Elsevier

Placenta

Volume 35, Issue 9, September 2014, Pages 763-771
Placenta

Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling

https://doi.org/10.1016/j.placenta.2014.06.369Get rights and content

Highlights

  • Adenosine A2B receptor agonist NECA reduces trophoblast migration.

  • A2B adenosine receptor stimulation decreases phosphorylation of ERK1/2 and SAPK/JNK.

  • ProMMP-2 activity and MMP-2 mRNA decrease after A2B adenosine receptor activation.

Abstract

Introduction

Shallow trophoblast invasion of the maternal spiral arteries contributes to impaired placental perfusion and is hypothesized to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine.

Methods

We investigated the effects of hypoxia and A2B adenosine receptor signaling on migration, invasion, proteolytic activity of matrix metalloproteinase (MMP)-2, expression of MMP-2 and vascular endothelial growth factor (VEGF) mRNA, and production of human chorionic gonadotropin (hCG) in trophoblast cells (HTR-8/SVneo, BeWo).

Results

The adenosine A2B receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) reduced trophoblast (HTR-8/SVneo and BeWo) migration at 2%, 8% and 21% O2 compared to untreated control cells. A2B adenosine receptor stimulation decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) at all three O2 concentrations. ProMMP-2 activity, MMP-2 mRNA levels and hCG levels were markedly decreased after A2B adenosine receptor activation in trophoblast cells. Adenosine receptor A2B stimulation decreased VEGF expression at 2% and 8% O2 but led to increased levels at 21% O2.

Conclusions

These data indicate A2B receptor activation blunts trophoblast migration possibly as a result of reduced activation of the MAPK signaling pathway and lower proMMP-2 levels. These data suggest a role for adenosine receptor A2B in placental development and possibly in the pathophysiology of preeclampsia.

Introduction

Adenosine is a purine nucleoside and involved in numerous physiological processes [1]. Extracellular adenosine levels increase in response to hypoxia, ischemia and inflammation, preventing tissue damage during instances of cellular stress or injury [2], [3]. Adenosine receptors are transmembrane spanning G protein-coupled receptors and include A1, A2A, A2B, and A3 subtypes [4]. Adenosine and adenosine receptors are involved in angiogenesis, endothelial cell proliferation, migration and blood vessel formation in various vascular beds [5], [6]. Adenosine also promotes neovascularization [2], [5] and under hypoxic conditions human endothelial and smooth muscle cells modulate adenosine receptors toward an A2B “angiogenic” phenotype [7].

Preeclampsia is a pregnancy-specific syndrome that affects 3–5% of pregnant women worldwide [8] and a leading cause of maternal and fetal morbidity and mortality [9]. Preeclampsia is clinically characterized by new onset hypertension and proteinuria after 20 weeks of gestation [10]. The pathophysiology of the syndrome is not fully understood. However, placental hypoxia as a result of impaired trophoblast invasion is proposed to be a central component of the pathophysiology of preeclampsia [11], [12]. Hypoxia is a potent stimulus for the release of adenosine [3], [4] and patients with preeclampsia exhibit higher concentrations of adenosine in the maternal and fetal circulation compared to uncomplicated pregnancies [13], [14]. Maternal adenosine concentrations increase with the severity of the syndrome [15]. Furthermore, adenosine receptor expression in the human placenta is higher in pregnancies complicated by preeclampsia [16]. However, the role of adenosine receptor A2B in the placenta, a receptor influenced by hypoxic conditions, is unclear.

The focus of this study was to investigate the effect of adenosine receptor A2B stimulation in placental function using a model system of trophoblast cells (HTR-8/SVneo and BeWo trophoblast cell line), and specifically to investigate the role of adenosine receptor A2B in trophoblast cell migration, invasion and the cell signaling pathways. We hypothesized that hypoxia through adenosine receptor A2B activation may adversely affect trophoblast function and thereby contribute to placental dysfunction and the pathophysiology of preeclampsia.

Section snippets

Cell culture

We used the human, invasive extravillous cytotrophoblast HTR-8/SVneo cell line (HTR-8/SVneo, gift by Prof. Charles Graham, Queen's University, Kingston, ON, Canada), established from immortalized explant cultures of first trimester chorionic villi and the immortalized trophoblast cell line BeWo, which has the capability to fuse and is a well-established model for the syncytiotrophoblast (Cell Lines Service, Eppelheim, Germany). HTR-8/SVneo were cultured in RPMI 1640 media (Invitrogen, Germany)

Treatments

Trophoblast cells were incubated in the presence or absence of adenosine A2B receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA, 10 μM) or antagonist 8-[4-[((4-cyanophenyl) carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine hydrate (MRS 1754, 1 μM), both purchased from Sigma–Aldrich (Steinheim, Germany). The A2B receptor is the least well-defined adenosine receptor subtype. NECA is the most potent nonselective adenosine A2B receptor agonist, with a concentration producing a half-maximal (EC

Adenosine A2B receptor activation inhibits migration of trophoblast cells

To elucidate the ability of trophoblast cells to migrate in the scratch wound under the conditions of stimulation or inhibition of adenosine receptor A2B we conducted migration assays with HTR-8/SVneo and BeWo trophoblast cells in presence or absence of adenosine A2B receptor agonist (NECA, 10 μM) and antagonist (MRS 1754, 1 μM), respectively. Stimulation of the adenosine A2B receptor with NECA significantly decreased HTR-8/SVneo trophoblast migration compared to untreated controls at 2% O2

Discussion

Early in pregnancy, the placenta develops in a state of low oxygen tension. The placental oxygen tension steeply rises between 8 and 12 weeks of gestation, reflecting the onset of maternal blood flow. Incomplete spiral artery remodeling by invasive cytotrophoblast cells has been described in pregnancies complicated by preeclampsia and fetal growth restriction [20], [21]. The mechanism(s) associated with this impaired trophoblast function is not well understood, however placental hypoxia is one

Conflict of interest

No conflicts of interest, financial or otherwise, are declared by the authors.

Author contributions

N.D., R.W.P. and F.V.-H. conception and design of research, A.S., and M.J.K. provided methodological support, N.D., A.S. and F.V.-H. analyzed data and interpreted results, N.D. and A.S. prepared figures, N.D. drafted manuscript, F.V.-H. and R.W.P. edited and revised manuscript, all authors approved the final version of the manuscript.

Acknowledgments

The studies mentioned herein have been supported by the German Research Foundation, (VE490/4-1) and the Preeclampsia Foundation. We thank Ms Brunhild Koepsell for technical support.

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