Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling
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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2021, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :Studies in vitro show that LOPE also associates with decreased A2AAR protein abundance, increased human cationic amino acid transporter 1 (hCAT-1)–mediated L-arginine transport, and lower endothelial nitric oxide synthase (eNOS) activity compared with healthy pregnancies [9,10]. A recent study showed that activation of A2BAR increased the expression of the hypoxia-inducible factor-1α, a transcription factor induced in preeclampsia [18], in human placental villous explants [7], and reduced the migration of human trophoblast cell lines [19]. Thus, it is likely that this receptor subtype is involved in the pathophysiology of preeclampsia.
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2018, Environment InternationalCitation Excerpt :Among these, several DMR were located in genes pointing towards preeclampsia, hypertensive and metabolic disorders. ADORA2B, encoding the Adenosine A2B receptor, was shown to play a role in placental development and possibly in the pathophysiology of hypoxia and preeclampsia in pregnant women (Acurio et al., 2014; Darashchonak et al., 2014; Jia et al., 2012), a pathology which has also been associated with air pollution exposure during pregnancy (Pedersen et al., 2014). In both mice and humans, it has been demonstrated that activation of ADORA2B signaling contributes to the pathogenesis of preeclampsia (Huang et al., 2017), to small fetuses, small placentas and fetal growth restriction (Iriyama et al., 2015).
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2018, Biochemical PharmacologyCitation Excerpt :In rat skeletal muscle, A2BAR activation was found not to modulate ERK1/2, although it stimulated cAMP accumulation [18]. In trophoblast cells, it was found that A2BAR downregulates ERK1/2 activity [19]. ERK1/2 activity stimulated by adenosine-5′-N-ethyluronamide (NECA) in A2BAR-expressing CHO is via PI3K, but not PKA [14].
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2017, Molecular Aspects of MedicineCitation Excerpt :Since some studies report changes in mRNA level by either not quantitative, semi-quantitative, or quantitative experimental approaches, the results regarding ARs expression level in preeclampsia may also be misinterpreted and should be taken with caution. Despite the fact that A2BAR activation in LOPE was suggested as a potential compensatory mechanism for this disease-associated foetoplacental endothelial dysfunction (Escudero et al., 2008), the effect of activating A2BAR on the capacity of trophoblast to migrate, proliferate, and invade in vitro is contradictory (Darashchonak et al., 2014a,b). Studies in HUVECs from preeclampsia (apparently from LOPE pregnancies) show impaired NO/VEGF signalling pathway in response to a general activator of A2BAR, thus hindering the capacity of these cells to proliferate and migrate (Acurio et al., 2014).