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• W2894096609 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. 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. The α-2 adrenoceptors (α2ARs), encoded by ADRA2 genes, are G-protein–coupled receptors with vital roles in cellular physiological processes and adaptation to stress.1Saunders C. Limbird L.E. Localization and trafficking of alpha2-adrenergic receptor subtypes in cells and tissues.Pharmacol Ther. 1999; 84: 193-205Crossref PubMed Scopus (109) Google Scholar Generally speaking, engagement of α2ARs by catecholamines (preferentially by norepinephrine) results in negative feedback inhibition of adenylate cyclase by G-protein subunit α, group i, resulting in reduced cAMP synthesis.2Brede M. Philipp M. Knaus A. Muthig V. Hein L. α2-adrenergic receptor subtypes – novel functions uncovered in gene-targeted mouse models.Biol Cell. 2004; 96: 343-348Crossref PubMed Scopus (81) Google Scholar, 3Tan C.M. Limbird L.E. The α2-adrenergic receptors: lessons from knockouts.in: Perez D.M. The Adrenergic Receptors: In the 21st Century. Humana Press Inc., Totowa, NJ2006: 241-265Crossref Google Scholar, 4Gilbach R. Hein L. Are the pharmacology and physiology of α2-adrenoceptors determined by α2-heteroreceptors and autoreceptors respectively?.Br J Pharmacol. 2012; 165: 90-102Crossref PubMed Scopus (60) Google Scholar α2AR agonists have been widely used for treatment of various medical conditions, including hypertension, attention-deficit/hyperactivity disorder, opioid withdrawal, and, more recently as, anesthesia adjuvants.5Crassous P.A. Denis C. Paris H. Sénard J.M. Interest of alpha2-adrenergic agonists and antagonists in clinical practice: background, facts and perspectives.Curr Top Med Chem. 2007; 7: 187-194Crossref PubMed Scopus (61) Google Scholar These uses result from the preponderant expression of α2ARs in vasculature and in the central nervous system. However, a number of studies have reported presence of α2AR on other cell types with important sympathetic reactive responses such as adipocytes, pancreatic β-cells, and even cancer epithelial cells where it was found that α2AR stimulation increases metastatic burden in the context of surgical stress.6Lavon H. Matzner P. Benbenishty A. Sorski L. Rossene E. Haldar R. Elbaz E. Cata J.P. Gottumukkala V. Ben-Eliyahu S. Dexmedetomidine promotes metastasis in rodent models of breast, lung, and colon cancers.Br J Anaesth. 2018; 120: 188-196Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar The relevance of the α2ARs in reproductive biology was demonstrated through the use of genetically engineered murine models in which the three known receptor subtypes (α2A, α2B, and α2C) were knocked out.7Philippe M. Brede M. Hadamek K. Gessler M. Lohse M.J. Hein L. Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo.Nat Genet. 2002; 31: 311-315Crossref PubMed Scopus (60) Google Scholar In mice in which all three α2ARs were deleted, lethality at embryonic day 10.5 was significantly increased because of defective vascular development.7Philippe M. Brede M. Hadamek K. Gessler M. Lohse M.J. Hein L. Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo.Nat Genet. 2002; 31: 311-315Crossref PubMed Scopus (60) Google Scholar Furthermore, it was shown that phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the placenta was lower than in wild-type animals. Of interest, several lines of evidence pointed toward placental α2BAR and not α2A or α2C AR subtypes as critical for embryonic survivability, at least in the mouse.7Philippe M. Brede M. Hadamek K. Gessler M. Lohse M.J. Hein L. Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo.Nat Genet. 2002; 31: 311-315Crossref PubMed Scopus (60) Google Scholar One proposed explanation was that co-expression of α2BAR and type 1 receptor for vascular endothelial growth factor (VEGF) by spongiotrophoblasts and trophoblast giant cells prevents the generation of soluble type 1 receptor for VEGF, the antagonist of VEGF.8Muthig V. Gilsbach R. Haubold M. Philipp M. Ivacevic T. Gessler M. Hein L. Upregulation of soluble vascular endothelial growth factor receptor 1 contributes to angiogenesis defects in the placenta of alpha2B-adrenoceptor deficient mice.Circ Res. 2007; 101: 682-691Crossref PubMed Scopus (21) Google Scholar Collectively, the above data provide baseline evidence that perturbation of placental α2AR expression and/or activity may contribute to pregnancy-specific pathologies, including fetal loss and defective angiogenesis. Aberrant placental growth, maturation, angiogenesis, and invasion are histopathologic hallmarks of both preeclampsia (PE) and intrauterine growth restriction (IUGR); however, the underlying molecular mechanisms remain elusive.9Reijnders I.F. Mulders A.G.M.G.J. Koster M.P.H. Placental development and function in women with a history of placenta-related complications: a systematic review.Acta Obstet Gynecol Scand. 2018; 97: 248-257Crossref PubMed Scopus (12) Google Scholar, 10Li H. Dakour J. Kaufman S. Guilbert L.J. Winkler-Lowen B. Morrish D.W. Adrenomedullin is decreased in preeclampsia because of failed response to epidermal growth factor and impaired syncytialization.Hypertension. 2003; 42: 895-900Crossref PubMed Scopus (27) Google Scholar, 11Newhouse S.M. Davidge S.T. Winkler-Lowen B. Demianczuk N. Guilbert L.J. In vitro differentiation of villous trophoblasts from pregnancies complicated by intrauterine growth restriction with and without pre-eclampsia.Placenta. 2007; 28: 999-1003Crossref PubMed Scopus (43) Google Scholar Trophoblast syncytialization is a highly coordinated cell-to-cell fusion process necessary for formation and function of the syncytiotrophoblast, which produces and secretes many hormones, including β-human chorionic gonadotropin (β-hCG).12Castellucci M. Scheper M. Scheffen I. Celona A. Kaufmann P. The development of the human placental villous tree.Anat Embryol (Berl). 1990; 181: 117-128Crossref PubMed Scopus (155) Google Scholar, 13Castellucci M. Kosanke G. Verdenelli F. Huppertz B. Kaufmann P. Villous sprouting: fundamental mechanisms of human placental development.Hum Reprod Update. 2000; 6: 485-494Crossref PubMed Scopus (127) Google Scholar, 14Loregger T. Pollheimer J. Knofler M. Regulatory transcription factors controlling function and differentiation of human trophoblast--a review.Placenta. 2003; 24: S104-S110Crossref PubMed Scopus (26) Google Scholar, 15Handwerger S. New insights into the regulation of human cytotrophoblast cell differentiation.Mol Cell Endocrinol. 2010; 323: 94-104Crossref PubMed Scopus (52) Google Scholar Several proteins, growth factors, and cytokines can modulate trophoblast syncytialization.16Knerr I. Schubert S.W. Wich C. Amann K. Aigner T. Vogler T. Jung R. Dötsch J. Rascher W. Hashemolhosseini S. Stimulation of GCMa and syncytin via cAMP mediated PKA signaling in human trophoblastic cells under normoxic and hypoxic conditions.FEBS Lett. 2005; 579: 3991-3998Crossref PubMed Scopus (105) Google Scholar, 17Orendi K. Gauster M. Moser G. Meiri H. Huppertz B. The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins.Reproduction. 2010; 140: 759-766Crossref PubMed Scopus (118) Google Scholar Chief among them are syncytin-1 and syncytin-2, which are considered hallmarks of terminal differentiation of placental trophoblast linage.18Soygur B. Sati L. The role of syncytins in human reproduction and reproductive organ cancers.Reproduction. 2016; 152: R167-R178Crossref PubMed Scopus (25) Google Scholar The synthesis and expression of syncytins depends on the cAMP/protein kinase A pathway as demonstrated through experiments that involve pharmacologic stimulation of cytotrophoblast-like (BeWo) cells with the adenylate cyclase activator and intracellular cAMP-elevating agent forskolin.16Knerr I. Schubert S.W. Wich C. Amann K. Aigner T. Vogler T. Jung R. Dötsch J. Rascher W. Hashemolhosseini S. Stimulation of GCMa and syncytin via cAMP mediated PKA signaling in human trophoblastic cells under normoxic and hypoxic conditions.FEBS Lett. 2005; 579: 3991-3998Crossref PubMed Scopus (105) Google Scholar, 19Wice B. Menton D. Geuze H. Schwartz A.L. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro.Exp Cell Res. 1990; 186: 306-316Crossref PubMed Scopus (255) Google Scholar From the above experimental data it is tempting to speculate that aberrant expression and activation of α2ARs could disrupt placental development, leading to pathologic processes. This concept has biological plausibility because trophoblast syncytialization is defective in both PE and IUGR.20Lee X. Keith Jr., J.C. Stumm N. Moutsatsos I. McCoy J.M. Crum C.P. Genest D. Chin D. Ehrenfels C. Pijnenborg R. van Assche F.A. Mi S. Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia.Placenta. 2001; 22: 808-812Crossref PubMed Scopus (148) Google Scholar, 21Roland C.S. Hu J. Ren C.E. Chen H. Li J. Varvoutis M.S. Leaphart L.W. Byck D.B. Zhu X. Jiang S.W. Morphological changes of placental syncytium and their implications for the pathogenesis of preeclampsia.Cell Mol Life Sci. 2016; 73: 365-376Crossref PubMed Scopus (76) Google Scholar, 22Ruebner M. Strissel P.L. Ekici A.B. Stiegler E. Dammer U. Goecke T.W. Faschingbauer F. Fahlbusch F.B. Beckmann M.W. Strick R. Reduced syncytin-1 expression levels in placental syndromes correlates with epigenetic hypermethylation of the ERVW-1 promoter region.PLoS One. 2013; 8: e56145Crossref PubMed Scopus (61) Google Scholar These two conditions may occur if heightened expression of α2ARs inhibits adenylyl cyclase, thereby reducing cAMP production and in turn the synthesis of syncytins. At least two α2AR subtypes are known to be present in human placenta, specifically α2AAR and α2BAR.23Falkay G. Kovács L. Expression of two alpha 2-adrenergic receptor subtypes in human placenta: evidence from direct binding studies.Placenta. 1994; 15: 661-668Crossref PubMed Scopus (21) Google Scholar This conclusion was reached from radioligand binding experiments and not by immunohistology, which, before the present study, has not been performed. Here, we tested the hypotheses that PE and IUGR are associated with differential expression of placental α2ARs and that α2ARs are key regulators of trophoblast syncytialization and migration. Placental tissues retrieved from 52 women were distributed in the following groups: i) PE with severe features [sPE; n = 8; means ± SEM gestational age (GA), 31 ± 1 weeks]; ii) sPE + IUGR (n = 9; mean GA, 30 ± 1 weeks); iii) idiopathic IUGR (n = 8; mean GA, 31 ± 1 weeks); iv) idiopathic preterm birth (iPTB; n = 16; mean GA, 31 ± 1 weeks), and v) healthy term controls (n = 11; mean GA, 39 ± 1 weeks) were analyzed. GA in all cases was established from last menstrual period and/or early ultrasound (<20 weeks).24American College of Obstetricians and GynecologistsACOG Practice Bulletin No. 101: ultrasonography in pregnancy.Obstet Gynecol. 2009; 113: 451-461Crossref PubMed Scopus (280) Google Scholar Preterm birth was defined as delivery before 37 weeks GA. For all iPTB cases intraamniotic infection was excluded based on negative results of amniotic fluid biochemical testing, negative bacterial cultures, and absence of histologic chorioamnionitis. iPTB tissues were the best possible control to identify potential GA regulation in the expression of α2ARs. This control was important because women with sPE and IUGR are frequently delivered at earlier GAs. Because patient recruitment and sample collection were completed before publication of the 2013 Task Force (May 2005 to July 2012), the definition of PE was based on American College of Obstetricians and Gynecologists criteria valid at the time of sample collections.25ACOG Committee on Practice Bulletins--ObstetricsACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002.Obstet Gynecol. 2002; 99: 159-167Crossref PubMed Google Scholar, 26American College of Obstetricians and GynecologistsTask Force on Hypertension in PregnancyHypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy.Obstet Gynecol. 2013; 122: 1122-1131Crossref PubMed Scopus (790) Google Scholar IUGR was defined ultrasonographically as an estimated fetal growth <10th percentile for GA.27American College of Obstetricians and GynecologistsACOG Practice bulletin no. 134: fetal growth restriction.Obstet Gynecol. 2013; 121: 1122-1133Crossref PubMed Google Scholar A diagnosis of idiopathic IUGR was established in babies without detectable structural anomalies, and when examination of the placenta and the work-up to rule out chromosomal aneuploidy did not provide a cause for lack of growth. Presence of fetal structural anomalies and antenatal viral infections (ie, HIV, hepatitis) were considered exclusion criteria. All women were delivered at Yale-New Haven Hospital and provided signed informed consent under protocols approved by the Human Investigation Committee of Yale University. Within minutes from the time of delivery of the placenta, a full-thickness biopsy was retrieved from the central portion of the placenta. Approximately one-half of the tissue was fixed in formalin. For the second half, decidua basalis was dissected from the villous trophoblast in sterile fashion, rinsed in sterile saline, frozen in liquid nitrogen, and kept at −80°C for RNA studies. Purified rabbit polyclonal antibodies were used to stain for α2AAR, α2CAR, and α2BAR (ab91648; Abcam Inc., Cambridge, MA).28Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Subtype-specific intracellular trafficking of alpha2-adrenergic receptors.Mol Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar The three polyclonal antisera are specific for their respective α2AR subtype and do not cross-react with other α2AR subtypes. The specificity of these antibodies has been established in several previously published studies.29Motawea H.K. Jeyaraj S.C. Eid A.H. Mitra S. Unger N.T. Ahmed A.A. Flavahan N.A. Chotani M.A. Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2.Am J Physiol Cell Physiol. 2013; 305: C829-C845Crossref PubMed Scopus (26) Google Scholar, 30Bailey S.R. Eid A.H. Mitra S. Flavahan S. Flavahan N.A. Rho kinase mediates cold-induced constriction of cutaneous arteries: role of alpha2C-adrenoceptor translocation.Circ Res. 2004; 94: 1367-1374Crossref PubMed Scopus (178) Google Scholar, 31Chotani M.A. Mitra S. Su B.Y. Flavahan S. Eid A.H. Clark K.R. Montague C.R. Paris H. Handy D.E. Flavahan N.A. Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells.Am J Physiol Heart Circ Physiol. 2004; 286: H59-H67Crossref PubMed Scopus (80) Google Scholar, 32Jeyaraj S.C. Unger N.T. Eid A.H. Mitra S. El-Dahdah N.P. Quilliam L.A. Flavahan N.A. Chotani M.A. Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.Am J Physiol Cell Physiol. 2012; 303: C499-C511Crossref PubMed Scopus (46) Google Scholar The anti-α2CAR antibody recognizes the approximately 70-kDa mature receptor form that translocates from the Golgi compartment to the cell surface.33Jeyaraj S.C. Chotani M.A. Mitra S. Gregg H.E. Flavahan N.A. Morrison K.J. Cooling evokes redistribution of alpha2C-adrenoceptors from Golgi to plasma membrane in transfected human embryonic kidney 293 cells.Mol Pharmacol. 2001; 60: 1195-1200Crossref PubMed Scopus (103) Google Scholar A rabbit monoclonal antibody was used to detect phospho-ERK1/2 (p-ERK1/2) (catalog number 4370; Cell Signaling Technology, Danvers, MA). Five-micron serial paraffin sections of fetal membranes and placental villous tissue were immunostained for α2ARs and p-ERK1/2 as previously described.34Oliver E.A. Buhimschi C.S. Dulay A.T. Baumbusch M.A. Abdel-Razeq S.S. Lee S.Y. Zhao G. Jing S. Pettker C.M. Buhimschi I.A. Activation of the receptor for advanced glycation end products system in women with severe preeclampsia.J Clin Endocrinol Metab. 2011; 96: 689-698Crossref PubMed Scopus (28) Google Scholar Term human myometrium was used as a positive control. After antigen retrieval with citrate buffer and pretreatment with 1% hydrogen peroxide (15 minutes), sections were incubated overnight (4°C) with affinity-purified rabbit polyclonal antibodies to α2AAR, α2BAR, or α2CAR (dilution 1:100)32Jeyaraj S.C. Unger N.T. Eid A.H. Mitra S. El-Dahdah N.P. Quilliam L.A. Flavahan N.A. Chotani M.A. Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.Am J Physiol Cell Physiol. 2012; 303: C499-C511Crossref PubMed Scopus (46) Google Scholar or p-ERK1/2 (dilution 1:100) then at room temperature (60 minutes) with biotinylated donkey anti-rabbit IgG (dilution 1:600; Jackson ImmunoResearch Laboratories, West Grove, PA). Negative controls consisted of tissue samples from the same patient that were processed identically but incubated with non-immune rabbit IgG (Jackson ImmunoResearch Laboratories). Sections were stained by using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) with VECTOR NovaRed chromogen, counterstained with hematoxylin, and subjectively scored, as previously described.34Oliver E.A. Buhimschi C.S. Dulay A.T. Baumbusch M.A. Abdel-Razeq S.S. Lee S.Y. Zhao G. Jing S. Pettker C.M. Buhimschi I.A. Activation of the receptor for advanced glycation end products system in women with severe preeclampsia.J Clin Endocrinol Metab. 2011; 96: 689-698Crossref PubMed Scopus (28) Google Scholar All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified. Specific staining in extravillous trophoblasts, villous cytotrophoblasts, syncytiotrophoblast, and wall of placental vascular structures was evaluated semiquantitatively in a blinded fashion by two independent observers (H.K.B.M. and I.A.B) from three random fields per slide. Staining intensity was scored on a scale from 0 (absent) to 5 (intense). The human choriocarcinoma BeWo cell line (gift from Dr. John M. Robinson, The Ohio State University, Columbus, OH) shares morphologic and biosynthetic features with human invasive trophoblasts and is widely used to study placental trophoblast function.17Orendi K. Gauster M. Moser G. Meiri H. Huppertz B. The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins.Reproduction. 2010; 140: 759-766Crossref PubMed Scopus (118) Google Scholar In this study BeWo cells were used for in vitro manipulation of α2CAR. BeWo cells were cultured in Ham's F12 medium supplemented with 10% fetal bovine serum and l-glutamine (2 mmol/L), containing 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Gaithersburg, MD), and were incubated under humidified atmosphere of 5% CO2 in air at 37°C. Cells were seeded at 0.5 to 1 × 106 in T25 25-cm2 flasks and passaged at 60% to 80% confluency by using 0.25% Trypsin (Gibco). For β-hCG measurements and for protein and RNA isolation, cells were seeded at 105 cells per well on 6-well plates. Cells were incubated overnight in culture media replaced daily with fresh media that contained 20 μmol/L forskolin or dimethyl sulfoxide (DMSO; vehicle control), as appropriate for the planned experiments. α2CAR was visualized with polyclonal rabbit antibody,32Jeyaraj S.C. Unger N.T. Eid A.H. Mitra S. El-Dahdah N.P. Quilliam L.A. Flavahan N.A. Chotani M.A. Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.Am J Physiol Cell Physiol. 2012; 303: C499-C511Crossref PubMed Scopus (46) Google Scholar followed by goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (A11037; Life Technologies, Carlsbad, CA). E-cadherin (marker of progenitor cytotrophoblasts) was visualized with mouse monoclonal anti–E-cadherin monoclonal antibody (HECD-1; dilution 1:200; Invitrogen, Carlsbad, CA), followed by goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 (A11001; Invitrogen). For gene expression studies, placental tissue was flash frozen in liquid nitrogen immediately after delivery and maintained at −80°C. BeWo cells were washed with ice-cold phosphate-buffered saline, 1 mL TRIzol was added to each well, and plates were stored at −80°C. Placental and BeWo cell RNA was extracted with TRIzol and quantitated and reverse transcribed into cDNA with random hexamer primers by using standard procedures. RT-PCR was performed by using TaqMan (Applied Biosystems, Carlsbad, CA) chemistry in 20-μL reactions composed of 10 μL mastermix (TaqMan Fast Advanced Master Mix), 8 μL water, 1 μL cDNA template, and 1 μL PCR probe set (TaqMan Gene Expression Assays). The following probes (Applied Biosystems) were used: α2AAR (ADRA2A; Hs01099503_s1; reference sequence NM_000681.3), α2BAR (ADRA2B; Hs00265090_s1; reference sequence NM_000682.6), α2CAR (ADRA2C; Hs03044628_s1; reference sequence NM_000683.3), and for the housekeeping genes ribosomal protein L30 (RPL30; Hs00265497_m1; reference sequence NM_000989.3) and β–2-microglobulin (B2M; Hs99999907_m1; reference sequence NM_004048.2). Unique probes for experiments with BeWo cells were syncytin-1 (ERVW-1; Hs00205893_m1; reference sequence NM_001130925.1) and syncytin-2 (ERVFRD-1; Hs01652148_m1; reference sequence NM_207582.2) with RPL30 as housekeeping gene. Placental mRNA expression of α2ARs was normalized to RPL30 and B2M by using cDNA derived from pools of RNA extracted from iPTB and term control placenta tissue, and BeWo expression was normalized to RPL30. Amplification was performed in duplicate reactions for each target in a two-step cycle (denaturation, 95°C for 15 seconds; annealing/extension at 62°C for 60 seconds) for 40 cycles in the StepOnePlus Real-Time PCR System (Applied Biosystems). Post-processing calculations were performed by using StepOnePlus Software version 2.1 (Applied Biosystems, Foster City, CA). Ct values for α2AR were normalized to the geometric mean of the endogenous control RNAs by using calculations of dCt (Ct of the target − Ct mean of endogenous controls). Calculation of ddCt (dCt of individual sample − Ct of same target in a reference sample) used as reference sample a cDNA pool of all tissues. For BeWo cell experiments, RNA from cells transduced with the non-human target control vector was used as reference. α2CAR expression in BeWo cells was detected with affinity-purified rabbit polyclonal α2CAR antisera (dilution 1:500) from total cellular protein prepared as previously described.32Jeyaraj S.C. Unger N.T. Eid A.H. Mitra S. El-Dahdah N.P. Quilliam L.A. Flavahan N.A. Chotani M.A. Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.Am J Physiol Cell Physiol. 2012; 303: C499-C511Crossref PubMed Scopus (46) Google Scholar Cells were lyzed in buffer that contained protease inhibitor cocktail (Complete; Roche Applied Sciences, Indianapolis, IN), and 20 μg of total protein was boiled in reducing sample buffer and separated on 4% to 20% Mini Protean TGX gels (Bio-Rad, Hercules, CA). Intensity of the band that corresponded to α2CAR was quantified by using ImageQuant TL software version 8.1 (GE Healthcare, Pittsburgh, PA) and normalized to glyceraldehyde-3-phosphate dehydrogenase as loading control. Pharmacologic studies used the nonsubtype selective α2AR receptor agonist UK 14,304 [5-bromo-N-(4,5-dihydro-1H-imidazole-2-yl)-6-quinoxalinamine] or the antagonist MK912 that is more selective for α2CAR.35Uhlén S. Porter A.C. Neubig R.R. The novel alpha-2 adrenergic radioligand [3H]-MK912 is alpha-2C selective among human alpha-2A, alpha-2B and alpha-2C adrenoceptors.J Pharmacol Exp Ther. 1994; 271: 1558-1565PubMed Google Scholar After 48 hours, cells were treated in a dose-dependent fashion with UK 14,304 (0.01 to 10 nmol/L) or DMSO for 15 minutes. In another set of experiments, cells were treated with UK 14,304 for 15 minutes after a 30-minute pretreatment with 1 nmol/L MK912 or corresponding volume of water (vehicle control for MK912). β-hCG secretion in cell culture media was assayed by enzyme-linked immunosorbent assay (BioVendor Research and Diagnostic Products, Asheville, NC). BeWo cells were seeded 105 per well and incubated overnight. In the morning, culture medium was removed and replaced with fresh solution. BeWo cells were transduced with lentiviral particles at a multiplicity of infection = 1 and were subjected to puromycin selection according to the supplier's protocol (Sigma-Aldrich). Transduced cells underwent three consecutive passages under puromycin selection (9 days total) before use in knockdown assays. α2CAR expression was knocked down in BeWo cells by using a lentiviral shRNA construct that targeted ADRA2C (TRCN0000008077; 5′-CCGGCAACGACGAGACCTGGTACATCTCGAGATGTACCAGGTCTCGTCGTTGTTTTT-3′; Sigma-Aldrich). BeWo cells transduced with a non-human target control, pLKO.1-puro (Sigma-Aldrich), served as negative control. Cells were incubated in the presence or absence of 20 μmol/L forskolin to induce differentiation. RNA was extracted, and expression of α2CAR and of syncytin-1 and syncytin-2 mRNA was measured by quantitative real-time RT-PCR. Culture media were collected before and at 24, 48, and 72 hours after 15-minute exposure to UK 14,304 or DMSO (vehicle control). Media were stored at −80°C until β-hCG measurement. The migration potential of BeWo cells was assessed by wound healing assay as previously described.36Ali M. Heyob K. Jacob N.K. Rogers L.K. Alterative expression and localization of profilin 1/VASPpS157 and cofilin 1/VASPpS239 regulates metastatic growth and is modified by DHA supplementation.Mol Cancer Ther. 2016; 15: 2220-2231Crossref PubMed Scopus (18) Google Scholar A single wound gap was induced on each well by scratching the cell monolayer with a pipette tip. BeWo cells were pretreated with 1 nmol/L MK912 or H2O for 15 minutes, followed by incubation with 10 mmol/L UK 14,304 or DMSO for 60 minutes. Images of five wounds for each treatment condition were analyzed at 0 and 24 hours after treatment, and percentage of migration at 24 hours was calculated from three points per wound. ImageJ software version 1.46r (NIH, Bethesda, MD; https://imagej.nih.gov/ij) was used for image analysis. Statistical analyses were performed with SigmaPlot statistical software version 12.5 (Systat Software, San Jose, CA). After distribution testing by using Shapiro-Wilk test, data were compared with t-tests, one-, two-, or three-way analysis of variance or Kruskal-Wallis analysis of variance on ranks as appropriate." @default.
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• W2894096609 date "2018-12-01" @default.
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• W2894096609 title "Human Placenta Expresses α2-Adrenergic Receptors and May Be Implicated in Pathogenesis of Preeclampsia and Fetal Growth Restriction" @default.
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• W2894096609 doi "https://doi.org/10.1016/j.ajpath.2018.08.011" @default.
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