Elsevier

Pregnancy Hypertension

Volume 13, July 2018, Pages 225-234
Pregnancy Hypertension

Impaired Wnt5a signaling in extravillous trophoblasts: Relevance to poor placentation in early gestation and subsequent preeclampsia

https://doi.org/10.1016/j.preghy.2018.06.022Get rights and content

Highlights

  • Wnt5a expression is decreased in the first trimester placentas which later cause PE.

  • Wnt5a expression is decreased in PE placentas compared with normal term placentas.

  • Impaired Wnt5a signaling impairs the invasive and tube forming capabilities of EVT.

  • Impaired Wnt5a signaling is associated with poor placentation and subsequent PE.

Abstract

Background

Defective decidual endovascular trophoblast invasion and subsequent impaired spiral artery remodeling is highly associated with the pathogenesis of preeclampsia (PE). Since there are scant and conflicting data regarding the function of Wnt5a signaling in extravillous trophoblasts (EVT), the aim of this study was to investigate whethere impaired Wnt5a signaling affects the invasive and tube forming capabilities of EVT.

Methods

Expression levels of Wnt ligands were compared between first trimester chorionic villi of women who later developed PE and women with unaffected pregnancies using publicly available microarray data (GSE12767). Wnt5a expression was examined in placentas using quantitative RT-PCR, Western blot analysis and immunohistochemistry. The function of Wnt5a signaling in EVT was investigated in an immortalized first trimester EVT cell line, HTR-8/SVneo, using small-interfering RNAs, recombinant human Wnt5a (rhWnt5a), and inhibitors of JNK or PKC.

Results

Microarray data analysis of the first trimester placentas showed that, among Wnt ligands, Wnt5a expression was significantly lower in women who later developed PE. The mRNA and protein expression levels of Wnt5a were significantly decreased in PE placentas compared with normal term placentas. Wnt5a knockdown significantly suppressed invasion and tube formation of HTR-8/SVneo cells, while the addition of rhWnt5a augmented the cell migration, invasion, and tube formation. Repression of Wnt5a/PKC signaling in HTR-8/SVneo cells inhibited cell invasion, but did not alter cell tube formation. In contrast, inhibition of Wnt5a/JNK signaling attenuated rhWnt5a-induced invasion and tube formation capabilities.

Conclusions

These findings suggest that impaired Wnt5a signaling is associated with poor placentation and subsequent PE.

Introduction

Preeclampsia (PE) is estimated to complicate 2–8% of all pregnancies, and is a leading cause of maternal and neonatal morbidity and mortality [1]. It has been postulated that PE occurs in two stages, i.e. that poor placentation typified by insufficient remodeling of spiral arteries (first stage) results in the release of excessive amounts of placental materials that lead to an excessive maternal inflammatory response and endothelial dysfunction (second stage) [2]. Placentation is a highly complex, regulated process that is crucial for normal fetal growth and the maintenance of a healthy pregnancy. During normal placentation, extravillous trophoblasts (EVT) invade into the decidua and the inner one third of the myometrium [3], [4]. A subset of EVT, i.e. endovascular trophoblasts, invade maternal vessels, disrupt the endothelium and the smooth muscle layer, and replace the vascular wall [3], [4]. These conversions allow spiral arteries to become widely dilated independently of vasomotor control, thereby providing a sufficient blood supply in intervillous space to meet the requirements of the fetus. On the other hand, defective decidual endovascular trophoblast invasion and subsequent impaired spiral artery remodeling are thought to result in less dilated vessels and a lack of adequate placental perfusion, leading to the development of PE [3], [4]. Although early placentation has long been the focus of intense research efforts, the mechanism of impaired spiral artery remodeling has not been fully elucidated.

Accumulating evidence suggests that Wingless (Wnt) signaling plays an important role in placental development and human trophoblast differentiation [5], [6]. Wnt proteins comprise a family of 19 secreted glycoproteins that act as ligands via 12 putative cell-surface receptors and co-receptors [7]. Wnt signaling is a complex pathway that modulates a number of signal transduction pathways and regulates diverse biological functions in a highly cell- and tissue-dependent manner [7], [8], [9]. Wnt signaling is broadly divided into two main categories, i.e. beta-catenin-dependent and beta-catenin-independent signaling. The former signaling pathway regulates target gene expression via beta-catenin stabilization and its translocation to the nucleus, and is closely associated with cell proliferation, cell fate determination, and differentiation [7], [9]. On the other hand, the latter includes activation of c-Jun NH2-terminal kinase (JNK) and protein kinase C (PKC), which regulate cell functions such as cell movement and polarity [7], [8]. Of the 19 known Wnt ligands, 14 have been reported to be expressed in the first trimester placenta [10]. However, the exact role of Wnt ligands in early placentation, especially in spiral artery remodeling, remains controversial. We hypothesized that Wnt signaling is associated with poor placentation and subsequent PE. The aim of this study was therefore to explore the role and underlying mechanisms of Wnt signaling in impaired early placentation and PE.

Section snippets

Microarray data mining for Wnt ligands in early placentation

Publicly available gene expression data of chorionic villus sampling were obtained from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/gds/) as series matrix files. There was only one data set (GSE12767) that was composed of the first trimester chorionic villi of women who later developed PE (n = 4) and women with unaffected pregnancies (n = 8). Expression values of Wnt ligands were log2 transformed, and probe sets whose maximum expression value across samples was less than 5

Analysis of microarray data reveals Wnt5a as a plausible candidate gene relevant to poor placentation

In order to identify Wnt ligands potentially relevant to early placentation in PE, a total of 25 probes corresponding to Wnt ligand genes, whose maximum expression value across samples was more than 5, were detected in a microarray data set of chorionic villous sampling (GSE12767) that was composed of first trimester chorionic villi of women who later developed PE (n = 4) and women with normal pregnancies (n = 8). Among 25 probes, two probes, both of which represent Wnt5a, showed significantly

Discussion

In this study, we first showed that Wnt5a expression is suppressed in the early placentas of women before developing PE. Moreover, down-regulated Wnt5a was also observed in placentas of PE compared with normal first and term placentas. Furthermore, Wnt5a via PKC and JNK signaling was involved in EVT cell invasion and tube formation. All these findings suggest that dysregulated Wnt5a signaling can be partly attributed to impaired early placentation and the subsequent development of PE.

The

Conclusion

Impaired Wnt5a signaling may be relevant to poor placentation and subsequent PE, and inhibition of Wnt5a/JNK signaling attenuated rhWnt5a-induced invasion and tube formation capabilities. Therefore, modulation of Wnt5a signaling can be a promising therapeutic strategy for the prevention of PE.

Author’s roles

Kondoh E, Matsumura N, and Konishi I designed this study and supervised the project. Ujita M, Chigusa Y, Mogami H, Kawasaki K, Kiyokawa H, Takai H, Sato M, and Kawamura Y performed the in vitro experiments. Ujita M and Kondoh E analyzed data and wrote the paper. Chigusa Y, Mogami H, Horie A, and Baba T edited the manuscript. Mandai M finally approved the version to be published. All authors discussed the results and implications and commented on the manuscript at all stages.

Study funding

Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology, Japan (No. 26670720).

Acknowledgments

We thank Ms. Teruko Yano for her outstanding secretarial assistance.

Conflict of interest

The authors have no conflicts of interest to declare.

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