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. 2018 Dec;188(12):2774-2785.
doi: 10.1016/j.ajpath.2018.08.011. Epub 2018 Sep 29.

Human Placenta Expresses α 2-Adrenergic Receptors and May Be Implicated in Pathogenesis of Preeclampsia and Fetal Growth Restriction

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Human Placenta Expresses α 2-Adrenergic Receptors and May Be Implicated in Pathogenesis of Preeclampsia and Fetal Growth Restriction

Hanaa K B Motawea et al. Am J Pathol. .
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Abstract

α2-Adrenergic receptors (α2ARs) are G-protein-coupled receptors involved in catecholamine signaling by extracellular regulated protein kinase 1 and 2 (ERK1/2) pathways. We examined placental expression and function of α2AR subtypes in women with severe preeclampsia (sPE) with and without intrauterine growth restriction (IUGR). Placental biopsies were analyzed from 52 women with i) sPE (n = 8); ii) sPE + IUGR (n = 9); iii) idiopathic IUGR (n = 8); iv) idiopathic preterm birth (n = 16); and v) healthy term controls (n = 11). Expression of α2AR subtypes (α2A, α2B, α2C) and phospho-ERK1/2 (receptor activation marker) was investigated by immunohistochemistry and/or quantitative real-time RT-PCR. The effects of α2CAR knockdown on syncytialization (syncytin-1 and -2) and β-human chorionic gonadotropin secretion were examined in BeWo cells stimulated with forskolin. The effects of α2AR agonist UK 14,304 and specific α2CAR antagonist were tested, using a trophoblast migration assay. All three α2ARs were expressed and functionally active in human placenta with site-specific localization. Highest α2BAR and α2CAR mRNA expression was identified in sPE + IUGR. α2CAR knockdown increased expression of syncytin-1 and -2 but decreased secretion of β-human chorionic gonadotropin. UK 14,304 impaired trophoblast migration. The observed α2AR expression pattern suggests different function for each subtype. α2CAR modulates trophoblast syncytialization and migration and may carry pathogenic role in sPE + IUGR.

Figures

Figure 1
Figure 1
Immunohistochemical staining of α2-adrenergic receptor subtype A (α2AAR), α2BAR, α2CAR, and phospho-extracellular signal-regulated protein kinase (p-ERK)1/2 in human placental tissue. A: Representative micrographs stained for α2AAR from pregnancies complicated by idiopathic preterm birth (iPTB), severe preeclampsia (sPE), sPE with intrauterine growth restriction (sPE + IUGR), and IUGR alone. The imaged zones are representative for the areas scored semiquantitatively. B: α2AAR staining was much stronger in the media of blood vessels in SV and IV than in TV. C: Staining for α2BAR predominated in villous cytotrophoblasts (CYTs) and extravillous trophoblasts (EVTs). There was an irregular pattern of stromal staining in sPE + IUGR. D: Overall no significant differences were observed in signal intensity among groups. E: Staining for α2CAR was most prominent in EVT, CYT, and syncytiotrophoblast (SCT). F: Results of histologic scoring indicated significantly higher α2CAR staining of EVT and SCT associated with sPE + IUGR. Insets display regions of interest at higher magnification. Representative p-ERK1/2 staining from a patient with sPE + IUGR indicated activation in placental vasculature and EVTs. Data are expressed as means ± SEM. n = 6 to 15 patients in each group. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, determined by three-way analysis of variance. Scale bars: 100 μm (A, C, and E); 25 μm (insets). SV, stem villi; IV, intermediate villi; TV, terminal villi; VS, villous stroma.
Figure 2
Figure 2
Relative abundance of α2-adrenergic receptor subtype A (α2AAR), α2BAR, and α2CAR mRNA in placenta. A: Relative expression of α2AAR, α2BAR, and α2CAR mRNA in placental tissue of idiopathic preterm birth (iPTB) and term placenta are displayed. Relative quantitation (RQ) of different receptor subtypes was reported relative to expression of housekeeping genes β2-microglobulin and ribosomal protein L30 by using dCt. B–D: Estimate of relative placental mRNA abundance (ddCT) for α2AAR (B), α2BAR (C), and α2CAR (D) for pregnancies complicated by iPTB, severe preeclampsia (sPE), sPE with intrauterine growth restriction (sPE + IUGR), and IUGR, and term controls. ddCt RQ values are reported relative to a reference RNA pool of all tissues. Data are expressed as means ± SEM. n = 4 to 6 samples in each group. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Figure 3
Figure 3
Effect of α2-adrenergic receptor (α2AR) agonist activation on secretory and migratory function of BeWo cells. A: Levels of β-human chorionic gonadotropin (β-hCG) secreted from BeWo cells stimulated with forskolin (FSK; 20 μmol/L) and exposed to increasing concentrations of α2AR agonist UK 14,304. B: β-hCG secreted from FSK-stimulated BeWo cells treated with UK 14,304 for 15 minutes after pretreatment with the α2CAR antagonist MKI912. C: Representative images of in vitro wound healing scratch assay assessed BeWo cell migration at 24 hours after treatment with α2AR agonist UK 14,304 alone or after pretreatment with α2CAR antagonist MK912. Control wells (CRL) were treated with corresponding volume of vehicle (water and dimethyl sulfoxide). D: Quantification of the gap after 24 hours showed that α2AR activation with UK 14,304 resulted in a significant reduction in cell migration and that pretreatment with α2CAR antagonist MK912 blocked the UK 14,304–mediated inhibition. Dashed lines mark baseline level. Data are expressed as means ± SEM. n = 3 independent experiments expressed as percentage of baseline level before agonist (A); n = 3 experiments (B); n = 3 independent experiments (D). P < 0.05 versus baseline (one-way repeated measures analysis of variance); P < 0.05 (one-way analysis of variance).
Figure 4
Figure 4
Effect of ADRA2C knockdown on expression of syncytins and secretory function of BeWo cells. A: mRNA levels of α2-adrenergic receptor subtype C (α2CAR) after lentiviral knockdown of ADRA2C (KD) or of a non-human target (CRL). B and C: Syncytin-1 (B) and syncytin-2 (C) mRNA after stimulation with forskolin (FSK). D and E: Time course of levels of secreted β-human chorionic gonadotropin (β-hCG) from stably transduced ADRA2C knockdown cells (black bars) and non-human target knockdown cells (control; gray bars) exposed to dimethyl sulfoxide (DMSO; D) as vehicle control or FSK (20 μmol/L; E) to induce syncytialization. F: Levels of β-hCG secreted from FSK-stimulated (20 μmol/L for 48 hours) ADRA2C knockdown cells exposed to increasing concentrations of α2AR agonist UK 14,304. G: Levels of β-hCG secreted from FSK (20 μmol/L) stimulated non-human target knockdown cells treated with UK 14,304. Dashed lines mark baseline level. Data are expressed as means ± SEM. n = 3 stably transduced ADRA2C knockdown cells (D and E); n = 3 non-human target knockdown cells (D and E); n = 3 experiments (F); n = 3 independent experiments (G). P < 0.05 (t-test); P < 0.05 (two-way repeated measures analysis of variance); P < 0.05 versus baseline (one-way repeated measures analysis of variance).
Supplemental Figure S1
Supplemental Figure S1
Immunolocalization of α2-adrenergic receptor (α2AR) subtypes in placenta of women with idiopathic preterm birth (iPTB) and uncomplicated deliveries at term. A–F: Representative immunostaining patterns for α2AAR, α2BAR, and α2CAR of placenta from women with iPTB (A–C) or uncomplicated term pregnancy (Term; D–F). In iPTB α2AAR was primarily localized in the wall of the blood vessels of stem villi (sv) and intermediate villi (iv). Terminal villi (tv) were almost free of staining. α2BARs were primarily localized to villous cytotrophoblast (cyt). Villous stroma (vs) and extravillous trophoblast (evt) showed only weak staining. α2CAR immunostaining was most intense in evt, cyt, and syncytiotrophoblast (sct). At term, staining was reduced for all α2AR. G–I: Results of the semiquantitative analysis of staining intensity in iPTB and term placentas for the regions highlighted above. Data are expressed as means ± SEM. n = 5 to 8 patients in each group. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (two-way repeated measure analysis of variance). Scale bars = 100 μm.
Supplemental Figure S2
Supplemental Figure S2
Identification of α2-adrenergic receptor subtype C (α2CAR) by Western blot analysis and immunofluorescence. A: Immunoblot of α2CAR expression in BeWo cells treated with dimethyl sulfoxide (DMSO) or forskolin (FSK; 20 μmol/L). B: Densitometric quantification of the α2CAR band of BeWo cells treated with DMSO or FSK. Data analysis indicated a significant increase in response to FSK stimulation compared with DMSO, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C and D: Indirect immunofluorescence of BeWo cells stained with anti-α2CAR (red), anti–E-cadherin (green), and DAPI (blue) treated with DMSO or 20 μmol/L FSK for 72 hours. Data are expressed as means ± SEM. n = 4 FSK stimulation (B); n = 5 DMSO stimulation (B). P < 0.05 (t-test).

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