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Decreased expression of Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 may be involved in the development of pre-eclampsia

  • Author Footnotes
    1 These authors contributed equally to this project and should be considered as co-first author.
    Juan Li
    Footnotes
    1 These authors contributed equally to this project and should be considered as co-first author.
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Author Footnotes
    1 These authors contributed equally to this project and should be considered as co-first author.
    Xin Luo
    Footnotes
    1 These authors contributed equally to this project and should be considered as co-first author.
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Xiaoqiu Xiao
    Affiliations
    Laboratory of Lipid and Glucose Research, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Xuemei Zhang
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Hongbo Qi
    Correspondence
    Corresponding author.
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Xiru Liu
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Hua Zhang
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Li Gao
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Zhongmei Yang
    Affiliations
    Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, People’s Republic of China
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  • Author Footnotes
    1 These authors contributed equally to this project and should be considered as co-first author.
Published:August 28, 2013DOI:https://doi.org/10.1016/j.rbmo.2013.07.015

      Abstract

      Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2) is a protein that mediates actin cytoskeletal reorganization and lamellipodia protrusion formation, which are required for cell migration and invasion. The primary purpose of this study was to determine whether there is an association between reactive oxygen species (ROS) and WAVE2 in pre-eclampsia, and whether WAVE2 expression in trophoblast cells is vulnerable to oxidative stress. This study observed excessive generation of ROS and decreased expression of WAVE2 in pre-eclamptic placentas compared with normotensive controls. Moreover, there was a significant negative correlation between ROS and WAVE2 protein in pre-eclamptic placenta (P < 0.001). An in-vitro model of hypoxia–reoxygenation (H/R) was used to imitate oxidative stress in placental trophoblasts, and it was found that the expression of WAVE2 protein in trophoblasts was decreased after H/R treatment. Additionally, compared with normoxia, decreased cell proliferation, higher cell apoptosis and attenuated cell migration and invasion were detected in trophoblasts exposed to H/R. In conclusion, the findings strongly suggest that excessive oxidative stress can decrease WAVE2 expression in trophoblasts and that the decreased expression of WAVE2 in trophoblast cells may be involved in the development of pre-eclampsia.
      It is postulated that pre-eclampsia is due to shallow trophoblast invasion and insufficient remodelling of uterine spiral artery. Oxidative stress, resulting in over-production of reactive oxygen species (ROS), plays an important role in this process. However, the way that placental oxidative stress results in the reduced trophoblasts invasion and migration remains enigmatic. WAVE2 is a protein that mediates actin cytoskeletal reorganization and lamellipodia protrusion formation which are required for cell migration and invasion. We observed excessive generation of ROS and decreased expression of WAVE2 in pre-eclamptic placentas compared with normotensive controls. Moreover, there was a significant negative correlation between ROS and WAVE2 protein in pre-eclamptic placenta. We utilized an in-vitro model of hypoxia–reoxygenation (H/R) to imitate oxidative stress in placental trophoblasts, and we found that the expression of WAVE2 protein in trophoblasts was decreased followed by H/R treatment. Additionally, compared with normoxia condition, decreased cell proliferation, higher cell apoptosis and attenuated cell migration and invasion were detected in trophoblasts exposed to H/R. Our findings highlight that decreased WAVE2 expression is associated with oxidative stress and, thus, may be involved in the pathogenesis of pre-eclampsia.

      Keywords

      Introduction

      Pre-eclampsia is a pregnancy-specific obstetric complication characterized not only by hypertension but also by a high level of proteinuria after 20 weeks of gestation. It is a major cause of maternal and fetal mortality and morbidity and occurs in 3–14% of all pregnancies worldwide (
      • Campbell O.M.
      • Graham W.J.
      Lancet Maternal Survival Series Steering Group
      Strategies for reducing maternal mortality: getting on with what works.
      ,
      • Irminger-Finger I.
      • Jastrow N.
      • Irion O.
      Preeclampsia: a danger growing in disguise.
      ). The aetiopathogenesis of pre-eclampsia is unclear, but excessive oxidative stress and abnormal trophoblast invasion seem to be major contributors to the development of this pregnancy disorder.
      Pregnancy is a state of oxidative stress arising from increased production of reactive oxygen species (ROS). However, a heightened level of oxidative stress is encountered in pre-eclampsia (
      • Myatt L.
      • Cui X.
      Oxidative stress in the placenta.
      ). Excessive oxidative stress is associated with shallow trophoblast invasion of maternal endometrial spiral arteries, and ROS generation is considered to be an important intermediate event in this process (
      • Murata M.
      • Fukushima K.
      • Takao T.
      • Seki H.
      • Takeda S.
      • Wake N.
      Oxidative stress produced by xanthine oxidase induces apoptosis in human extravillous trophoblast cells.
      ). In addition to causing oxidative damage to DNA, lipid membranes and proteins (
      • Dhar-Mascareño M.
      • Cárcamo J.M.
      • Golde D.W.
      Hypoxia-reoxygenation-induced mitochondrial damage and apoptosis in human endothelial cells are inhibited by vitamin C.
      ), intracellular ROS accumulation can also alter cell signalling as well as gene expression in ways that are similar to those seen in pre-eclampsia (
      • Cindrova-Davies T.
      • Yung H.W.
      • Johns J.
      • Spasic-Boskovic O.
      • Korolchuk S.
      • Jauniaux E.
      • Burton G.J.
      • Charnock-Jones D.S.
      Oxidative stress, gene expression, and protein changes induced in the human placenta during labor.
      ). However, the molecular mechanisms underlying the role of ROS in trophoblast invasion are still unknown.
      Cell migration and invasion are induced by rearrangement of the actin cytoskeleton at the leading edge of cells and are accompanied by the formation of specific cellular structures termed lamellipodia and filopodia (
      • Rottner K.
      • Stradal T.E.
      Actin dynamics and turnover in cell motility.
      ). WAVE2 protein is required for lamellipodium formation and mediates cell invasion and migration (
      • Pocha S.M.
      • Cory G.O.
      WAVE2 is regulated by multiple phosphorylation events within its VCA domain.
      ). The depletion of WAVE2 by RNAi significantly attenuated the invasion and metastatic phenotypes of murine melanoma cells (
      • Kurisu S.
      • Suetsugu S.
      • Yamazaki D.
      • Yamaguchi H.
      • Takenawa T.
      Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells.
      ). Moreover, the depletion of WAVE2 by RNAi abrogated both human breast cancer cell invasion and intensive F-actin accumulation at the invasion site (
      • Takahashi K.
      • Suzuki K.
      WAVE2, N-WASP, and Mena facilitate cell invasion via phosphatidylinositol 3-kinase-dependent local accumulation of actin filaments.
      ). Furthermore, a high concentration of WAVE2 was thought to be correlated with a poor clinical outcome in a variety of tumours, such as human hepatocellular carcinoma, lung squamous cell carcinoma and breast adenocarcinomas (
      • Cai X.
      • Xiao T.
      • James S.Y.
      • Da J.
      • Lin D.
      • Liu Y.
      • Zheng Y.
      • Zou S.
      • Di X.
      • Guo S.
      • Han N.
      • Lu Y.J.
      • Cheng S.
      • Gao Y.
      • Zhang K.
      Metastatic potential of lung squamous cell carcinoma associated with HSPC300 through its interaction with WAVE2.
      ,
      • Iwaya K.
      • Norio K.
      • Mukai K.
      Coexpression of Arp2 and WAVE2 predicts poor outcome in invasive breast carcinoma.
      ,
      • Yang L.Y.
      • Tao Y.M.
      • Ou D.P.
      • Wang W.
      • Chang Z.G.
      • Wu F.
      Increased expression of Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 correlated with poor prognosis of hepatocellular carcinoma.
      ). A recent study demonstrated that WAVE2 works as an intermediate that links ROS signalling to actin cytoskeletal reorganization in the invasion and metastasis of mouse melanoma cell lines (
      • Park S.J.
      • Kim Y.T.
      • Jeon Y.J.
      Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott–Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells.
      ).
      This study detected the intracellular location and expression of WAVE2 in placentas and simultaneously examined the correlation between ROS and WAVE2 in normotensive and pre-eclamptic placentas to determine whether there is a relationship between ROS and WAVE2 in pre-eclampsia. In addition, since placental oxidative stress could be the consequence of fluctuations in oxygen concentrations after hypoxia–reoxygenation (H/R) through the actions of ROS (
      • Burton G.J.
      • Yung H.W.
      Endoplasmic reticulum stress in the pathogenesis of early-onset pre-eclampsia.
      ), and given that H/R represents a suitable model system for investigating the generation of placental oxidative stress in pre-eclampsia (
      • Hung T.H.
      • Burton G.J.
      Hypoxia and reoxygenation: a possible mechanism for placental oxidative stress in preeclampsia.
      ), this study used the human first-trimester extravillous trophoblast cell line HTR8/SVneo and exposed these cells to H/R to determine whether WAVE2 expression in trophoblast cells is vulnerable to oxidative stress and to evaluate the relationship between WAVE2 concentrations and trophoblast invasion activity.

      Materials and methods

      Patients and sample collection

      The study is under the permission of Ethical Committee of Human Experimentation in Chongqing (licence no. 2013-04) in February 2013. A total of 40 patients were selected: 20 women were pre-eclamptic patients and 20 women with uncomplicated pregnancies were the controls. All patients were recruited in the Department of Obstetrics and Gynecology at the First Affiliated Hospital of Chongqing Medical University from September 2011 to February 2012. The definition and criteria of pre-eclampsia were based on the diagnostic criteria outlined by the American College of Obstetrics and Gynecology (
      ACOG Practice Bulletin
      Diagnosis and management of preeclampsia and eclampsia.
      ). All patients delivered by Caesarean section. The control group was undergoing Caesarean section for breech presentation or maternal request prior to the onset of labour. All subjects with diabetes mellitus, chronic renal diseases, chronic hypertension or other metabolic diseases were excluded from this study.
      Placental tissues were collected immediately after delivery. The tissues were dissected from the centre of the placenta, avoiding areas of infarction, calcification or haematoma. The general dimension of each sample was approximately 1–2 cm3, with a wet weight of 100 mg. After being washed in phosphate-buffered saline (PBS), one biopsy was immediately fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry. The other two biopsies were snap frozen in liquid nitrogen and stored at −80°C for RNA and protein isolation. Peripheral venous blood (5 ml) was sampled before surgery. After centrifugation at 2000g for 10 min at 4°C, serum samples were obtained and stored at −80°C until use.

      Immunohistochemistry

      The sections (5 μm) were de-waxed, rehydrated and incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity. After heat-induced antigen retrieval in an autoclave, sections were incubated overnight at 4°C with rabbit anti-WAVE2 (1:200; Santa Cruz, CA, USA) and were then incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:1000; Santa Cruz). Staining was completed by incubation with diaminobenzidine chromogen solution (DAB). Haematoxylin was used as a nuclear counterstain. A negative control was performed using nonimmune rabbit IgG. The immunoreactivity was detected on a light microscope. For each sample, experiments were performed at least three times, and comparable results were obtained each time.

      Quantitative real-time PCR

      Total RNA was extracted from the placental tissues homogenized in TRIzol reagent (TaKaRa, Japan) and was purified according to the manufacturer’s instructions. Reverse transcription was performed using the Primescript RT reagent kit (TaKaRa). The primers included WAVE2 (forward: 5′-TACCACCACCGCTTTCTGATAC-3′; reverse: 5′-ACATCCCGCTTCTCTTGTTCC-3′) and GAPDH (forward: 5′-CTTTGGTATCGTGGAAGGACTC-3′; reverse: 5′-GTAGAGGCAGGGATGATGTTCT-3′). Quantitative real-time PCR (qRT-PCR) was performed using a real-time PCR instrument (Bio-Rad) with the SYBR Green Realtime PCR Master Mix kit (TaKaRa). PCR proceeded with an initial preincubation step at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 min). The CT values were normalized to GADPH and the relative mRNA concentrations of WAVE2 were analysed with the 2−ΔΔCT method (
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method.
      ). Experiments were performed in triplicate and repeated at least three times.

      Western blot analysis

      Proteins were extracted from placental tissues and HTR-8/SVneo cells by the Total Protein Extraction Kit (KeyGEN Biotech, Nanjing, China) following the manufacturer’s instructions. A BCA(bicinchoninic acid) assay (Beyotime, Shanghai, China) was used to determine the protein concentration of the lysates. Equal amounts of total protein (40 μg) were subjected to electrophoresis on 8% SDS-PAGE using a BioRad Power-Pac 200 apparatus and transferred to a 0.45 μm polyvinylidene difluoride membrane using a BioRad Mini-Transblotter apparatus. The membrane was then blocked for 1 h and incubated with the rabbit anti-WAVE2 (1:500; Santa Cruz). GADPH (1:3000; Santa Cruz) was used as a loading control. Immunodetection was accomplished using goat anti-rabbit secondary antibodies and an enhanced chemiluminescence detection kit (ECL; Beyotime). The experiment was performed in triplicate.

      ROS determination

      When ROS are present in placental tissue, they can react with the colour reagent xylenol orange in an acidic solution with sorbitol and ammonium iron sulphate, producing a purple colouration in proportion to the concentration of ROS in the sample. In brief, placental tissue (approximately 100 mg) was homogenized to collect the fresh tissue supernatant and the colour reagent was added following the manufacturer’s instructions (KeyGEN Biotech). After mixing for 10 s and incubating for 30 min at room temperature, the optical density of the mixture was measured at 550 nm. The experiment was performed in triplicate.

      Cell culture and H/R application

      A first-trimester extravillous trophoblast cell line (HTR8/SVneo cells), kindly provided by CH Graham (Kingston, Ontario, Canada), was grown in RPMI 1640 (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL). The cells were maintained under standard culture conditions as the normoxic control (5% CO2/balanced air). H/R intervention (8 h at 2% oxygen, followed by 16 h at standard culture conditions, two cycles) was performed as previously described (
      • Hung T.H.
      • Chen S.F.
      • Li M.J.
      • Yeh Y.L.
      • Hsieh T.T.
      Differential effects of concomitant use of vitamins C and E on trophoblast apoptosis and autophagy between normoxia and hypoxia-reoxygenation.
      ).

      Flow cytometric analysis of apoptosis and intracellular ROS formation

      The apoptotic rate of HTR-8/SVneo cells was measured by an Annexin V–FITC and PI Apoptosis Detection Kit (KeyGEN Biotech). This method was carried out as described previously (
      • Luo X.
      • Yao Z.W.
      • Qi H.B.
      • Liu D.D.
      • Chen G.Q.
      • Huang S.
      • Li Q.S.
      Gadd45alpha as an upstream signaling molecule of p38 MAPK triggers oxidative stress-induced sFlt-1 and sEng upregulation in preeclampsia.
      ). In brief, after H/R exposure for 48 h, the cells were stained with binding buffer (1 μg/ml propidium iodide and 1 μg/ml fluorescein isothiocyanate, FITC, -labelled annexin-V) and were then gently vortexed and incubated in the dark for 30 min at room temperature. Apoptosis rates were quantified with a FACS Vantage SE flow cytometer (BD Biosciences, San Jose, CA, USA). Experiments were performed five times.
      Intracellular ROS was assessed by means of an oxidation-sensitive fluorescent probe (dichlorodihydrofluorescein diacetate, DCFH-DA; Sigma, St Louis, MO, USA). Briefly, 1 × 106 cells from each sample were collected and treated with 20 μmol/l DCFH-DA. Then, cells of each group were incubated at 37°C for 1 h. After incubation, cells were analysed using a flow cytometer to determine the cellular fluorescence intensity at excitation and emission wavelengths of 488 nm and 525 nm. The fluorescence intensity of DCFH reflected any enhanced oxidative stress. Experiments were performed in triplicate.

      Cell proliferation assay

      Cell proliferation was examined by a cell counting kit-8 (CCK8; Sigma). Briefly, 5 × 103 cells were placed in H/R or normal conditions for 8 h, 24 h, 32 h, 48 h, 56 h and 72 h. Then, 10 μl CCK8 was added to each well, and cells were incubated at 37°C for 2 h. The optical density of each well was measured using a microculture plate reader (model 550; BioRad) at a wavelength of 450 nm. Experiments were performed in triplicate.

      Matrigel invasion assay

      The invasion assay was performed in 24-well plates with membrane inserts. Insert membranes were pre-coated with 100 ml Matrigel (BD Biosciences) at 1 mg/ml for 3 h at 37°C. A total of 1 × 105 HTR-8/SVneo cells were plated in the upper compartment of the Matrigel matrix. Then, medium with 10% FBS was added to the lower compartment. After 48 h of incubation, cells that had invaded to the other side of the insert were fixed with 3% paraformaldehyde and were then stained with crystal violet, counted and photographed with a microscope in 10 random fields at a magnification ×200. Finally, the presented data were expressed as a percentage of the control values. Experiments were performed in triplicate.

      Trans-well migration assay

      Methods performed to determine the migratory ability of HTR8/SVneo cells were similar to Matrigel invasion assay except that the trans-well insert was not coated with Matrigel. Experiments were performed in triplicate.

      Cell immunofluorescence

      After 48 h of incubation, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and blocked in 1% bovine serum albumin. Then, cells were incubated with rabbit anti-WAVE2 (1:20; Santa Cruz), washed in PBS and incubated in FITC-conjugated anti-rabbit IgG (1:100; Zhongsan Golden Bridge Crop, Beijing, China) for 2 h at 37°C. Nuclei were stained with propidium iodide. Images were obtained using a laser-scanning confocal microscope. Experiments were performed in triplicate.

      ELISA

      The concentrations of WAVE2 in the maternal serum and supernatants of the HTR-8/SVneo cells were determined by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Uscn Life Science, Wuhan, China) following the manufacturer’s instructions. The experiment was performed at least three times with triplicate samples.

      Statistical analysis

      All statistical analyses were performed using Statistical Package for Social Sciences Graduate Pack version 17.0 statistical software (SPSS, Chicago, IL, USA). All data fitted the Gaussian distribution and are presented as mean ± standard deviation. Significant differences between the two groups were evaluated by independent t-tests. The Pearson’s correlation coefficient was used for correlation analysis. Differences were considered significant at P < 0.05.

      Results

      Clinical characteristics

      As shown in Table 1, there was no significant difference in gestational age, maternal age and body mass index between the pre-eclampsia and control groups. However, the birthweight of infants from pre-eclamptic mothers was significantly reduced (P < 0.05). In addition, women from the pre-eclampsia group had significantly lower placental weight (P < 0.05) and higher systolic blood pressure, diastolic blood pressure and 24-hour proteinuria relative to normal pregnancies (all P < 0.01).
      Table 1Clinical characteristics of the pre-eclampsia and control groups.
      VariablePre-eclampsia (n =20)Control (n =20)
      Age of patients (years)30.12 ± 3.1229.32 ± 2.23
      Length of gestation (weeks)35.26 ± 2.1237.52 ± 1.43
      Body mass index (kg/m2)24.12 ± 1.7825.34 ± 1.89
      Proteinuria (g/24 h)1.96 ± 0.24
      P<0.01.
      0.08 ± 0.05
      Systolic blood pressure (mmHg)154 ± 13
      P<0.01.
      98 ± 11
      Diastolic blood pressure (mmHg)105 ± 14
      P<0.01.
      69 ± 8
      Birthweight (g)2446 ± 187
      P<0.05.
      3207 ± 233
      Placental weight (g)432 ± 53
      P<0.05.
      536 ± 67
      low asterisk P < 0.05.
      low asterisklow asterisk P < 0.01.

      Immunohistochemical localization of WAVE2 in normal and pre-eclampsia placentas

      The localization of WAVE2 protein in placentas was investigated by immunohistochemistry. WAVE2 protein was located in the cytoplasm of syncytiotrophoblasts and vascular endothelial cells. In comparison to the normal trophoblast cells, the positive WAVE2 signals were lower in the pre-eclampsia trophoblast cells (Figure 1).
      Figure thumbnail gr1
      Figure 1Immunohistochemistry for WAVE2 protein concentrations and locations in normal (n = 20) and pre-eclampsia placentas (n = 20). (A) Representative immunostaining of WAVE2 protein in normotensive placentas (brown); WAVE2 protein located in the syncytiotrophoblasts (Sc, arrow) and vascular endothelial cells (Ec, arrowhead); (A inset), higher magnification view. (B) Representative immunostaining of WAVE2 protein in pre-eclampsia placentas; the positive signals for WAVE2 are lower in the pre-eclampsia trophoblast cells than in the normal trophoblast cells. (C) Negative control using non-immune rabbit IgG. Bars = 25 μm (A–C), 6.25 μm (A inset).

      Down-regulation of WAVE2 expression in pre-eclampsia

      The differences of WAVE2 expression levels between the normal and pre-eclampsia patients were examined by qRT-PCR, Western blot and ELISA. As shown in Figure 2B, the molecular weight of WAVE2 is approximately 85 kDa. The expression of WAVE2, at both the mRNA (Figure 2A) and protein (Figure 2B) levels, was significantly lower in the pre-eclampsia placentas compared with the control group (P < 0.05). Additionally, the concentration of WAVE2 protein in the sera of patients with pre-eclampsia was also significantly lower than in the healthy controls (P < 0.05; Figure 2C).
      Figure thumbnail gr2
      Figure 2WAVE2 expression in placentas and maternal serum in normal (n = 20) and pre-eclampsia patients (n = 20). (A) Ratio of WAVE2 mRNA concentration obtained from placentas (P < 0.05). (B) Representative immunoblotting of WAVE2 normalized with GADPH (upper panel) and comparison of protein concentrations in different groups (lower panel; P < 0.05). (C) WAVE2 protein concentrations in maternal blood serum of patients with pre-eclampsia and healthy controls (P < 0.05).

      The negative correlation between WAVE2 and ROS in pre-eclamptic placentas

      ROS concentrations were higher in pre-eclamptic placentas than in normotensive placentas (P < 0.05; Figure 3A). Interestingly, there was a negative correlation between WAVE2 and ROS concentrations in pre-eclamptic placentas (r = −0.726, P < 0.001; Figure 3B). However, this negative correlation could not be found in normotensive placentas (data not shown).
      Figure thumbnail gr3
      Figure 3The correlation between WAVE2 and ROS concentrations in pre-eclamptic placentas. (A) Comparison of ROS concentrations in normal (n = 20) and pre-eclampsia patients (n = 20; P < 0.05. (B) Negative correlation between WAVE2 and ROS concentrations in pre-eclamptic placentas (r = −0.726, P < 0.001).

      Higher ROS generation in the HTR-8/SVneo cells after exposure to H/R

      H/R-induced ROS accumulation in HTR-8/SVneo cells was evaluated by monitoring the change in fluorescence intensity of DCFH (Figure 4A, a representative picture). As shown in Figure 4B, HTR-8/SVneo cells exposed to H/R displayed a significant increase in fluorescence intensity compared with control cells without H/R exposure (n = 3, P < 0.05).
      Figure thumbnail gr4
      Figure 4Effects of hypoxia–reoxygenation (H/R) on intracellular reactive oxygen species formation. (A) Representative picture showing a dramatic increase in fluorescence intensity in the H/R group compared with the control group. (B) ROS concentrations in the H/R and control groups (n = 3; P < 0.05). DCFH = dichlorodihydrofluorescein diacetate.

      Decreased cell proliferation, higher cell apoptosis and attenuated cell invasion and migration in the HTR-8/SVneo cells in H/R-induced oxidative stress

      The effect of H/R on apoptosis in HTR-8/SVneo cells was quantitatively detected by flow cytometric analysis. Figure 5A is a representative flow cytometric picture of apoptosis in trophoblastic cells with or without H/R exposure, showing that 2.05% of control cells (without H/R exposure) excluded propidium iodide and were positive for annexin V–FITC binding, which were identified as early apoptotic cells. However, the number of early apoptotic cells rose to 34.14% after exposure to H/R for 48 h. The mean early apoptotic cells in the H/R and control groups were 30.6 ± 2.3% and 2.9 ± 0.6%, respectively (n = 5, P < 0.01; Figure 5B).
      Figure thumbnail gr5
      Figure 5Effects of hypoxia–reoxygenation (H/R)-induced oxidative stress on proliferation, apoptosis and migration and invasion in HTR-8/SVneo cells. (A) The effect of H/R-induced oxidative stress on apoptosis in HTR-8/SVneo cells assessed by flow cytometry. (B) Mean percentage of early apoptosis from independent experiments; each experiment was run five times; significant differences were observed for H/R-induced oxidative-stress-mediated early apoptosis in HTR-8/SVneo cells. (C) Inhibition of proliferation in HTR-8/SVneo cells of the H/R group was significantly higher than that of the control group (the data in control group was 100%, n = 3) (P < 0.05). (D) Invaded cells in the invasion assay (upper panel) and summary of three independent experiments that assessed the effect of H/R-induced oxidative stress on invasion of HTR-8/SVneo cells (lower panel). (E) Migrated cells in the trans-well migration assay (upper panel) and summary of three independent experiments that assessed the effect of H/R-induced oxidative stress on migration of HTR-8/SVneo cells (lower panel). PI = propidium iodide. Bars = 25 μm. P < 0.05, ∗∗P < 0.01.
      As shown in Figure 5C, with the prolonged exposure time, the inhibition of proliferation in HTR-8/SVneo cells following the H/R treatment was significantly higher than that of the control group based on CCK8 measurement (n = 3, P < 0.05).
      To assess the influence of H/R-induced oxidative stress on the invasion and migration of trophoblasts, the trans-well matrigel invasion assay and migration assay in vitro were used. The results showed that after exposure to the H/R environment for 48 h, both the invasion and migration of HTR-8/SVneo cells were decreased compared with the control group (P < 0.05; Figure 5D, E).

      Down-regulation of WAVE2 expression in HTR-8/SVneo cells exposed to H/R

      To study the effects of H/R-induced oxidative stress on in-vitro expression of WAVE2 in trophoblast cells, cell immunofluorescence, Western blotting and ELISA were performed. As shown in Figure 6A, WAVE2 protein was located in the cytoplasm of HTR-8/SVneo cells, and cells exposed to H/R displayed weaker staining compared with the control group. The relative expression of WAVE2 protein in HTR-8/SVneo cells after exposure to H/R was reduced by approximately 45% compared with that of the in control group (P < 0.05; Figure 6B). Simultaneously, WAVE2 protein in the cell supernatant obtained from H/R was also lower than in the control group (P < 0.05; Figure 6C).
      Figure thumbnail gr6
      Figure 6Expression of WAVE2 protein in HTR-8/SVneo cells exposed to hypoxia–reoxygenation (H/R). (A) Immunofluorescent detection of WAVE2 protein (green) and nuclei (red, propidium iodide, PI) in HTR-8/SVneo cells placed in H/R for 48 h or unexposed. WAVE2 protein was located in the cytoplasm of HTR-8/SVneo cells, and cells demonstrated weaker staining after exposure to H/R for 48 h compared with the control group (n = 3). (B) Relatively reduced expression of WAVE2 protein in HTR-8/SVneo cells after exposure to H/R for 48 h (n = 3). (C) WAVE2 protein concentrations in the cell supernatant of the H/R and control groups, respectively (n = 3). Bars = 25 μm. P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

      Discussion

      This study has focused on WAVE2 protein in connection with a pathological condition of pregnancy, pre-eclampsia and confirmed the presence of WAVE2 protein in the placenta. WAVE2 is mainly found in the cytoplasm of trophoblasts and vascular endothelial cells. Moreover, the WAVE2 mRNA and protein expression in the pre-eclamptic placenta as well as maternal blood serum WAVE2 protein concentration of pre-eclamptic patients were significantly lower than in the controls. Previous studies have shown that WAVE2 expression was very strong, especially in placenta during pregnancy (
      • Sossey-Alaoui K.
      • Head K.
      • Nowak N.
      • Cowell J.K.
      Genomic organization and expression profile of the human and mouse WAVE gene family.
      ,
      • Suetsugu S.
      • Miki H.
      • Takenawa T.
      Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex.
      ). A previous study also demonstrated that WAVE2 was expressed in vascular endothelial cells during embryogenesis, and the decreased WAVE2 expression impaired angiogenesis in vivo (
      • Yamazaki D.
      • Suetsugu S.
      • Miki H.
      • Kataoka Y.
      • Nishikawa S.
      • Fujiwara T.
      • Yoshida N.
      • Takenawa T.
      WAVE2 is required for directed cell migration and cardiovascular development.
      ). Given the importance of WAVE2 in angiogenesis and embryogenesis during pregnancy, the findings of the present study suggest that the decreased expression of WAVE2 in the pre-eclamptic placenta may be involved in the development of pre-eclampsia.
      Oxidative stress, resulting from over-production of ROS, has been proposed as an important intermediary step in the pathogenesis of pre-eclampsia. A recent report revealed that WAVE2 expression is affected by ROS concentrations (
      • Park S.J.
      • Kim Y.T.
      • Jeon Y.J.
      Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott–Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells.
      ). Therefore, the present study detected ROS concentrations and WAVE2 expression in normotensive and pre-eclamptic placentas and simultaneously examined the correlation between ROS concentrations and WAVE2 expression. In accordance with previous studies (
      • Al-Gubory K.H.
      • Fowler P.A.
      • Garrel C.
      The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes.
      ,
      • Zhou X.
      • Zhang G.Y.
      • Wang J.
      • Lu S.L.
      • Cao J.
      • Sun L.Z.
      A novel bridge between oxidative stress and immunity: the interaction between hydrogen peroxide and human leukocyte antigen G in placental trophoblasts during preeclampsia.
      ), the data revealed that ROS concentrations were higher in pre-eclamptic placentas than in normotensive placentas. This result further confirmed the direct effect of oxidative stress on placental function in pre-eclampsia. In addition, there is a significant negative correlation between ROS concentrations and WAVE2 protein expression in pre-eclamptic placentas. This negative correlation suggests that an increase in ROS coincides with a decline in the expression of WAVE2 protein. However, this negative correlation could not be found in normotensive placentas. Therefore, the decreased expression of WAVE2 was closely associated with excessive oxidative stress in pre-eclamptic placenta.
      Reduced invasion of trophoblasts and incomplete conversion of spiral arteries are key pathological features of pre-eclampsia (
      • Verlohren S.
      • Geusens N.
      • Morton J.
      • Verhaegen I.
      • Hering L.
      • Herse F.
      • Dudenhausen J.W.
      • Muller D.N.
      • Luft F.C.
      • Cartwright J.E.
      • Davidge S.T.
      • Pijnenborg R.
      • Dechend R.
      Inhibition of trophoblast-induced spiral artery remodeling reduces placental perfusion in rat pregnancy.
      ). Many studies have indicated that excessive oxidative stress will lead to higher cell apoptosis, increased cell proliferation inhibition and decreased cell invasion in trophoblasts (
      • Hung T.H.
      • Skepper J.N.
      • Charnock-Jones D.S.
      • Burton G.J.
      Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia.
      ,
      • Li H.
      • Gu B.
      • Zhang Y.
      • Lewis D.F.
      • Wang Y.
      Hypoxia-induced increase in soluble Flt-1 production correlates with enhanced oxidative stress in trophoblast cells from the human placenta.
      ,
      • Seeho S.K.
      • Park J.H.
      • Rowe J.
      • Morris J.M.
      • Gallery E.D.
      Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration.
      ). Since WAVE2 works as an intermediate that links ROS signalling to actin cytoskeletal reorganization and is essential for cell invasion and migration (
      • Park S.J.
      • Kim Y.T.
      • Jeon Y.J.
      Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott–Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells.
      ), oxidative stress might attenuate trophoblast migration and invasion in pre-eclampsia through regulating the expression of WAVE2 in trophoblasts.
      Experiments on placental ischaemia/reperfusion intervention have provided evidence that H/R would stimulate trophoblastic cells to generate ROS sharply and was a possible mechanism for placental oxidative stress in pre-eclampsia (
      • Burton G.J.
      • Yung H.W.
      Endoplasmic reticulum stress in the pathogenesis of early-onset pre-eclampsia.
      ,
      • Hung T.H.
      • Burton G.J.
      Hypoxia and reoxygenation: a possible mechanism for placental oxidative stress in preeclampsia.
      ). Therefore, the present study used the human first-trimester extravillous trophoblast cell line HTR8/SVneo and exposed these cells to H/R to imitate an ischaemia/reperfusion injury of the placental trophoblast and to assess whether oxidative stress affects WAVE2 expression in extravillous trophoblast cells. The relationship between WAVE2 concentrations and trophoblast invasion activity was also evaluated.
      The results demonstrated that HTR8/SVneo cells treated with H/R displayed a dramatic increase in ROS concentrations compared with the non-treated group. Moreover, increased inhibition of proliferation, higher apoptosis, invasion and migration were found in HTR-8/SVneo cells when exposed to H/R. The previous study (
      • Hung T.H.
      • Skepper J.N.
      • Burton G.J.
      In vitro ischemia-reperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies.
      ) demonstrated that ROS generated from H/R may initially act as intracellular signalling molecules regulating various cellular functions, including proliferation and apoptosis of extravillous trophoblast cells. If the condition of H/R repeats and persists, the generation of ROS exceeds the ability of antioxidant defences, and cellular dysfunction, growth arrest or cell death will result. Increased apoptosis and/or decreased proliferation could reduce the number of extravillous trophoblasts, leading to impaired adaptation of uteroplacental arteries during pregnancy (
      • Huppertz B.
      • Gauster M.
      • Orendi K.
      • König J.
      • Moser G.
      Oxygen as modulator of trophoblast invasion.
      ).
      ROS have also been shown to act as signalling molecules to regulate gene expression (
      • Bedard K.
      • Krause K.H.
      The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.
      ). The present results indicated that WAVE2 protein was located in the cytoplasm of HTR-8/SVneo cells and that cells exposed to H/R displayed weaker staining compared with the control group. Moreover, the expression of WAVE2 protein in HTR-8/SVneo cells and the concentration of WAVE2 in cell supernatant were decreased after H/R treatment. These findings revealed that the high ROS concentrations could regulate WAVE2 expression in extravillous trophoblast cells. In other words, the expression of WAVE2 in extravillous trophoblast cells is vulnerable to oxidative stress. The decreased expression of WAVE2 in extravillous trophoblast cells might change intracellular rearrangement of the actin cytoskeleton and impair migration and invasion of extravillous trophoblast cells and, thus, may be involved in the development of pre-eclampsia. Oxidative stress inhibits WAVE2 synthesis in the trophoblasts through an unclear mechanism. However, studies have demonstrated that H/R-induced oxidative stress will cause increased release of endothelin 1 by the placenta, which leads to endoplasmic reticulum stress in trophoblasts. Endoplasmic reticulum stress contributes to the pathogenesis of pregnancy complications, such as pre-eclampsia. One consequence of endoplasmic reticulum stress is the inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor subunit α (
      • Jain A.
      • Olovsson M.
      • Burton G.J.
      • Yung H.W.
      Endothelin-1 induces endoplasmic reticulum stress by activating the PLC-IP3 pathway.
      ). Therefore, reduced production of WAVE2 under oxidative stress is possibly through inhibition of protein synthesis due to endoplasmic reticulum stress. This needs to be confirmed by future study.
      In summary, this study has demonstrated that decreased expression of WAVE2 is associated with oxidative stress in pre-eclamptic placentas and, thus, may be involved in the pathogenesis of pre-eclampsia. Further studies are needed to confirm the direct role of ROS in WAVE2 expression and trophoblast invasion, the direct effect of WAVE2 on trophoblast invasion and the exact signalling pathway of oxidative stress in regulation of WAVE2 expression.

      Acknowledgements

      This work was supported by Grant No. 81070502 from the National Natural Science Foundation of China and the Key Laboratory for Major Obstetric Diseases of Guangdong Province. The authors thank the women who donated tissue for this study and Dr Charles Graham from Queen’s University, Kingston, Ontario, Canada for providing the HTR-8/SVneo cell line. They also thank the English language professional service of ELSEVIER WebShop for reading and correcting this manuscript.

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