The features of the E-cadherin/β-catenin signaling pathway in the placenta and peripheral blood of pregnant women with fetal growth restriction

According to the modern concept, fetal growth restriction (FGR) is considered as polyetiologic pathological condition, when the fetus is unable  to reach its genetically programmed growth potential and has a high risk of perinatal complications [1]. According to data from varisous sources, FGR complicates up to 10–15% of all pregnancies every year. Despite the heterogeneity of this complication, it is believed that it is of placental origin,  that is the most common cause of its development [2]. Placental dysfunction is defined as the pathophysiological basis, and manifests itself in placental dysfunction with preceding cascade of pathological processes at several levels of regulation [3, 4].

It is known that the physiology of placentation is based on the balanced differentiation of different types of cells. The canonical Wnt signaling pathway, which regulates embryogenesis, cell differentiation, etc. plays the central role in normal placental development [5–7]. Activation of Wnt signaling leads to a complex cascade of intracellular reactions, resulting in stabilization and migration of β-catenin into the cell nucleus. β-catenin molecule acts as a cofactor of the signaling pathway and regulates the processes of cell proliferation, differentiation, and apoptosis. It is believed that activation of the canonical Wnt signaling occurs with formation of a specific binding interaction between the WNT ligand and the Frizzled receptor (FZD) [8, 9]. At least ten WNT ligands were identified in the placenta. Among them, WNT2 showed the highest levels of expression  [10]. The canonical Wnt signaling pathway is a mechanism associated with stabilization of the key intracellular element of this signaling pathway – the cytoplasmic protein β-catenin (Wnt/β-catenin signaling pathway). The transmission of Wnt signals is activated by ligands. Also, one of the regulators of the Wnt signaling pathway is the cytoplasmic protein β-catenin. After binding of the Wnt ligand to the transmembrane receptor Frizzled and co-receptors –  lipoprotein receptor-related proteins 5 (LRP5) and 6 (LRP6),  the cytoplasmic protein DVL activates  and promotes degradation  of  the APC/Axin/CK1a (casein kinase 1a)/GSK3β (glycogen synthase kinase 3 β) complex leading to stabilization and accumulation of  the active form of β-catenin unphosphorylated in the cytoplasm.  The accumulated β-catenin migrates to the nucleus. It binds to the TCF/LEF family – a group of transcription factors (T-cell factor/lymphoid enhancer-binding factor), which subsequently activate the transcription of target genes. In the absence of Wnt ligands, when the canonical  Wnt pathway is not activated, the APC/Axin/CK1a/GSK3β complex binds to β-catenin and causes phosphorylation, that is inactivation of β-catenin and its proteasomal degradation.

The cell adhesion molecule E-cadherin plays an important role in regulation of the nuclear translocation of β-catenin. It binds to β-catenin in the perimembrane region and forms a strong complex that blocks the intracellular migration of this protein. These data indicate that E-cadherin can act as a regulator of β-catenin-dependent gene transcription  [11, 12]. Our previous studies showed that E-cadherin expression level is lower in FGR placental villi, that can be critical in the expression of β-catenin-dependent genes, such as  MMP-9, CCND1, and BIRC5 [13].

In addition, the previous studies  suggested that decreased E-cadherin levels can be directly associated with increased cellular pro-apoptotic activity [14], that can be due to smaller placental size in FGR.

The objective of the study was to identify the features of the E-cadherin/β-catenin signaling pathway in the placenta and blood of pregnant women by determination of expression of MMP-9, CCND1 and BIRC5 genes induced by nuclear translocation of β-catenin, as well as expression of WNT2 gene and to explore the apoptosis of the placental cells.

Materials and methods

The study was carried out at the Academician V.I. Kulakov National MedicalResearch Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia. The study included 82 women, who were divided into 2 groups. The main group consisted of 46 patients with the postnatal diagnosis of fetal growth restriction without  hypertension complications. The control group consisted of 36 patients with normal  pregnancy.

The diagnosis of fetal growth restriction was made in accordance with the clinical recommendations approved by the Minstry of Health of Russia “Fetal growth restriction, requiring maternal healthcare” (slowing of fetal growth velocity;  the estimated fetal weight (EFW) and/or abdominal circumference (AC) <10th percentile in combination with abnormal blood flow according to Doppler ultrasound,  or EFW and/or AC <3rd percentile [1].

 Inclusion criteria in the study were the following: the age of pregnant women 22–45 years;  35–40 weeks of pregnancy; singleton pregnancy complicated with FGR  (the main group); normal pregnancy (the control group);  obtaining of informed consent to participate in the study.

Exclusion criteria were ages below 22 years and over 45 years; multiple pregnancy; pregnancy achieved  by using assisted reproductive technologies; decompensated extragenital pathology; acute infectious, oncological diseases, and mental disorders; hypertensive complications during pregnancy; antiphospholipid syndrome; fetal malformations.

All women enrolled in  the study underwent standard examination, including  collection and evaluation of somatic, obstetric and gynecological anamnesis, laboratory and instrumental tests.

Investigation of the features of the E-cadherin/β-catenin signaling pathway in pregnant women was carried out by determining the expression of MMP-9, CCND1 and BIRC5 genes caused by the nuclear β-catenin translocation,  expression of the WNT2 gene in the placental tissue and the peripheral blood, as well as by studying the features of the apoptosis of placental cells.

Detection of gene expression in the peripheral blood and the placental tissue

Venous blood samples were collected from the cubital vein in the morning, on an empty stomach, in the Vacuette blood collection tubes with an anticoagulant. The samples were processed within 30 minutes after material collection. Then 200 µl of ExtractRNA lysis buffer (“Evrogen”, Russia) was added to 200 µl of whole blood. The samples were subsequently stored at -80°C.

Placental tissue samples were collected immediately following childbirth. Small pieces of villous placental tissue (0.5×0.5 cm) were collected in the area about 3 cm from the umbilical cord.  The fragments of placental tissue were placed in cryo-tube containing RNAlater solution and then stored at -80°C.

Total RNA was extracted from the samples of peripheral blood and placental tissue using phenol extraction. The degree of purification and concentration of the isolated RNA were determined using the DeNovix fluorimeter (“TermoFisher”, USA). Each isolated RNA sample was converted to cDNA using the OT-1 reverse transcription kit (“Syntol”, Russia).

Amplification of RNA for gene expression of MMP-9, CCND1, BIRC5, WNT2 genes and  housekeeping gene HPRT1 by real-time polymerase chain reaction (RT-PCR) with specific primers and probes for the analyzed gene regions was performed using the CFX-96 device (“Bio-Rad”, USA). Primer and probe sequences are represented in Table 1.

RT-PCR  protocol was the following:  95°С – 5 minutes; 40 cycles: 95°С – 10 sec, 60°С – 25 sec.

Analysis of the features of the apoptosis of placental cells

Placental tissue was fixed for 24 h in 4% paraformaldehyde solution in  phosphate-buffered saline (PBS) at 4°C. Then without washing,  they were transferred into 30% sucrose for another 24 hours at 4°C. Subsequently, the tissue was frozen in the cryo embedding medium using Tissue-Tek OCT Compound. (“Sakura Finetek”, USA). Sections (12 µm thick) were obtained in the Microm HM525 cryostat (“Thermo Scientific”, Great Britain). The TUNEL reaction was performed using In Situ Cell Death Detection Kit, Fluorescein (“Roche”, Germany) according to the manufacturer’s protocols. The sections were examined using a Leica SP5 confocal microscope (Germany). Each placental sample was examined  using 5 fields of view. The number of apoptotic cells was represented as a percentage of the total number.

Conjugated E-cadherin antibodies were used to differentiate trophoblast cells from stromal cells (“R&D systems”, USA).

Apoptosis assessment was performed in all E-cadherin-positive trophoblast cells.

Statistical analysis

Statistical data processing was perfomed using Microsoft Excel, OriginPro 8 и SPSS Statistics 17. The data are represented as median (Мe) and upper and lower quartiles (Q25%; Q75%). The quantitative data were compared using the Mann–Whitney U test. Fisher’s exat test was used for comparison of categorical variables between the groups. The threshold was considered statistically significant at p<0.05.

Results

The clinical characteristics of patiens is represented in Table 2.

There were no differences between the groups of patients in BMI  and age. However, impaired uteroplacental blood flow was significantly more often in the group of patients with FGR. Gestational age was significantly less in the main group versus the control group. In the main group, pretern birth was in 11/46 women (23.9%), and term birth was in the others.

Analysis of gene expression in the placental tissue and peripheral bollod found that  MMP-9 and BIRC5 expression levels in placental tissue were significantly higher in patients in the main group compared with women in the control group (р=0.03 and р=0.02, respectively). There were no significant differences between the groups in the expression of the CCND1, WNT2 genes (Fig. 1). The comparison of patients with preterm and term birth in the main group found no significant differences in the expression levels of the MMP-9 and  BIRC5 genes in the placental tissue; the values for MMP-9 were 2.4 (1.1; 6.9) and  2 (1.4; 7), respectively, р=0.62; and for BIRC5 – 1.6 (1.5;3.2) and 1.5 (1.2; 4.7), respectively, р=0.7.

The comparison of gene expression in the maternal blood flow showed low expression levels of MMP-9 in the main group versus the control group (р=0.001). There were no significant differences between the groups in the expression levels of  CCND1 and BIRC5. It should be noted that there was no expression of the  WNT2 gene in patients in both groups (Fig.2).

Assessment of the apoptosis in the placental cells showed higher apoptotic activity in patients in the main group compared with the control group; the values were 0.16 (0.08; 0.23)% and 0.09 (0.04; 0.16)%, respectively (р=0.03). At the same time, in the presence of FGR, trophoblast apoptosis increased in the placentas versus stromal cells. The comparative analysis of the apoptotic activity of trophoblast cells showed that in FGR  apoptosis was more pronounced compared with normal pregnancy –  0.15 (0.04; 0.24)% and 0.07 (0,04; 0,15)%, respectively (р=0.017) (Fig. 3).

Discussion

In recent decades, the scientists have focused on the effects of the Wnt signaling pathway, which plays a key role in cell proliferation, differentiation and apoptosis during the embryonic development. [5–7].

Previously conducted foreign studies indicate the activation of Wnt signaling in FGR. The study by Sola I.M. et al. [15] showed higher expression of WNT5A protein and β-catenin in the placental tissue of patients with FGR compared with   women with normal pregnancy. These results are consistent with the previously published results [16], demonstrating higher Dishevelled family proteins (DVL1-3) expression in IUGR-related placentas. Being Wnt signaling members, these proteins promote accumulation of β-catenin in the cytoplasm [17].

The data obtained in our study does not exclude the results of foreigh researchers. Expression level of the MMP-9 и BIRC5 genes was significantly higher in the placental tissue of patients in the main group, that indicates an increase in the translocation of a portion of β-catenin into the nucleus with subsequent triggering of activation cascade of transcription factors and genes.  At the same time the WNT2 gene, the protein product of which is a key player in the canoninc signaling pathway in the placenta [10], did not demonstrate significant differences between the groups in the expression level. The absence of significant differences in expression  of  WNT2 suggests that the Wnt signaling pathway is not involved in the development of  FGR. However, increased expression of β-catenin-dependent genes (MMP-9 and BIRC5) , can be explained  by reduced expression of E-cadherin in FGR, as we have previously established [13], and as a consequence, reduced expression of  E-cadherin/β-catenin complexes and the appearance of free β-catenin.

Gene expression in the maternal blood flow was assessed to identify noninvasive diagnostic markers. Comparative analysis found no association between gene expression in the placental tissue and systemic circulation. At the same time, significantly low expression level of MMP-9 was identified  in women in the main group. According to literature data, MMP-9 I in leukocytes participates in various inflammatory reactions, such as permeability of physical barriers, leukocyte migration cytokine secretion [18]. Therefore,  reduced MMP-9 expression in women with FGR may indicate dysregulation of the immune response.

It is known that with the development of placental complications during pregnancy, there is elevated  activation of villous trophoblast apoptosis compared with normal pregnancy, that indicates the importance of normal regulation of these processes during trophoblast invasion and subsequent development [19]. The data represented by us is partially consistent with the data in the studies by  Smith S.C. et al. [20] and Ishihara N. et al. [21], which also demonstrated increased placental cell apoptosis in the development of FGR. However, their studies showed no differences in the ratio of the apoptosis in the villous trophoblast to placental stroma.

At the same time, the mechanisms of  intensification of apoptosis in FGR remain unclear. Han H.J. et al.[14] noted that  reduced levels of E-cadherin can be directly associated with increased cellular pro-apoptotic activity. Taking into account previously identified decreased levels of transmembrane and soluble E-cadherin in patients with FGR, there are reasons to believe that this mechanism underlies the increased number of trophoblastic cells with the signs of apoptosis in women with this pregnancy pathology. Our study expanded the knowledge of the pathophysiological mechanisms of placental complications during pregnancy. In particular, it was found that E-cadherin is a regulator of the WNT signaling pathway. However, regulatory mechanisms cadherin expression are not fully understood, that indicates the prospects for further research in this area.

Conclusion

Thus, the identified placental  MMP-9 and BIRC5 overexpression caused by the nuclear β-catenin translocation, as well as development of apoptosis of placental trophoblast cells in patients with FGR can be associated with reduced expression of  E-cadherin/β-catenin complexes. Reduced MMP-9 expression in the peripheral blood of women with FGR may indicate dysregulation of the immune response. In view of the above, it seems appropriate to conduct additional in-depth studies to expand understanding of the pathophysiological mechanisms of FGR at the molecular level.