGF Induction of Integrin ITGAV/ITGB3 Subunit Aggregation via FLT1 Signaling

To address whether or not VEGF-induced ITGAV/ ITGB3 subunit expression is functional, we incubated TCL1 cells with VEGF and performed immunofluorescence analysis against the ITGAV/ITGB3 subunit (Fig. 2, a–c). The aggregation of integrin ITGAV/ITGB3 subunit was observed after incubation with 10 (Fig. 2b) or 100 (Fig. 2c) ng/ml VEGF for 8 h. In contrast, exponentially growing TCL1 cells did not show these aggregations (Fig. 2a).



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  		FIG. 2. VEGF induces ITGAV/ITGB3 integrin subunit aggregation via FLT1 signaling. Asynchronously growing TCL1 cells transformed with vector (a–c) or sFLT (d–f) were incubated for 8 h with VEGF, then fixed, stained, and observed with an original magnification x400. Bars = 20 µm. Arrowheads indicate aggregation

Next, we transfected soluble FLT1 DNA (sFLT1), an inhibitor against VEGF and its receptors, FLT1 and KDR (kinase insert domain receptor, also known as VEGF receptor 2) [24] into TCL1 cells and incubated the cells with VEGF. When sFLT1 was introduced (Fig. 2, d–f), aggregation of the ITGAV/ITGB3 subunit was not observed, even after incubation with 100 ng/ml VEGF (Fig. 2f).

TNF Induction of ITGAV/ITGB3 Integrin Subunit Aggregation via FLT1 Signaling

Because TNF induced ITGAV and ITGB3 expression, we investigated whether TNF could induce ITGAV/ITGB3 subunit aggregation in the same manner as VEGF had done (Fig. 3A). Nuclear staining analysis revealed that TNF induced apoptosis in about 40% of TCL1 cells seeded on a poly-L-lysine coated dish after 8 h of incubation (Fig. 3Aa). When stained with anti-ITGAV/ITGB3 antibody, TCL1 was shown to induce ITGAV/ITGB3 subunit aggregation (Fig. 3Ac). Surprisingly, sFLT1 suppressed ITGAV/ITGB3 subunit aggregation induced by TNF in the same way it had suppressed aggregation induced by VEGF (Fig. 3, Ab and Ad).



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  		FIG. 3. A) TNF strengthens ITGAV/ITGB3 adhesive function through FLT1. Asynchronously growing TCL1 cells transformed with vector (a, b) or sFLT (c, d) were incubated for 8 h with 100 pg/ml TNF, then stained and observed with an original magnification x400. Bars = 100 µm. The arrows indicate apoptotic nuclei, and arrowheads indicate integrin ITGAV/ITGB3 subunit aggregation. B) TNF induces VEGF expression in TCL1 cells. Asynchronously growing cells were incubated with 100 pg/ml TNF and incubated for the indicated time; then cellular proteins were extracted, electrophoresed, and transferred onto Immobilon-P before analysis by immunoblotting using anti-VEGF antibody (A20) (lower); Coomassie brilliant blue-stained SDS-PAGE gel as a protein control (upper). C) Neutralization of VEGF inhibits TNF-induced ITGAV/ITGB3 subunit aggregation. Asynchronously growing TCL1 were incubated for 8 h with 100 pg/ml TNF cells without (a, b) or with (c, d) 20 µg/ml anti-VEGF antibody (JH121), then stained and observed with an original magnification x400. Bars = 20 µm

Next, to investigate whether TNF could induce VEGF in TCL1 cells, we performed immunoblot analysis. As shown in Figure 3B, TNF induced synthesis of VEGF in TCL1 cells. Because it was assumed that the effect of TNF on ITGAV/ITGB3 subunit aggregation was induced via VEGF, we tried to neutralize VEGF using anti-VEGF antibody (Fig. 3C). When VEGF was neutralized, the aggregation of ITGAV/ITGB3 subunit was not observed.

Anti-ITGAV/ITGB3 Integrin Antibody Suppression of TNF-Induced Apoptosis in TCL1 Cells

We have previously reported that ECM rescues TNF-induced apoptosis through integrin receptor signaling [7]. To investigate the effect of the ITGAV/ITGB3 subunit on TNF-induced apoptosis, micro-colony formation assay (Fig. 4a) and nuclear staining with Hoechst (Fig. 4b) were performed. The number of colonies surviving on IgG and anti-ITGAV/ITGB3 antibody-coated dishes were 11.7 ± 4.1 and 36.8 ± 4.3, respectively. In nuclear staining experiments, the ratios of apoptotic cells on IgG and anti-ITGAV/ITGB3 antibody-coated coverslips were 45.5 ± 4.2% and 31.0 ± 4.5%, respectively.



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  		FIG. 4. Inhibition of TNF-induced apoptosis with anti-ITGAV/ITGB3 integrin antibodies. A) Colony survival assay. Number of TCL1 cell colonies after incubation with TNF was counted microscopically (x100). Data are represented as mean ± standard deviation of three independent trials. B) Nuclear staining. Hoechst 33852 staining of TCL1 cells exposed to IgG or anti-ITGAV/ITGB3 antibody followed by 12-h incubation with TNF reveals fragmented cells (see Fig. 3) that were counted microscopically (x100). Data are represented as mean ± standard deviation of three independent trials

Tube-like Formation of TCL1 Cells on Matrigel

To assess whether TCL1 shows morphology characteristic of endothelial cells, we seeded TCL1 cells on growth factor-reduced Matrigel. Two hours after initiation of incubation with Matrigel, TCL1 cells started to show tube-like formation, and the cells formed complete, typical, tube-like structures after 12 h of incubation (Fig. 5b). In contrast, JEG3 cells, which are derived from human choriocarcinoma, did not show typical tube-like formation even after 24 h of incubation (Fig. 5c).



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  		FIG. 5. TCL1 cells show tube-like formation on Matrigel. Tubes formed by TCL1 cells (a, b) or JEG3 cells (c) were examined at different magnifications (x40–x400). Bars = 100 µm

Endovascular Differentiation in TCL1 Cells and FLT1 Signaling

To clarify the role of ITGAV/ITGB3 subunit on tube-like formation in TCL1 cells, we examined the effect of IgG and anti-ITGAV/ITGB3 antibodies on tube-like formation (Fig. 6A). When incubated with anti-ITGAV/ITGB3 antibodies (Fig. 6, Ac and Ad), TCL1 cells showed impaired tube-like structure formation compared with that of controls (Fig. 6, Aa and Ab). The number of capillary networks (arrow) after 4 h of incubation in control and anti-ITGAV/ITGB3 antibodies was 17.8 ± 4.0 and 10.8 ± 3.7, respectively; after 8 h of incubation in control and anti-ITGAV/ITGB3 antibodies, the numbers were 20.8 ± 3.5 and 12.0 ± 3.6, respectively (Fig. 6B).



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  		FIG. 6. Tube-like formation in TCL1 cells requires signaling via ITGAV/ITGB3 subunits. A) Cells seeded on Matrigel with IgG (a, b) or anti-ITGAV/ ITGB3 antibody (c, d) were observed at 4 h (a, c) or 8 h (b, d). Bars = 100 µm. B) The number of capillary networks (arrow) per 1 mm2 surface area was counted at magnification x400. Data are represented as mean ± SD in three independent trials

Next, we investigated the effect of sFLT1 on tube-like formation in TCL1 cells (Fig. 7A). Tube-like structure formation was impaired in sFLT1-introduced cells (Fig. 7, Ad and Af) compared with control cells (Fig. 7, Ac and Ae). The numbers of capillary networks after 4 h of incubation in control and with anti-ITGAV/ITGB3 antibodies were 20.0 ± 4.0 and 11.0 ± 4.1. After 8 h of incubation in control and with anti-ITGAV/ITGB3 antibodies, the numbers of networks were 22.2 ± 5.8 and 12.4 ± 3.5, respectively (Fig. 7B).



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  		FIG. 7. Soluble FLT DNA inhibits tube-like formation in TCL1 cells. A) Cells transformed with vector (a, c, and e) or sFLT (b, d, and f) were seeded at 36 h after transformation on Matrigel and incubated at 37°C. B) The number of capillary networks per 1 mm2 was counted at magnification x400. Data are represented as mean ± SD of three independent trials. Bars = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
EVT cells alter their integrin expression in parallel with varied ECM distribution during invasion [3, 4]. Pre-eclampsia, one of the leading causes of maternal and neonatal morbidity, highlights the significance of integrin switching during EVT differentiation. Aberrant integrin expression with failure to up-regulate ITGA1/ITGB1 and to down-regulate ITGA6/ITGB4 may contribute to the inability of EVT to invade the uterus in pre-eclampsia [5, 6]. Not only interstitial invasion, but also endovascular invasion, is consistently undeveloped with this disorder. Consequently, maternal vessels contain fewer EVT. The EVT cells fail to switch on ITGAV/ITGB3, which is normally expressed by the most differentiated and invasive population of cytotrophoblast [11, 12]. Thus, it has been suggested that alteration of integrin expression during the differentiation of EVT may be responsible for mediating distinct signals upon adhesion to ECM. The mechanism that regulates this integrin conversion has been unclear; however, we have shown that TNF is involved in apoptosis and ITGA6/ITGA1 switching, both of which are considered to be related to invasiveness, in TCL1 cells [7].

In the present experiments, we demonstrated that antibodies against ITGAV/ITGB3 suppressed tube-like formation in TCL1. Along with endovascular differentiation, the adhesion molecule repertoires change in a comprehensive manner mimicking that of vascular cells, particularly endothelial cells [11]. Zhou et al. [12] reported that EVT in vivo showed reduced staining for adhesion receptors that was characteristic of stable epithelial monolayers, and showed enhanced staining of adhesion molecules that was characteristic of endothelial cells, including ITGAV/ITGB3 integrin, which is believed to play an important role in angiogenesis [9, 10]. Inhibitors of ITGAV/ITGB3 have shown antiangiogenic activity in many models [14]. Indeed, a humanized version of LM609, a monoclonal antibody that blocks ITGAV/ITGB3 signaling, has entered clinical trials as an antiangiogenic agent [25]. Therefore, ITGAV/ITGB3 integrins could play an important role in endovascular differentiation in EVT similar to that of endothelial cells. Many studies suggest an important role for apoptosis in pregnancy [26, 27]. Current results also demonstrate that ITGAV/ITGB3 integrin is involved in cellular survival in EVT cells in a manner similar to that of ITGA6 and ITGA1 [7].

VEGF plays a crucial role in angiogenesis [13, 14]. VEGF family members and their receptors seem to have important actions during early vasculogenesis and angiogenesis [14]. Witmer et al. [15] reported that VEGF up-regulates ITGAV/ITGB3 integrins. In patients with severe pre-eclampsia, FLT1 staining is decreased in placental tissues and cytotrophoblast secretion of sFLT in vitro is also increased. When FLT1 is inhibited, cell invasion, survival, and integrin switching are affected [28]. Ahmad and Ahmed [29] reported that elevated levels of sFLT1 in pre-eclampsia are responsible for inhibiting angiogenesis and that dysregulation of the VEGF axis is involved in pathogenesis of pre-eclampsia. Herein, we showed that VEGF up-regulated expression of ITGAV/ITGB3 integrin subunits and strengthened adhesive function in TCL1 cells. Soluble FLT1 DNA inhibited ITGAV/ITGB3 subunit aggregation and tube-like formation in TCL1 cells. In addition, neutralization of VEGF inhibited ITGAV/ITGB3 subunit aggregation. Therefore, it can be assumed that signals via VEGF axis play an important role in EVT differentiation. Because both FLT1 and KDR are expressed in placental villous endothelium, the distinct roles of each VEGF receptor in placental development is still controversial [30, 31]. Further evidence is necessary to define the role of each VEGF receptor in EVT differentiation.

A second issue is the role of TNF in endovascular differentiation of TCL1 cells. Serum TNF levels are elevated [32] and placental expression of TNF is increased in pre-eclampsia [33]. TNF is involved in gene transcription control and cellular differentiation through nuclear factor ?B activation [34], and it also induces apoptosis in a wide range of cells. Therefore, it is possible in pre-eclampsia that abnormal placental levels of TNF induce inappropriate EVT integrin expression, consequently leading to abnormal apoptosis, failure of differentiation, or both. TNF induces VEGF protein expression in TCL1 cells as well as other kinds of cells [35, 36]. In the present results, neutralization of VEGF inhibited TNF-induced ITGAV/ITGB3 subunit aggregation. Thus it seems that at least some of the effect of TNF was induced through the VEGF-VEGFR system. Of interest, recent evidence suggests that TNF{alpha} also stimulates release of sFLT from placental explants [29]. We should note the microenvironments surrounding EVT in early pregnancy [37]. Taking hypoxic environments into account, there might be sequential and synergistic effects between TNF and VEGF. Hypoxia induces proinflammatory cytokines, including TNF [38]. Placental VEGF expression is also up-regulated by hypoxia [39]. Indeed, when cultured under hypoxic conditions, cytotrophoblast fails to differentiate [40]. If excessive hypoxia or inflammation (or both) develop, such regulation might be impaired with resulting failure of both cellular survival and EVT differentiation. This failure, not only in invasiveness but also in endovascular differentiation, could cause later clinical complications such as pre-eclampsia.

In summary, we have shown that VEGF induces ITGAV/ ITGB3 expression and strengthens adhesive function in TCL1 cells, which have the potential to differentiate into endothelial cells. TNF showed same biological effects thorough VEGF induction. The ITGAV/ITGB3 integrin subunits seem to be involved in responding to both survival signals from ECM and endovascular morphological change. Although TCL1 cells show a very similar phenotype to EVT, our experiments should be verified in cells obtained during the first trimester and also in vivo. Even with the need to confirm our findings with other models, we conclude that TCL1 cells provide a potential model to elucidate functional differentiation of EVT cells in vitro as has been discussed elsewhere [7]. Thus, the current results suggest that TNF and VEGF, together with ECM, collaboratively regulate biological behavior of EVT through integrin signaling in early human pregnancy.


   FOOTNOTES
 
1 Supported in part by Grant-in-Aid 16790609 from the Ministry of Education of Japan, the Uehara Memorial Foundation, and the Inamori Foundation. Back

2 Correspondence: Kotaro Fukushima, Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. FAX: 81 92 642 5414; kfuku{at}gynob.med.kyushu-u.ac.jp Back

Received: 3 January 2005.

First decision: 31 January 2005.

Accepted: 17 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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