- Email: [email protected]
Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester
Placenta xxx (2015) 1e8
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester Q. Luo, X. Liu, Y. Zheng, Y. Zhao, J. Zhu, L. Zou* Department of Obstetrics and Gynecology, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 14 February 2015
Introduction: Maternal spiral artery remodeling after embryo implantation is a crucial process for successful pregnancy and rely on well-controlled trophoblast functions. Ephrin-B2 is found to be of great importance in various cell functions in both benign human tissue and tumors. However, its role in the regulation of trophoblast remains unknown. This study is conducted to investigate the role of ephrin-B2induced trophoblast functions related to artery remodeling. Methods: Trophoblast cell line HTR-8/SVneo was used to investigate the effects of ephrin-B2 inhibition on cell proliferation, apoptosis, migration, invasion and tube formation. Placental-decidual co-culture (PDC) system was conducted to verify ephrin-B2-induced trophoblast functions ex vivo. Factors involving in artery remodeling process, such as matrix metalloproteinases (MMPs), placental growth factors (PlGF) and vascular endothelial growth factor (VEGF) were tested at transcriptional level. Results: Inhibition of ephrin-B2 suppressed cell proliferation and induced cell apoptosis in HTR-8/SVneo cells. Down-regulation of ephrin-B2 impaired migration/invasion capabilities of HTR-8/SVneo cells and significantly decreased gene expression of MMPs. Also, a worse tube formation and a decrease in gene expression of PlGF was observed after down-regulation of ephrin-B2. However gene expression of VEGFA did not show significantly statistical difference. These effects were further confirmed by PDC system showing an inadequate trophoblast invasion and spiral artery remodeling. Discussion: Ephrin-B2 might be act as a positive regulator in maternal artery remodeling via both trophoblast invasion and endovascular formation. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Ephrin-B2 Trophoblast Placental-decidual co-culture (PDC) Artery remodeling
1. Introduction Appropriate spiral artery remodeling in early pregnancy is pivotal for successful fetal development and pregnancy outcomes. After embryo implantation, villous cytotrophoblasts may either fuse to form syncytothophoblasts or differentiate to extra villous trophoblast (EVT) cells growing out of villi and invading into maternal tissue. EVT cells are divided into two subtypes: (1) interstitial EVTs (inEVTs) invading into maternal decidual stroma and part of myometrium; (2) endovascular EVTs (enEVTs) migrating into spiral arteries and transforming into endothelial phenotype. Both subtypes are involved in the conversion of maternal arteries into vessels characterized with high perfusion and low resistance which ensure abundant nutrient and blood supply to the fetus [1]. Any abnormality during this process is
* Corresponding author. Tel.: þ86 13607166321. E-mail address: [email protected] (L. Zou).
highly associated with pregnancy-related complications such as early pregnancy loss, preeclampsia and fetal growth restriction [2e4]. Thus, studies in the mechanism of trophoblast invasion and vascular transformation are of great value for early diagnosis and treatment of pregnant complications. Ephrin-B2 is a membrane-anchored ligand for Eph receptors belonging to the biggest family of receptor tyrosine kinases. This highly conserved molecule family widely express in both benign human tissue and tumors [5]. In mammals, there are nine glycosylphosphatidylinositol-linked EphA ligands and three EphB transmembrane ligands [6]. Ephrins/Ephs mediate bidirectional signaling pathways as both of them are membrane bounded [7] and are demonstrated to be essential in cell migration, proliferation and angiogenesis regulation [8]. The special pattern of ephrin-B2 and EprhinB4 in vascular development implicated their essential roles in placentation. The expression of eprhin-B2/EphrinB4 at maternalefetal interface has been demonstrated in previews studies [9e11]. While EphrinB4 has been demonstrated to play a critical role in directing trophoblast invasion [12], there has been no research
http://dx.doi.org/10.1016/j.placenta.2015.02.009 0143-4004/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
2
Q. Luo et al. / Placenta xxx (2015) 1e8
focusing on ephrin-B2-induced trophoblast functions in pregnancy. The purpose of this study was to unveil the role of ephrin-B2 in the regulation of trophoblast involving in spiral artery remodeling. 2. Methods 2.1. Tissue collection Villous and decidual tissues were obtained from women undergoing elective termination of pregnancy in first trimester in Wuhan Union Hospital. Recruited women aged from 18 to 32y with an average of 24.1 ± 1.0y. The average gravity and parity was 2.0 ± 0.2 and 0.5 ± 0.1, respectively. Pregnancy was diagnosed by ultrasound examination as intrauterine and single. Exact gestational age was calculated by last menstruation period or ultrasound report. Gestational age at termination ranged from 6 to 9 weeks with an average of 49.0 ± 1.1 days. Pregnant women with one or more of the following situations were excluded from sample collection: complications such as hypertension, heart disease or other chronic diseases, previous obstetric history with pregnant complications (e.g. preeclampsia) and/or adverse pregnant outcomes (e.g. abortion), uterine surgery history including cesarean section, confirmed abortions and severe reproductive tract infection. Tissue samples were collected and washed in 0.9% saline and were later used for immunohistochemistry or placental-decidual co-culture. All procedures were approved by the local research ethics committee with written informed consent documents in advance. Tissues from 10 pregnant women were collected for immunohistochemistry (IHC) staining and another 12 for co-culture. 2.2. Cell culture and transfection The human EVT cell line HTR-8/SVneo was a kind gift from Dr. Charles Graham (Queen's University, Canada). Cells were cultured in RPMI 1640 (Hyclone, USA) with 10% fetal bovine serum (FBS, Gibco-BRL-Life Technologies, USA), 100IU/ml penicillin and 100 mg/ml streptomycin at 37 C in 5% CO2. Culture medium was changed every 48 h. Cells were transfected with DNA plasmid against ephrin-B2 expression (sh-ephrin-B2) or a non-specific sequence as negative control (shNC) (GenePharma, Shanghai, China) after being sowed into 6-well plates with a total number of 4 105 cells for each well and cultured overnight. Transfection was conducted using Neofect™ (Neofect Biotechnologies, Beijing, China) according to the manufacturer's instruction. Transfected cells were harvested 48 h after treatment to test the inhibition efficiency via quantitative real-time polymerase chain reaction (RT-PCR) and western blotting. 2.3. RNA extraction and RT-PCR RNA isolation, reverse transcription, RT-PCR assay and data analysis were performed using the method described previously [13]. Sequence of primers were as follows: GAPDH forward primer: 50 -ACAAACATAAGCAAGGCACAGT-30 , reverse primer: 50 -GGTCGGAGTCAACGGATTTG-30 ; ephrin-B2 forward primer: 50 -GGT GGTCCTCTTGCTGAAGT-30 , reverse primer: 50 -CGCTGACCTTCTCGTAGTGA-30 ; VEGF forward primer: 50 -GCCTCGGGCTTGTCACATTTT-30 , reverse primer: 50 -CCCTGATGA GATCGAGTACATCT-30 ; PlGF forward primer: 50 -GGGGAAGAGGAGGAGAGAGA-30 , reverse primer: 50 -CTCTCACGTTGTTGAAGGCA-30 ; MMP-2 forward primer: 50 -TGAT CTTGACCAGAATACCATCGA-30 , reverse primer: 50 -GGCTTGCGAGGGAAGAAGTT-30 . MMP-9 forward primer: 50 -TTTGAGTCCGGTGGACGATG-30 , reverse primer: 50 -GC TCCTCAAAGACCGAGTCC-30 . The PCR conditions were as follows: 95 C for 30 s, 40 cycles at 95 C for 30s, 65 C for 30 s and extension at 72 C for 60 s using StepOne™
Fig. 2. Transfection efficiency of ephrin-B2 in HTR-8/SVneo cells. A. Relative expression of ephrin-B2 after different treatment determined by RT-PCR. The expression of ephrin-B2 mRNA did not show a statistical difference between shNC group and controls while decreasing by 80% in sh-ephrin-B2 groups. B. Western blot showed the expression of ephrin-B2 after treatment with different DNA plasmid. C. Relative expression of ephrin-B2 after different treatment determined by Western blot. (NS: not significant, **: P < 0.01, ***: P < 0.001) (n ¼ 3 in triplicate for RT-PCR and n ¼ 5 in triplicate for Western blotting).
Real-Time PCR System. Comparative Ct method (2(△△Ct) method) was used to analyze relative gene expressions with GAPDH as the internal control. 2.4. Western blotting Total proteins of cell cultures were extracted using RIPA and total protein concentrations were determined using BCA method. 50 mg total proteins of each sample were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Membranes were incubated with antibodies against ephrin-B2 (1:500, Abcam) or GADPH (1:1000, Affinity) at 4 C overnight. Incubated membranes were developed by horseradish peroxidase (HRP)-
Fig. 1. Expression of ephrin-B2 in first human placenta. A. Ephrin-B2 was localized in trophoblasts in human placental villi. B CK-7 staining of trophoblasts in placental villi. C. Negative control of IHC staining in placental villi. D. Expression of ephrin-B2 in maternal decidua. E. HLA-G staining of EVTs in maternal decidua. F. Negative control of IHC staining in maternal decidua. Villous and decidual samples were collected from 10 pregnant women.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Q. Luo et al. / Placenta xxx (2015) 1e8 conjugated goat anti-rabbit IgG (1:1000, Affinity) for 1 h at room temperature the next day. Proteins were visualized by enhanced chemiluminescence (ECL) procedure. 2.5. Cell proliferation assay Proliferation capacity was assessed by Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). Transfected cell were replanted to 96-well plates at 5 104 cells per well with 100 mL complete medium and cultured for 24 h. 100 mL of CCK-8 solution was added into each well, and incubated at 37 C in 5% CO2for another 2 h. Absorbance was measured at 450 nm using a micro-plate reader. 2.6. Hoechst 33258 staining Hoechst 33258 staining was performed to examine cell apoptosis. Transfected cells were fixed overnight at 4 C. After rinsing in PBS, Hoechst 33258 solution (Beyotime, Jiangsu, China) was added to each well. Slides were observed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) at a magnification of 200. The apoptotic cells were identified with condensed chromatin and nuclear fragments. The number of apoptotic cells in ten random fields for each 6-well plate was counted. 2.7. Caspase-3 colorimetric assay Cell apoptosis was further examined by Caspase-3 colorimetric assay kit (KeyGEN BioTECH, Nanjing, China). Assay procedure was conducted according to the
3
manufacture's instruction. Briefly, 200 mg total proteins extracted from transfected cells were diluted in 50 mL lysis buffer. 50 mL 2 Reaction Buffer and 5 mL Caspase-3 Substrate was added to each sample sequentially. All samples were incubated at 37 C for 4 h in dark. Absorbance was measured at 405 nm using a micro-plate reader. Comparison of the absorbance implicated Caspase-3 activity.
2.8. Matrigel invasion and migration assay A total of 2 105 transfected cells were resuspended in 200 mL serum-free RPMI1640 and added to 24-well transwell insert (pore size 8 mm, Corning, USA) with 600 mL complete culture medium in the lower chamber. After cultured at 37 C for 24 h, inserts were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet. Migrating cells were counted under an inverted phase-contrast microscope (Olympus, Japan) at a magnification of 200. Average number of five random fields was calculated for each insert. Similar method was applied to invasion assay except each insert was pre-coated with 50 mL Matrigel (BD Biosciences, San Jose, CA, USA). 2.9. Network formation assay in vitro HTR-8/SVneo is possible to show endothelial cell-like behavior in the form of tube-like network formation on Matrigel substance [14]. 96-well plates were coated with 100 mL Matrigel and allowed to gel formation at 37 C for 1 h. A total of 1 104 transfected cells in medium containing 1% FBS were plated into the upper layer of the formed gel. After incubation for 6 h at 37 C, plates were observed under
Fig. 3. Inhibition of ephrin-B2 reduced cell proliferation and promoted cell apoptosis in HTR8/SVneo cells. A. CCK-8 assay showed that relative rate of cell proliferation was slightly decreased in sh-ephrin-B2 inhibition group. B. Hoechst 33258 staining for cell apoptosis analysis at a magnification of 200. Typical apoptotic cells were visualized with condensed chromatin and nuclear fragments (arrows). C. Cells with down-regulation of ephrin-B2 showed an increase in relative apoptotic rate. D. Relative absorbance rate decreased in shephrin-B2 treated cells in caspase-3 colorimetric assay. (*: P < 0.05) (n ¼ 3 in triplicate).
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
4
Q. Luo et al. / Placenta xxx (2015) 1e8
microscope and photographed. The number of branching points generating at least three tubules were counted. 2.10. Placental-decidual co-culture (PDC) The establishment of PDC was modified on the method described previously [15,16]. Decidual tissue was dissected into 3 mm2 cubes and placed in the lower chamber of co-culture dishes pre-coated with 200 mL pre-coated phenol red-free Matrigel in DMEM/F-12 media with 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin. Dissected villus from the same pregnant woman were placed into the upper compartments (pore size 8 mm, Corning, USA) with serum-free DMEM/F12 media containing 1 mg sh-ephrin-B2 or shNC plasmid. PDC system were cultured at 37 C in 5% CO2. Culture medium changed every 48 h. Co-culture tissues were fixed and embedded in paraffin wax on the 6th day.
were incubated for 1 h at room temperature with primary antibody (cytokeratin7 1:400, CD34 1:400, a-SMA 1:400 all from Boster, China, ephrin-B2 1:50, Affinity, HLAG 1:800 Agent) after blocking. Slides were incubated for another 30 min with HRPconjugated goat anti-rabbit IgG (1:1000; Santa Cruz). Slides were then developed with diaminobenzidine for 5 min and hematoxylin for 2 min. 2.12. Statistical analysis The data was shown as mean and SD (standard deviation) and statistical analysis was performed using GraphPad Prism 5. Differences between two groups were analyzed by t-test. A P value <0.05 was considered statistically significant.
3. Results
2.11. Immunohistochemistry
3.1. Expression of ephrin-B2 in human first trimester placenta
5 mm paraffin sections were used for immunohistochemistry. Rehydrated paraffins were heated in citrate buffer (pH ¼ 6.0) for antigen retrieval for 15 min. Endogenous peroxidase activity was quenched by incubation in 3% H2O2 for 20 min. Sections
The expression of ephrin-B2 in human first trimester placental villi and maternal decidua was shown by IHC staining. Ephrin-B2
Fig. 4. Ephrin-B2 regulated migration and invasion capability of HTR8/SVneo cells. A. Representative images of migration and invasion assay of HTR-8/SVneo cells at a magnification of 200. B&C. Statistical analysis of migration and invasion assays. Down-regulation of ephrin-B2 in HTR-8/SVneo impaired cell migration and invasion capability. D&E The relative gene expression of MMP-2/9 was decreased after sh-ephrin-B2 treatment. (*: P < 0.05, **: P < 0.01, ***: P < 0.001) (n ¼ 3 in triplicate).
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Q. Luo et al. / Placenta xxx (2015) 1e8
5
was found to be expressed in trophoblasts in placental villi (Fig. 1AeC). In maternal decidua, ephrin-B2 was found to be expressed in cells including EVTs (Fig. 1DeF).
sh-ephrin-B2 cells compared to controls (Fig. 3D, P < 0.05), indicating higher caspase-3 activity after down-regulation of ephrin-B2 in HTR-8/SVneo cells.
3.2. Inhibition of ephrin-B2 in HTR-8/SVneo cells
3.4. Down-regulation of ephrin-B2 suppressed HTR-8/SVneo migration and invasion by regulating MMPs
HTR-8/SVneo cell were transfected with plasmids specially targeting ephrin-B2. Transfection efficiency was shown by fluorescence microscope and further assessed by RT-PCR. The inhibition efficiency was more than 80% compared to controls (Fig. 2A, P < 0.001). Reduction of ephrin-B2 was identified by Western Blot in HTR-8/ SVneo cells compared with sh-NC group (Fig. 2B and C, P < 0.01). 3.3. Inhibition of ephrin-B2 reduced cell proliferation and promoted cell apoptosis Proliferation of HTR-8/SVneo was slightly reduced after downregulation of ephrin-B2 compared to negative controls (Fig. 3A P < 0.05). Cell apoptosis was tested via Hoechst staining and caspase-3 colorimetric assay. Down-regulation of ephrin-B2 in HTR-8/SVneo cells was observed with more apoptotic cells (Fig. 3B) and increased relative apoptotic rate (Fig. 3C, P < 0.05). Caspase-3 colorimetric assay showed a higher ratio of relative absorbance in
In transwell models, mobility ability was significantly reduced in the cells transfected with sh-ephrin-B2 (Fig. 4A and B, P < 0.05). Invasion assay showed the same tendency with migration assay (Fig. 4A and C, P < 0.01). MMP-2 and MMP-9 play an important role in trophoblast migration and invasion. In current study, transcriptional levels of MMP-2 and MMP-9 were reduced after downregulation of ephrin-B2 in HTR-8/SVneo cells (Fig. 4D, P < 0.01 and Fig. 4E, P < 0.001). 3.5. Inhibition of ephrin-B2 impaired trophoblast tube formation and suppressed PlGF expression Transfected cells were cultured in Matrigel pre-coated plates in order to assess angiogenesis capability of HTR-8/SVneo after different treatment. Down regulation of ephrin-B2 resulted in a decrease in branching points pre field (magnification 100)
Fig. 5. Ephrin-B2 inhibition prevented tube formation in HTR-8/SVneo cells. A. Tube formation of transfected cells replanted in conditional medium cultured for 6 h on Matrigel (magnification 100). B. Average number of branch points in random fields decreased in sh-ephrin-B2 groups. C. The expression of PlGF mRNA showed a dramatic decreased after ephrin-B2 in HTR-8/SVneo cells. D. VEGF mRNA level did not have a significant change between sh-ephrin-B2 group and negative controls in HTR-8/SVneo cells. (NS: not significant, *: P < 0.05) (n ¼ 3 in triplicate).
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
6
Q. Luo et al. / Placenta xxx (2015) 1e8
compared to negative controls (Fig. 5A and B, P < 0.05). Trophoblast angiogenesis has a close relationship with several angiogenesis factors such as VEGF and PlGF. RT-PCR showed that inhibition of ephrin-B2 significantly suppressed the expression of PlGF at transcriptional level (Fig. 5C, P < 0.05) while the change in VEGF-A mRNA level did not show any statistical difference in HTR-8/ SVneo cells (Fig. 5D). 3.6. Knockout of ephrin-B2 impaired maternal artery remodeling ex vivo PDC system was conducted to further investigate the effects of ephrin-B2 inhibition Unremodelled arteries were characterized with multiple layers of vascular smooth muscle cells (VSMCs), endothelial cell (EC) layer and no EVT (Fig. 6AeC). Active remodeling was characterized with EVTs, endothelial desquamation and disruption of VSMCs (Fig. 6GeI). Veins and glands were excluded by identification with IHC staining (Fig. 6DeF and JeL). After coculture for 6 days, shNC treatment groups showed more advanced remodeling and deeper invasion than sh-ephrin-B2 transfection groups (Fig. 6MeS). 4. Discussion Ephrin-B2 was initially recognized to be critically important for embryonic vasculature [17]. Later studies highlighted the instrumental role of ephrin-B2 in vasculature remodeling [18] and tumorgenesis [19]. Since it is well known that regulation of trophoblast functions shares similar mechanism with tumorgenesis
[20], it is reasonable to hypothesize that ephrin-B2 may also play an important role in the regulation of trophoblast function. Previous studies postulated the role of ephrin-B2 in placentation by IHC staining [9e11]. To date, ephrin-B2-induced trophoblast function in maternal spiral artery remodeling has not been investigated. Current study localized eprhin-B2 in human first trimester placenta villi as well as in maternal decidua. We demonstrated the effects of ephrin-B2 on trophoblast by down-regulation of its expression in trophoblast cell line. Invasion and tube formation of HTR-8/SVneo were significantly impaired after inhibition of ephrinB2 via regulation of MMPs and angiogenic factor, PlGF. Furthermore, we conducted an ex vivo model to verify the effects after ephrin-B2 knock-down. Inadequate invasion of trophoblast and endovascular transformation were observed in the co-culture system. Taking together these results indicate the important role of ephrin-B2 in maternal spiral artery remodeling in first trimester. Trophoblast invasion contributes to the success of maternal spiral artery remodeling. Mounting evidences suggest that aberration in maternal vascular modification resulting from shallow invasion of trophoblast is associated with obstetric complications [3,4]. In this study, we found a dramatic reduction in migration and invasion with small changes in cell proliferation and apoptosis after inhibition of ephrin-B2 in HTR-8/SVneo cells, especially invasion. Our results are in accordance with other study in tumor [21]. Trophoblast invasion in early pregnancy requires the anticipation of MMP-2 and MMP-9 [22]. Our study showed that down-regulation of ephrin-B2 inhibited the expression of both MMP-2 and MMP-9 at transcriptional level. However, Kwan et al. reported reduction of MMPs in response to ephrin-B2 stimulation [23]. This
Fig. 6. Immunohistochemistry staining of placental-decidual co-culture system. AeC. Intact arteries had no trophoblast present (A) and possessed multiple layers of vascular muscle (B) as well as an endothelial layer (C). DeF. Advanced remodeling arteries had invading trophoblasts (D), lost vascular muscle layers (E) and isolated endothelial cells (F). GeI: Veins showed a thin layer of endothelial cell (I), sporadic vascular muscle cell (H) and absence of trophoblast (I). JeL: Glands in maternal decidua were detective with CK-7 (G) but negative of a-SMA and CD 34 staining (K and L). M-S. IHC staining of PDC with different treatment on the 6th day. Sh-ephrin-B2 treatment samples showed an early remodeling with vascular muscle disruption at superficial area (O) and intact arteries (arrows in M, O, Q). Sh-NC treated samples showed remodeled arteries in deeper area (arrows in N, P, S). Samples were collected from 12 pregnant women in all.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Q. Luo et al. / Placenta xxx (2015) 1e8
contradiction may result from the unique mechanism of bidirectional signal pathways of eprhin-B2/Ephrin B4. For instance, EphB/ephrin-B2 signaling in ECs promotes cell adhesion [24], whereas the reverse signaling induces the opposite effect [25]. Endovascular trophoblast replacement is another crucial process of spiral artery remodeling [26]. EVTs differentiate into endovascular trophoblasts after they invade into maternal decidua, establishing the conversion of spiral arteries into low resistance vessels during pregnancy. Our study showed a worse network formation in HTR-8/SVneo cells with sh-ephrin-B2 treatment in vitro. Therefore, inadequate conversion of maternal spiral arteries resulting from ephrin-B2 inhibition may contribute to the pathogenesis of diseases related to impaired placentation. Reduced gene expressions of angiogenic factors in first trimester are related to the onset of placental vascular disorders in later term [27]. We further investigated the expression of angiogenic factors, PlGF and VEGF-A. The reduced gene expressions of both angiogenic factors are related to the onset of preeclampsia [27]. A reduction in PlGF transcription after down-regulation of ephrin-B2 was observed. However, mRNA level of VEGF-A increased after inhibition of ephrin-B2 without statistically significant difference. We speculate that this is probably because ephrin-B2 inhibits VEGF-induced angiogenesis by regulating VEGF2 and VEGF3 expression without directly targeting at VEGF-A [28]. We found that down-regulation of eprhin-B2 in placental villi resulted in shallow invasion and damaged conversion of maternal spiral arteries in ex vivo mode of artery remodeling, consistent with the findings of the effect of inhibition of eprhin-B2 in HTR-8/SVneo cells. Our study demonstrated the possible role of ephrin-B2 as a new target for placental vascular disorders. Regarding the possible molecular pathways, we speculate that eprhin-B2 may induce trophoblast functions in spiral artery remodeling via extracellular regulated ERK-1/2 and AKT signaling pathways. Erk 1/2 and AKT are the common pathways involved in trophoblast invasion [29,30] and angiogenesis [31]. Previous studies demonstrated that ephrin-B2 is able to active these pathways in hepatic stellate cells [32] and T cells [33]. Taken together, we hypothesize that similar mechanism works in ephrin-B2induced trophoblast function. We will investigate this issue in our future studies. In conclusion, our study identified the role of ephrin-B2 in trophoblast invasion and endovascular formation. Therefore, it may be an important positive regulator in trophoblast-dependent maternal spiral artery remodeling. Conflict of interest statement All the authors declare that they have no conflict of interest. Acknowledgment We wish to express sincere thanks to Dr. Charles Graham (Queen's University, Kingston, Ontario, Canada) for kindly providing the trophoblast cell line. This work was supported by National Natural Science Foundation of China (No. 81170584 and 81370733 to L Zou; No. 81100442 to Y. Zhao). References [1] Pijnenborg R, Vercruysse L, Hanssens M. The uterine spiral arteries in human pregnancy: facts and controversies. Placenta 2006 Sep-Oct;27(9e10):939e58. [2] Orozco AF, Jorgez CJ, Ramos-Perez WD, Popek EJ, Yu X, Kozinetz CA, et al. Placental release of distinct DNA-associated micro-particles into maternal circulation: reflective of gestation time and preeclampsia. Placenta 2009 Oct;30(10):891e7.
7
[3] Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod 2003 Jul;69(1):1e7. [4] van den Brule F, Berndt S, Simon N, Coulon C, Le Goarant J, Munaut C, et al. Trophoblast invasion and placentation: molecular mechanisms and regulation. Chem Immunol Allergy 2005;88:163e80. [5] Hafner C, Schmitz G, Meyer S, Bataille F, Hau P, Langmann T, et al. Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers. Clin Chem 2004 Mar;50(3):490e9. [6] Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 2010 Mar;10(3):165e80. [7] Klein R. Eph/ephrin signalling during development. Development 2012 Nov;139(22):4105e9. [8] Mosch B, Reissenweber B, Neuber C, Pietzsch J. Eph receptors and ephrin ligands: important players in angiogenesis and tumor angiogenesis. J Oncol 2010;2010:135285. [9] Chennakesava CS, Di Santo S, Ziemiecki A, Schneider H, Andres AC. Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development. Placenta 2006 SepeOct;27(9e10): 959e67. [10] Goldman-Wohl D, Greenfield C, Haimov-Kochman R, Ariel I, Anteby EY, Hochner-Celnikier D, et al. Eph and ephrin expression in normal placental development and preeclampsia. Placenta 2004 Aug;25(7):623e30. [11] Zhang J, Dong H, Wang B, Zhu S, Croy BA. Dynamic changes occur in patterns of endometrial EFNB2/EPHB4 expression during the period of spiral arterial modification in mice. Biol Reprod 2008 Sep;79(3):450e8. [12] Red-Horse K, Kapidzic M, Zhou Y, Feng KT, Singh H, Fisher SJ. EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation. Development 2005 Sep;132(18):4097e106. [13] Zhao Y, Zheng YF, Luo QQ, Yan T, Liu XX, Han L, et al. Edaravone inhibits hypoxiainduced trophoblast-soluble Fms-like tyrosine kinase 1 expression: a possible therapeutic approach to preeclampsia. Placenta 2014 Jul;35(7):476e82. [14] Highet AR, Zhang VJ, Heinemann GK, Roberts CT. Use of Matrigel in culture affects cell phenotype and gene expression in the first trimester trophoblast cell line HTR8/SVneo. Placenta 2012 Jul;33(7):586e8. [15] Dunk C, Petkovic L, Baczyk D, Rossant J, Winterhager E, Lye S. A novel in vitro model of trophoblast-mediated decidual blood vessel remodeling. Lab Invest 2003 Dec;83(12):1821e8. [16] Hazan AD, Smith SD, Jones RL, Whittle W, Lye SJ, Dunk CE. Vascular-leukocyte interactions: mechanisms of human decidual spiral artery remodeling in vitro. Am J Pathol 2010 Aug;177(2):1017e30. [17] Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 1998 May 29;93(5):741e53. [18] Davy A, Soriano P. Ephrin-B2 forward signaling regulates somite patterning and neural crest cell development. Dev Biol 2007 Apr 1;304(1):182e93. [19] Abengozar MA, de Frutos S, Ferreiro S, Soriano J, Perez-Martinez M, Olmeda D, et al. Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth. Blood 2012 May 10;119(19): 4565e76. [20] Holtan SG, Creedon DJ, Haluska P, Markovic SN. Cancer and pregnancy: parallels in growth, invasion, and immune modulation and implications for cancer therapeutic agents. Mayo Clin Proc 2009 Nov;84(11): 985e1000. [21] Meyer S, Hafner C, Guba M, Flegel S, Geissler EK, Becker B, et al. Ephrin-B2 overexpression enhances integrin-mediated ECM-attachment and migration of B16 melanoma cells. Int J Oncol 2005 Nov;27(5):1197e206. [22] Staun-Ram E, Goldman S, Gabarin D, Shalev E. Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion. Reprod Biol Endocrinol 2004 Aug 4;2:59. [23] Kwan Tat S, Pelletier JP, Amiable N, Boileau C, Lavigne M, MartelPelletier J. Treatment with ephrin B2 positively impacts the abnormal metabolism of human osteoarthritic chondrocytes. Arthritis Res Ther 2009;11(4):R119. [24] Oike Y, Ito Y, Hamada K, Zhang XQ, Miyata K, Arai F, et al. Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells. Blood 2002 Aug 15;100(4):1326e33. [25] Swift MR, Weinstein BM. Arterial-venous specification during development. Circulat Res 2009 Mar 13;104(5):576e88. [26] Whitley GS, Cartwright JE. Cellular and molecular regulation of spiral artery remodelling: lessons from the cardiovascular field. Placenta 2010 Jun;31(6): 465e74. [27] Farina A, Sekizawa A, De Sanctis P, Purwosunu Y, Okai T, Cha DH, et al. Gene expression in chorionic villous samples at 11 weeks' gestation from women destined to develop preeclampsia. Prenat Diagn 2008 Oct;28(10): 956e61. [28] Wang Y, Nakayama M, Pitulescu ME, Schmidt TS, Bochenek ML, Sakakibara A, et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 2010 May 27;465(7297):483e6. [29] Qiu Q, Yang M, Tsang BK, Gruslin A. Both mitogen-activated protein kinase and phosphatidylinositol 3-kinase signalling are required in epidermal growth factor-induced human trophoblast migration. Mol Hum Reprod 2004 Sep;10(9):677e84.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
8
Q. Luo et al. / Placenta xxx (2015) 1e8
[30] Fitzgerald JS, Busch S, Wengenmayer T, Foerster K, de la Motte T, Poehlmann TG, et al. Signal transduction in trophoblast invasion. Chem Immunol Allergy 2005;88:181e99. [31] Selvaraj SK, Giri RK, Perelman N, Johnson C, Malik P, Kalra VK. Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor. Blood 2003 Aug 15;102(4):1515e24. [32] Das A, Shergill U, Thakur L, Sinha S, Urrutia R, Mukhopadhyay D, et al. Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF
production and sinusoidal endothelial cell recruitment. Am J Physiol Gastrointest Liver Physiol 2010 Jun;298(6):G908e15. [33] Kawano H, Katayama Y, Minagawa K, Shimoyama M, Henkemeyer M, Matsui T. A novel feedback mechanism by Ephrin-B1/B2 in T-cell activation involves a concentration-dependent switch from costimulation to inhibition. Eur J Immunol 2012 Jun;42(6):1562e72.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester Q. Luo, X. Liu, Y. Zheng, Y. Zhao, J. Zhu, L. Zou* Department of Obstetrics and Gynecology, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 14 February 2015
Introduction: Maternal spiral artery remodeling after embryo implantation is a crucial process for successful pregnancy and rely on well-controlled trophoblast functions. Ephrin-B2 is found to be of great importance in various cell functions in both benign human tissue and tumors. However, its role in the regulation of trophoblast remains unknown. This study is conducted to investigate the role of ephrin-B2induced trophoblast functions related to artery remodeling. Methods: Trophoblast cell line HTR-8/SVneo was used to investigate the effects of ephrin-B2 inhibition on cell proliferation, apoptosis, migration, invasion and tube formation. Placental-decidual co-culture (PDC) system was conducted to verify ephrin-B2-induced trophoblast functions ex vivo. Factors involving in artery remodeling process, such as matrix metalloproteinases (MMPs), placental growth factors (PlGF) and vascular endothelial growth factor (VEGF) were tested at transcriptional level. Results: Inhibition of ephrin-B2 suppressed cell proliferation and induced cell apoptosis in HTR-8/SVneo cells. Down-regulation of ephrin-B2 impaired migration/invasion capabilities of HTR-8/SVneo cells and significantly decreased gene expression of MMPs. Also, a worse tube formation and a decrease in gene expression of PlGF was observed after down-regulation of ephrin-B2. However gene expression of VEGFA did not show significantly statistical difference. These effects were further confirmed by PDC system showing an inadequate trophoblast invasion and spiral artery remodeling. Discussion: Ephrin-B2 might be act as a positive regulator in maternal artery remodeling via both trophoblast invasion and endovascular formation. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Ephrin-B2 Trophoblast Placental-decidual co-culture (PDC) Artery remodeling
1. Introduction Appropriate spiral artery remodeling in early pregnancy is pivotal for successful fetal development and pregnancy outcomes. After embryo implantation, villous cytotrophoblasts may either fuse to form syncytothophoblasts or differentiate to extra villous trophoblast (EVT) cells growing out of villi and invading into maternal tissue. EVT cells are divided into two subtypes: (1) interstitial EVTs (inEVTs) invading into maternal decidual stroma and part of myometrium; (2) endovascular EVTs (enEVTs) migrating into spiral arteries and transforming into endothelial phenotype. Both subtypes are involved in the conversion of maternal arteries into vessels characterized with high perfusion and low resistance which ensure abundant nutrient and blood supply to the fetus [1]. Any abnormality during this process is
* Corresponding author. Tel.: þ86 13607166321. E-mail address: [email protected] (L. Zou).
highly associated with pregnancy-related complications such as early pregnancy loss, preeclampsia and fetal growth restriction [2e4]. Thus, studies in the mechanism of trophoblast invasion and vascular transformation are of great value for early diagnosis and treatment of pregnant complications. Ephrin-B2 is a membrane-anchored ligand for Eph receptors belonging to the biggest family of receptor tyrosine kinases. This highly conserved molecule family widely express in both benign human tissue and tumors [5]. In mammals, there are nine glycosylphosphatidylinositol-linked EphA ligands and three EphB transmembrane ligands [6]. Ephrins/Ephs mediate bidirectional signaling pathways as both of them are membrane bounded [7] and are demonstrated to be essential in cell migration, proliferation and angiogenesis regulation [8]. The special pattern of ephrin-B2 and EprhinB4 in vascular development implicated their essential roles in placentation. The expression of eprhin-B2/EphrinB4 at maternalefetal interface has been demonstrated in previews studies [9e11]. While EphrinB4 has been demonstrated to play a critical role in directing trophoblast invasion [12], there has been no research
http://dx.doi.org/10.1016/j.placenta.2015.02.009 0143-4004/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
2
Q. Luo et al. / Placenta xxx (2015) 1e8
focusing on ephrin-B2-induced trophoblast functions in pregnancy. The purpose of this study was to unveil the role of ephrin-B2 in the regulation of trophoblast involving in spiral artery remodeling. 2. Methods 2.1. Tissue collection Villous and decidual tissues were obtained from women undergoing elective termination of pregnancy in first trimester in Wuhan Union Hospital. Recruited women aged from 18 to 32y with an average of 24.1 ± 1.0y. The average gravity and parity was 2.0 ± 0.2 and 0.5 ± 0.1, respectively. Pregnancy was diagnosed by ultrasound examination as intrauterine and single. Exact gestational age was calculated by last menstruation period or ultrasound report. Gestational age at termination ranged from 6 to 9 weeks with an average of 49.0 ± 1.1 days. Pregnant women with one or more of the following situations were excluded from sample collection: complications such as hypertension, heart disease or other chronic diseases, previous obstetric history with pregnant complications (e.g. preeclampsia) and/or adverse pregnant outcomes (e.g. abortion), uterine surgery history including cesarean section, confirmed abortions and severe reproductive tract infection. Tissue samples were collected and washed in 0.9% saline and were later used for immunohistochemistry or placental-decidual co-culture. All procedures were approved by the local research ethics committee with written informed consent documents in advance. Tissues from 10 pregnant women were collected for immunohistochemistry (IHC) staining and another 12 for co-culture. 2.2. Cell culture and transfection The human EVT cell line HTR-8/SVneo was a kind gift from Dr. Charles Graham (Queen's University, Canada). Cells were cultured in RPMI 1640 (Hyclone, USA) with 10% fetal bovine serum (FBS, Gibco-BRL-Life Technologies, USA), 100IU/ml penicillin and 100 mg/ml streptomycin at 37 C in 5% CO2. Culture medium was changed every 48 h. Cells were transfected with DNA plasmid against ephrin-B2 expression (sh-ephrin-B2) or a non-specific sequence as negative control (shNC) (GenePharma, Shanghai, China) after being sowed into 6-well plates with a total number of 4 105 cells for each well and cultured overnight. Transfection was conducted using Neofect™ (Neofect Biotechnologies, Beijing, China) according to the manufacturer's instruction. Transfected cells were harvested 48 h after treatment to test the inhibition efficiency via quantitative real-time polymerase chain reaction (RT-PCR) and western blotting. 2.3. RNA extraction and RT-PCR RNA isolation, reverse transcription, RT-PCR assay and data analysis were performed using the method described previously [13]. Sequence of primers were as follows: GAPDH forward primer: 50 -ACAAACATAAGCAAGGCACAGT-30 , reverse primer: 50 -GGTCGGAGTCAACGGATTTG-30 ; ephrin-B2 forward primer: 50 -GGT GGTCCTCTTGCTGAAGT-30 , reverse primer: 50 -CGCTGACCTTCTCGTAGTGA-30 ; VEGF forward primer: 50 -GCCTCGGGCTTGTCACATTTT-30 , reverse primer: 50 -CCCTGATGA GATCGAGTACATCT-30 ; PlGF forward primer: 50 -GGGGAAGAGGAGGAGAGAGA-30 , reverse primer: 50 -CTCTCACGTTGTTGAAGGCA-30 ; MMP-2 forward primer: 50 -TGAT CTTGACCAGAATACCATCGA-30 , reverse primer: 50 -GGCTTGCGAGGGAAGAAGTT-30 . MMP-9 forward primer: 50 -TTTGAGTCCGGTGGACGATG-30 , reverse primer: 50 -GC TCCTCAAAGACCGAGTCC-30 . The PCR conditions were as follows: 95 C for 30 s, 40 cycles at 95 C for 30s, 65 C for 30 s and extension at 72 C for 60 s using StepOne™
Fig. 2. Transfection efficiency of ephrin-B2 in HTR-8/SVneo cells. A. Relative expression of ephrin-B2 after different treatment determined by RT-PCR. The expression of ephrin-B2 mRNA did not show a statistical difference between shNC group and controls while decreasing by 80% in sh-ephrin-B2 groups. B. Western blot showed the expression of ephrin-B2 after treatment with different DNA plasmid. C. Relative expression of ephrin-B2 after different treatment determined by Western blot. (NS: not significant, **: P < 0.01, ***: P < 0.001) (n ¼ 3 in triplicate for RT-PCR and n ¼ 5 in triplicate for Western blotting).
Real-Time PCR System. Comparative Ct method (2(△△Ct) method) was used to analyze relative gene expressions with GAPDH as the internal control. 2.4. Western blotting Total proteins of cell cultures were extracted using RIPA and total protein concentrations were determined using BCA method. 50 mg total proteins of each sample were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Membranes were incubated with antibodies against ephrin-B2 (1:500, Abcam) or GADPH (1:1000, Affinity) at 4 C overnight. Incubated membranes were developed by horseradish peroxidase (HRP)-
Fig. 1. Expression of ephrin-B2 in first human placenta. A. Ephrin-B2 was localized in trophoblasts in human placental villi. B CK-7 staining of trophoblasts in placental villi. C. Negative control of IHC staining in placental villi. D. Expression of ephrin-B2 in maternal decidua. E. HLA-G staining of EVTs in maternal decidua. F. Negative control of IHC staining in maternal decidua. Villous and decidual samples were collected from 10 pregnant women.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Q. Luo et al. / Placenta xxx (2015) 1e8 conjugated goat anti-rabbit IgG (1:1000, Affinity) for 1 h at room temperature the next day. Proteins were visualized by enhanced chemiluminescence (ECL) procedure. 2.5. Cell proliferation assay Proliferation capacity was assessed by Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). Transfected cell were replanted to 96-well plates at 5 104 cells per well with 100 mL complete medium and cultured for 24 h. 100 mL of CCK-8 solution was added into each well, and incubated at 37 C in 5% CO2for another 2 h. Absorbance was measured at 450 nm using a micro-plate reader. 2.6. Hoechst 33258 staining Hoechst 33258 staining was performed to examine cell apoptosis. Transfected cells were fixed overnight at 4 C. After rinsing in PBS, Hoechst 33258 solution (Beyotime, Jiangsu, China) was added to each well. Slides were observed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) at a magnification of 200. The apoptotic cells were identified with condensed chromatin and nuclear fragments. The number of apoptotic cells in ten random fields for each 6-well plate was counted. 2.7. Caspase-3 colorimetric assay Cell apoptosis was further examined by Caspase-3 colorimetric assay kit (KeyGEN BioTECH, Nanjing, China). Assay procedure was conducted according to the
3
manufacture's instruction. Briefly, 200 mg total proteins extracted from transfected cells were diluted in 50 mL lysis buffer. 50 mL 2 Reaction Buffer and 5 mL Caspase-3 Substrate was added to each sample sequentially. All samples were incubated at 37 C for 4 h in dark. Absorbance was measured at 405 nm using a micro-plate reader. Comparison of the absorbance implicated Caspase-3 activity.
2.8. Matrigel invasion and migration assay A total of 2 105 transfected cells were resuspended in 200 mL serum-free RPMI1640 and added to 24-well transwell insert (pore size 8 mm, Corning, USA) with 600 mL complete culture medium in the lower chamber. After cultured at 37 C for 24 h, inserts were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet. Migrating cells were counted under an inverted phase-contrast microscope (Olympus, Japan) at a magnification of 200. Average number of five random fields was calculated for each insert. Similar method was applied to invasion assay except each insert was pre-coated with 50 mL Matrigel (BD Biosciences, San Jose, CA, USA). 2.9. Network formation assay in vitro HTR-8/SVneo is possible to show endothelial cell-like behavior in the form of tube-like network formation on Matrigel substance [14]. 96-well plates were coated with 100 mL Matrigel and allowed to gel formation at 37 C for 1 h. A total of 1 104 transfected cells in medium containing 1% FBS were plated into the upper layer of the formed gel. After incubation for 6 h at 37 C, plates were observed under
Fig. 3. Inhibition of ephrin-B2 reduced cell proliferation and promoted cell apoptosis in HTR8/SVneo cells. A. CCK-8 assay showed that relative rate of cell proliferation was slightly decreased in sh-ephrin-B2 inhibition group. B. Hoechst 33258 staining for cell apoptosis analysis at a magnification of 200. Typical apoptotic cells were visualized with condensed chromatin and nuclear fragments (arrows). C. Cells with down-regulation of ephrin-B2 showed an increase in relative apoptotic rate. D. Relative absorbance rate decreased in shephrin-B2 treated cells in caspase-3 colorimetric assay. (*: P < 0.05) (n ¼ 3 in triplicate).
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
4
Q. Luo et al. / Placenta xxx (2015) 1e8
microscope and photographed. The number of branching points generating at least three tubules were counted. 2.10. Placental-decidual co-culture (PDC) The establishment of PDC was modified on the method described previously [15,16]. Decidual tissue was dissected into 3 mm2 cubes and placed in the lower chamber of co-culture dishes pre-coated with 200 mL pre-coated phenol red-free Matrigel in DMEM/F-12 media with 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin. Dissected villus from the same pregnant woman were placed into the upper compartments (pore size 8 mm, Corning, USA) with serum-free DMEM/F12 media containing 1 mg sh-ephrin-B2 or shNC plasmid. PDC system were cultured at 37 C in 5% CO2. Culture medium changed every 48 h. Co-culture tissues were fixed and embedded in paraffin wax on the 6th day.
were incubated for 1 h at room temperature with primary antibody (cytokeratin7 1:400, CD34 1:400, a-SMA 1:400 all from Boster, China, ephrin-B2 1:50, Affinity, HLAG 1:800 Agent) after blocking. Slides were incubated for another 30 min with HRPconjugated goat anti-rabbit IgG (1:1000; Santa Cruz). Slides were then developed with diaminobenzidine for 5 min and hematoxylin for 2 min. 2.12. Statistical analysis The data was shown as mean and SD (standard deviation) and statistical analysis was performed using GraphPad Prism 5. Differences between two groups were analyzed by t-test. A P value <0.05 was considered statistically significant.
3. Results
2.11. Immunohistochemistry
3.1. Expression of ephrin-B2 in human first trimester placenta
5 mm paraffin sections were used for immunohistochemistry. Rehydrated paraffins were heated in citrate buffer (pH ¼ 6.0) for antigen retrieval for 15 min. Endogenous peroxidase activity was quenched by incubation in 3% H2O2 for 20 min. Sections
The expression of ephrin-B2 in human first trimester placental villi and maternal decidua was shown by IHC staining. Ephrin-B2
Fig. 4. Ephrin-B2 regulated migration and invasion capability of HTR8/SVneo cells. A. Representative images of migration and invasion assay of HTR-8/SVneo cells at a magnification of 200. B&C. Statistical analysis of migration and invasion assays. Down-regulation of ephrin-B2 in HTR-8/SVneo impaired cell migration and invasion capability. D&E The relative gene expression of MMP-2/9 was decreased after sh-ephrin-B2 treatment. (*: P < 0.05, **: P < 0.01, ***: P < 0.001) (n ¼ 3 in triplicate).
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Q. Luo et al. / Placenta xxx (2015) 1e8
5
was found to be expressed in trophoblasts in placental villi (Fig. 1AeC). In maternal decidua, ephrin-B2 was found to be expressed in cells including EVTs (Fig. 1DeF).
sh-ephrin-B2 cells compared to controls (Fig. 3D, P < 0.05), indicating higher caspase-3 activity after down-regulation of ephrin-B2 in HTR-8/SVneo cells.
3.2. Inhibition of ephrin-B2 in HTR-8/SVneo cells
3.4. Down-regulation of ephrin-B2 suppressed HTR-8/SVneo migration and invasion by regulating MMPs
HTR-8/SVneo cell were transfected with plasmids specially targeting ephrin-B2. Transfection efficiency was shown by fluorescence microscope and further assessed by RT-PCR. The inhibition efficiency was more than 80% compared to controls (Fig. 2A, P < 0.001). Reduction of ephrin-B2 was identified by Western Blot in HTR-8/ SVneo cells compared with sh-NC group (Fig. 2B and C, P < 0.01). 3.3. Inhibition of ephrin-B2 reduced cell proliferation and promoted cell apoptosis Proliferation of HTR-8/SVneo was slightly reduced after downregulation of ephrin-B2 compared to negative controls (Fig. 3A P < 0.05). Cell apoptosis was tested via Hoechst staining and caspase-3 colorimetric assay. Down-regulation of ephrin-B2 in HTR-8/SVneo cells was observed with more apoptotic cells (Fig. 3B) and increased relative apoptotic rate (Fig. 3C, P < 0.05). Caspase-3 colorimetric assay showed a higher ratio of relative absorbance in
In transwell models, mobility ability was significantly reduced in the cells transfected with sh-ephrin-B2 (Fig. 4A and B, P < 0.05). Invasion assay showed the same tendency with migration assay (Fig. 4A and C, P < 0.01). MMP-2 and MMP-9 play an important role in trophoblast migration and invasion. In current study, transcriptional levels of MMP-2 and MMP-9 were reduced after downregulation of ephrin-B2 in HTR-8/SVneo cells (Fig. 4D, P < 0.01 and Fig. 4E, P < 0.001). 3.5. Inhibition of ephrin-B2 impaired trophoblast tube formation and suppressed PlGF expression Transfected cells were cultured in Matrigel pre-coated plates in order to assess angiogenesis capability of HTR-8/SVneo after different treatment. Down regulation of ephrin-B2 resulted in a decrease in branching points pre field (magnification 100)
Fig. 5. Ephrin-B2 inhibition prevented tube formation in HTR-8/SVneo cells. A. Tube formation of transfected cells replanted in conditional medium cultured for 6 h on Matrigel (magnification 100). B. Average number of branch points in random fields decreased in sh-ephrin-B2 groups. C. The expression of PlGF mRNA showed a dramatic decreased after ephrin-B2 in HTR-8/SVneo cells. D. VEGF mRNA level did not have a significant change between sh-ephrin-B2 group and negative controls in HTR-8/SVneo cells. (NS: not significant, *: P < 0.05) (n ¼ 3 in triplicate).
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
6
Q. Luo et al. / Placenta xxx (2015) 1e8
compared to negative controls (Fig. 5A and B, P < 0.05). Trophoblast angiogenesis has a close relationship with several angiogenesis factors such as VEGF and PlGF. RT-PCR showed that inhibition of ephrin-B2 significantly suppressed the expression of PlGF at transcriptional level (Fig. 5C, P < 0.05) while the change in VEGF-A mRNA level did not show any statistical difference in HTR-8/ SVneo cells (Fig. 5D). 3.6. Knockout of ephrin-B2 impaired maternal artery remodeling ex vivo PDC system was conducted to further investigate the effects of ephrin-B2 inhibition Unremodelled arteries were characterized with multiple layers of vascular smooth muscle cells (VSMCs), endothelial cell (EC) layer and no EVT (Fig. 6AeC). Active remodeling was characterized with EVTs, endothelial desquamation and disruption of VSMCs (Fig. 6GeI). Veins and glands were excluded by identification with IHC staining (Fig. 6DeF and JeL). After coculture for 6 days, shNC treatment groups showed more advanced remodeling and deeper invasion than sh-ephrin-B2 transfection groups (Fig. 6MeS). 4. Discussion Ephrin-B2 was initially recognized to be critically important for embryonic vasculature [17]. Later studies highlighted the instrumental role of ephrin-B2 in vasculature remodeling [18] and tumorgenesis [19]. Since it is well known that regulation of trophoblast functions shares similar mechanism with tumorgenesis
[20], it is reasonable to hypothesize that ephrin-B2 may also play an important role in the regulation of trophoblast function. Previous studies postulated the role of ephrin-B2 in placentation by IHC staining [9e11]. To date, ephrin-B2-induced trophoblast function in maternal spiral artery remodeling has not been investigated. Current study localized eprhin-B2 in human first trimester placenta villi as well as in maternal decidua. We demonstrated the effects of ephrin-B2 on trophoblast by down-regulation of its expression in trophoblast cell line. Invasion and tube formation of HTR-8/SVneo were significantly impaired after inhibition of ephrinB2 via regulation of MMPs and angiogenic factor, PlGF. Furthermore, we conducted an ex vivo model to verify the effects after ephrin-B2 knock-down. Inadequate invasion of trophoblast and endovascular transformation were observed in the co-culture system. Taking together these results indicate the important role of ephrin-B2 in maternal spiral artery remodeling in first trimester. Trophoblast invasion contributes to the success of maternal spiral artery remodeling. Mounting evidences suggest that aberration in maternal vascular modification resulting from shallow invasion of trophoblast is associated with obstetric complications [3,4]. In this study, we found a dramatic reduction in migration and invasion with small changes in cell proliferation and apoptosis after inhibition of ephrin-B2 in HTR-8/SVneo cells, especially invasion. Our results are in accordance with other study in tumor [21]. Trophoblast invasion in early pregnancy requires the anticipation of MMP-2 and MMP-9 [22]. Our study showed that down-regulation of ephrin-B2 inhibited the expression of both MMP-2 and MMP-9 at transcriptional level. However, Kwan et al. reported reduction of MMPs in response to ephrin-B2 stimulation [23]. This
Fig. 6. Immunohistochemistry staining of placental-decidual co-culture system. AeC. Intact arteries had no trophoblast present (A) and possessed multiple layers of vascular muscle (B) as well as an endothelial layer (C). DeF. Advanced remodeling arteries had invading trophoblasts (D), lost vascular muscle layers (E) and isolated endothelial cells (F). GeI: Veins showed a thin layer of endothelial cell (I), sporadic vascular muscle cell (H) and absence of trophoblast (I). JeL: Glands in maternal decidua were detective with CK-7 (G) but negative of a-SMA and CD 34 staining (K and L). M-S. IHC staining of PDC with different treatment on the 6th day. Sh-ephrin-B2 treatment samples showed an early remodeling with vascular muscle disruption at superficial area (O) and intact arteries (arrows in M, O, Q). Sh-NC treated samples showed remodeled arteries in deeper area (arrows in N, P, S). Samples were collected from 12 pregnant women in all.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
Q. Luo et al. / Placenta xxx (2015) 1e8
contradiction may result from the unique mechanism of bidirectional signal pathways of eprhin-B2/Ephrin B4. For instance, EphB/ephrin-B2 signaling in ECs promotes cell adhesion [24], whereas the reverse signaling induces the opposite effect [25]. Endovascular trophoblast replacement is another crucial process of spiral artery remodeling [26]. EVTs differentiate into endovascular trophoblasts after they invade into maternal decidua, establishing the conversion of spiral arteries into low resistance vessels during pregnancy. Our study showed a worse network formation in HTR-8/SVneo cells with sh-ephrin-B2 treatment in vitro. Therefore, inadequate conversion of maternal spiral arteries resulting from ephrin-B2 inhibition may contribute to the pathogenesis of diseases related to impaired placentation. Reduced gene expressions of angiogenic factors in first trimester are related to the onset of placental vascular disorders in later term [27]. We further investigated the expression of angiogenic factors, PlGF and VEGF-A. The reduced gene expressions of both angiogenic factors are related to the onset of preeclampsia [27]. A reduction in PlGF transcription after down-regulation of ephrin-B2 was observed. However, mRNA level of VEGF-A increased after inhibition of ephrin-B2 without statistically significant difference. We speculate that this is probably because ephrin-B2 inhibits VEGF-induced angiogenesis by regulating VEGF2 and VEGF3 expression without directly targeting at VEGF-A [28]. We found that down-regulation of eprhin-B2 in placental villi resulted in shallow invasion and damaged conversion of maternal spiral arteries in ex vivo mode of artery remodeling, consistent with the findings of the effect of inhibition of eprhin-B2 in HTR-8/SVneo cells. Our study demonstrated the possible role of ephrin-B2 as a new target for placental vascular disorders. Regarding the possible molecular pathways, we speculate that eprhin-B2 may induce trophoblast functions in spiral artery remodeling via extracellular regulated ERK-1/2 and AKT signaling pathways. Erk 1/2 and AKT are the common pathways involved in trophoblast invasion [29,30] and angiogenesis [31]. Previous studies demonstrated that ephrin-B2 is able to active these pathways in hepatic stellate cells [32] and T cells [33]. Taken together, we hypothesize that similar mechanism works in ephrin-B2induced trophoblast function. We will investigate this issue in our future studies. In conclusion, our study identified the role of ephrin-B2 in trophoblast invasion and endovascular formation. Therefore, it may be an important positive regulator in trophoblast-dependent maternal spiral artery remodeling. Conflict of interest statement All the authors declare that they have no conflict of interest. Acknowledgment We wish to express sincere thanks to Dr. Charles Graham (Queen's University, Kingston, Ontario, Canada) for kindly providing the trophoblast cell line. This work was supported by National Natural Science Foundation of China (No. 81170584 and 81370733 to L Zou; No. 81100442 to Y. Zhao). References [1] Pijnenborg R, Vercruysse L, Hanssens M. The uterine spiral arteries in human pregnancy: facts and controversies. Placenta 2006 Sep-Oct;27(9e10):939e58. [2] Orozco AF, Jorgez CJ, Ramos-Perez WD, Popek EJ, Yu X, Kozinetz CA, et al. Placental release of distinct DNA-associated micro-particles into maternal circulation: reflective of gestation time and preeclampsia. Placenta 2009 Oct;30(10):891e7.
7
[3] Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod 2003 Jul;69(1):1e7. [4] van den Brule F, Berndt S, Simon N, Coulon C, Le Goarant J, Munaut C, et al. Trophoblast invasion and placentation: molecular mechanisms and regulation. Chem Immunol Allergy 2005;88:163e80. [5] Hafner C, Schmitz G, Meyer S, Bataille F, Hau P, Langmann T, et al. Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers. Clin Chem 2004 Mar;50(3):490e9. [6] Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 2010 Mar;10(3):165e80. [7] Klein R. Eph/ephrin signalling during development. Development 2012 Nov;139(22):4105e9. [8] Mosch B, Reissenweber B, Neuber C, Pietzsch J. Eph receptors and ephrin ligands: important players in angiogenesis and tumor angiogenesis. J Oncol 2010;2010:135285. [9] Chennakesava CS, Di Santo S, Ziemiecki A, Schneider H, Andres AC. Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development. Placenta 2006 SepeOct;27(9e10): 959e67. [10] Goldman-Wohl D, Greenfield C, Haimov-Kochman R, Ariel I, Anteby EY, Hochner-Celnikier D, et al. Eph and ephrin expression in normal placental development and preeclampsia. Placenta 2004 Aug;25(7):623e30. [11] Zhang J, Dong H, Wang B, Zhu S, Croy BA. Dynamic changes occur in patterns of endometrial EFNB2/EPHB4 expression during the period of spiral arterial modification in mice. Biol Reprod 2008 Sep;79(3):450e8. [12] Red-Horse K, Kapidzic M, Zhou Y, Feng KT, Singh H, Fisher SJ. EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation. Development 2005 Sep;132(18):4097e106. [13] Zhao Y, Zheng YF, Luo QQ, Yan T, Liu XX, Han L, et al. Edaravone inhibits hypoxiainduced trophoblast-soluble Fms-like tyrosine kinase 1 expression: a possible therapeutic approach to preeclampsia. Placenta 2014 Jul;35(7):476e82. [14] Highet AR, Zhang VJ, Heinemann GK, Roberts CT. Use of Matrigel in culture affects cell phenotype and gene expression in the first trimester trophoblast cell line HTR8/SVneo. Placenta 2012 Jul;33(7):586e8. [15] Dunk C, Petkovic L, Baczyk D, Rossant J, Winterhager E, Lye S. A novel in vitro model of trophoblast-mediated decidual blood vessel remodeling. Lab Invest 2003 Dec;83(12):1821e8. [16] Hazan AD, Smith SD, Jones RL, Whittle W, Lye SJ, Dunk CE. Vascular-leukocyte interactions: mechanisms of human decidual spiral artery remodeling in vitro. Am J Pathol 2010 Aug;177(2):1017e30. [17] Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 1998 May 29;93(5):741e53. [18] Davy A, Soriano P. Ephrin-B2 forward signaling regulates somite patterning and neural crest cell development. Dev Biol 2007 Apr 1;304(1):182e93. [19] Abengozar MA, de Frutos S, Ferreiro S, Soriano J, Perez-Martinez M, Olmeda D, et al. Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth. Blood 2012 May 10;119(19): 4565e76. [20] Holtan SG, Creedon DJ, Haluska P, Markovic SN. Cancer and pregnancy: parallels in growth, invasion, and immune modulation and implications for cancer therapeutic agents. Mayo Clin Proc 2009 Nov;84(11): 985e1000. [21] Meyer S, Hafner C, Guba M, Flegel S, Geissler EK, Becker B, et al. Ephrin-B2 overexpression enhances integrin-mediated ECM-attachment and migration of B16 melanoma cells. Int J Oncol 2005 Nov;27(5):1197e206. [22] Staun-Ram E, Goldman S, Gabarin D, Shalev E. Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion. Reprod Biol Endocrinol 2004 Aug 4;2:59. [23] Kwan Tat S, Pelletier JP, Amiable N, Boileau C, Lavigne M, MartelPelletier J. Treatment with ephrin B2 positively impacts the abnormal metabolism of human osteoarthritic chondrocytes. Arthritis Res Ther 2009;11(4):R119. [24] Oike Y, Ito Y, Hamada K, Zhang XQ, Miyata K, Arai F, et al. Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells. Blood 2002 Aug 15;100(4):1326e33. [25] Swift MR, Weinstein BM. Arterial-venous specification during development. Circulat Res 2009 Mar 13;104(5):576e88. [26] Whitley GS, Cartwright JE. Cellular and molecular regulation of spiral artery remodelling: lessons from the cardiovascular field. Placenta 2010 Jun;31(6): 465e74. [27] Farina A, Sekizawa A, De Sanctis P, Purwosunu Y, Okai T, Cha DH, et al. Gene expression in chorionic villous samples at 11 weeks' gestation from women destined to develop preeclampsia. Prenat Diagn 2008 Oct;28(10): 956e61. [28] Wang Y, Nakayama M, Pitulescu ME, Schmidt TS, Bochenek ML, Sakakibara A, et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 2010 May 27;465(7297):483e6. [29] Qiu Q, Yang M, Tsang BK, Gruslin A. Both mitogen-activated protein kinase and phosphatidylinositol 3-kinase signalling are required in epidermal growth factor-induced human trophoblast migration. Mol Hum Reprod 2004 Sep;10(9):677e84.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009
8
Q. Luo et al. / Placenta xxx (2015) 1e8
[30] Fitzgerald JS, Busch S, Wengenmayer T, Foerster K, de la Motte T, Poehlmann TG, et al. Signal transduction in trophoblast invasion. Chem Immunol Allergy 2005;88:181e99. [31] Selvaraj SK, Giri RK, Perelman N, Johnson C, Malik P, Kalra VK. Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor. Blood 2003 Aug 15;102(4):1515e24. [32] Das A, Shergill U, Thakur L, Sinha S, Urrutia R, Mukhopadhyay D, et al. Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF
production and sinusoidal endothelial cell recruitment. Am J Physiol Gastrointest Liver Physiol 2010 Jun;298(6):G908e15. [33] Kawano H, Katayama Y, Minagawa K, Shimoyama M, Henkemeyer M, Matsui T. A novel feedback mechanism by Ephrin-B1/B2 in T-cell activation involves a concentration-dependent switch from costimulation to inhibition. Eur J Immunol 2012 Jun;42(6):1562e72.
Please cite this article in press as: Luo Q, et al., Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester, Placenta (2015), http://dx.doi.org/10.1016/j.placenta.2015.02.009