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Glyceryl Trinitrate Inhibits Hypoxia-Induced Release of Soluble fms-Like Tyrosine Kinase-1 and Endoglin from Placental Tissues

2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês

10.1016/j.ajpath.2011.02.013

ISSN

1525-2191

Autores

Ivraym B. Barsoum, Stephen J. Renaud, Charles H. Graham,

Tópico(s)

Fibroblast Growth Factor Research

Resumo

Preeclampsia is associated with increased circulating levels of proinflammatory molecules, such as soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng). On release by an inadequately perfused placenta into the maternal circulation, these molecules cause systemic endothelial dysfunction and the associated hypertension and proteinuria that characterize preeclampsia. We previously showed that glyceryl trinitrate (GTN) inhibits hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of term chorionic villi explants. Herein, we demonstrate that GTN inhibits the release of sFlt-1 and sEng from term chorionic villi explants exposed to hypoxia. Although transcript levels and secretion of sFlt-1 and sEng increased in explants exposed to hypoxia, low concentrations of GTN significantly inhibited the hypoxia-induced expression of these molecules at the mRNA and protein levels. Treatment of explants with GTN also prevented the hypoxia-induced accumulation of hypoxia-inducible factor-1α, a key mediator of cellular adaptations to hypoxia. Furthermore, knockdown of hypoxia-inducible factor-1α inhibited the hypoxia-induced secretion of sFlt-1 and sEng. This study provides evidence that hypoxia induces the release of sFlt-1 and sEng in the placenta via a mechanism that is inhibited by low concentrations of GTN. Our findings indicate that GTN may have potential applications in the treatment and/or prevention of preeclampsia. Preeclampsia is associated with increased circulating levels of proinflammatory molecules, such as soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng). On release by an inadequately perfused placenta into the maternal circulation, these molecules cause systemic endothelial dysfunction and the associated hypertension and proteinuria that characterize preeclampsia. We previously showed that glyceryl trinitrate (GTN) inhibits hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of term chorionic villi explants. Herein, we demonstrate that GTN inhibits the release of sFlt-1 and sEng from term chorionic villi explants exposed to hypoxia. Although transcript levels and secretion of sFlt-1 and sEng increased in explants exposed to hypoxia, low concentrations of GTN significantly inhibited the hypoxia-induced expression of these molecules at the mRNA and protein levels. Treatment of explants with GTN also prevented the hypoxia-induced accumulation of hypoxia-inducible factor-1α, a key mediator of cellular adaptations to hypoxia. Furthermore, knockdown of hypoxia-inducible factor-1α inhibited the hypoxia-induced secretion of sFlt-1 and sEng. This study provides evidence that hypoxia induces the release of sFlt-1 and sEng in the placenta via a mechanism that is inhibited by low concentrations of GTN. Our findings indicate that GTN may have potential applications in the treatment and/or prevention of preeclampsia. Preeclampsia is a leading cause of perinatal and maternal morbidity and mortality, affecting 3% to 7% of pregnant women worldwide.1Redman C.W. Sargent I.L. Latest advances in understanding preeclampsia.Science. 2005; 308: 1592-1594Crossref PubMed Scopus (2013) Google Scholar It is characterized by the development of maternal hypertension, proteinuria, edema, and systemic coagulopathies. Although the etiology of preeclampsia is not well understood, there is evidence that high levels of antiangiogenic molecules [ie, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng)] in the maternal circulation are linked to the endothelial dysfunction associated with this pregnancy complication.2Koga K. Osuga Y. Yoshino O. Hirota Y. Ruimeng X. Hirata T. Takeda S. Yano T. Tsutsumi O. Taketani Y. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia.J Clin Endocrinol Metab. 2003; 88: 2348-2351Crossref PubMed Scopus (336) Google Scholar, 3Levine R.J. Maynard S.E. Qian C. Lim K.H. England L.J. Yu K.F. Schisterman E.F. Thadhani R. Sachs B.P. Epstein F.H. Sibai B.M. Sukhatme V.P. Karumanchi S.A. Circulating angiogenic factors and the risk of preeclampsia.N Engl J Med. 2004; 350: 672-683Crossref PubMed Scopus (2760) Google Scholar, 4Venkatesha S. Toporsian M. Lam C. Hanai J. Mammoto T. Kim Y.M. Bdolah Y. Lim K.H. Yuan H.T. Libermann T.A. Stillman I.E. Roberts D. D'Amore P.A. Epstein F.H. Sellke F.W. Romero R. Sukhatme V.P. Letarte M. Karumanchi S.A. Soluble endoglin contributes to the pathogenesis of preeclampsia.Nat Med. 2006; 12: 642-649Crossref PubMed Scopus (1438) Google Scholar, 5Maynard S.E. Min J.Y. Merchan J. Lim K.H. Li J. Mondal S. Libermann T.A. Morgan J.P. Sellke F.W. Stillman I.E. Epstein F.H. Sukhatme V.P. Karumanchi S.A. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (3093) Google ScholarSecreted sFlt-1, usually a splice variant of Flt-1, is the soluble form of vascular endothelial growth factor receptor-1 (VEGFR-1). Soluble Flt-1 binds to angiogenic molecules (ie, placental growth factor and VEGF) and prevents them from interacting with VEGFR-1 and VEGFR-2 on the surface of endothelial cells.5Maynard S.E. Min J.Y. Merchan J. Lim K.H. Li J. Mondal S. Libermann T.A. Morgan J.P. Sellke F.W. Stillman I.E. Epstein F.H. Sukhatme V.P. Karumanchi S.A. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (3093) Google Scholar, 6Ahmad S. Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia.Circ Res. 2004; 95: 884-891Crossref PubMed Scopus (412) Google Scholar Consequently, any increase in blood sFlt-1 levels causes a decrease in bioavailable VEGF and placental growth factor, with corresponding inhibition of angiogenesis and vascular function. There is evidence that the placenta is a primary source of sFlt-1 in human pregnancy,7Banks R.E. Forbes M.A. Searles J. Pappin D. Canas B. Rahman D. Kaufmann S. Walters C.E. Jackson A. Eves P. Linton G. Keen J. Walker J.J. Selby P.J. Evidence for the existence of a novel pregnancy-associated soluble variant of the vascular endothelial growth factor receptor: Flt-1.Mol Human Reprod. 1998; 4: 377-386Crossref PubMed Scopus (104) Google Scholar and clinical studies have revealed that normal circulating sFlt-1 levels increase during gestation. However, compared with normal pregnancies, blood sFlt-1 levels are substantially higher in preeclamptic pregnancies.2Koga K. Osuga Y. Yoshino O. Hirota Y. Ruimeng X. Hirata T. Takeda S. Yano T. Tsutsumi O. Taketani Y. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia.J Clin Endocrinol Metab. 2003; 88: 2348-2351Crossref PubMed Scopus (336) Google Scholar Clinical signs of preeclampsia may be a direct outcome of high concentrations of serum sFlt-1.5Maynard S.E. Min J.Y. Merchan J. Lim K.H. Li J. Mondal S. Libermann T.A. Morgan J.P. Sellke F.W. Stillman I.E. Epstein F.H. Sukhatme V.P. Karumanchi S.A. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (3093) Google Scholar, 8Tsatsaris V. Goffin F. Munaut C. Brichant J.-F. Pignon M.-R. Noel A. Schaaps J.-P. Cabrol D. Frankenne F. Foidart J.-M. Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences.J Clin Endocrinol Metab. 2003; 88: 5555-5563Crossref PubMed Scopus (393) Google Scholar Furthermore, animal experimentation has revealed that abnormally high circulating levels of sFlt-1 lead to hypertension, proteinuria, and endothelial damage.5Maynard S.E. Min J.Y. Merchan J. Lim K.H. Li J. Mondal S. Libermann T.A. Morgan J.P. Sellke F.W. Stillman I.E. Epstein F.H. Sukhatme V.P. Karumanchi S.A. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (3093) Google Scholar, 9Makris A. Thornton C. Thompson J. Thomson S. Martin R. Ogle R. Waugh R. McKenzie P. Kirwan P. Hennessy A. Uteroplacental ischemia results in proteinuric hypertension and elevated sFlt-1.Kidney Int. 2007; 71: 977-984Crossref PubMed Scopus (259) Google ScholarEng is a cell membrane glycoprotein that functions as a coreceptor for transforming growth factor (TGF)-β signaling.10Cheifetz S. Bellon T. Cales C. Vera S. Bernabeu C. Massague J. Letarte M. Endoglin is a component of the transforming growth factor-β receptor system in human endothelial cells.J Biol Chem. 1992; 267: 19027Abstract Full Text PDF PubMed Google Scholar sEng competes with the binding of TGF-β1 to its corresponding cell surface receptors and, consequently, interferes with downstream TGF-β signaling in the vasculature.4Venkatesha S. Toporsian M. Lam C. Hanai J. Mammoto T. Kim Y.M. Bdolah Y. Lim K.H. Yuan H.T. Libermann T.A. Stillman I.E. Roberts D. D'Amore P.A. Epstein F.H. Sellke F.W. Romero R. Sukhatme V.P. Letarte M. Karumanchi S.A. Soluble endoglin contributes to the pathogenesis of preeclampsia.Nat Med. 2006; 12: 642-649Crossref PubMed Scopus (1438) Google Scholar Similar to signaling by VEGF and placental growth factor, TGF-β signaling plays an important role in the maintenance of vascular function and homeostasis. In preeclampsia, high levels of sEng in the maternal circulation correlate with increased expression of Eng in the placenta.4Venkatesha S. Toporsian M. Lam C. Hanai J. Mammoto T. Kim Y.M. Bdolah Y. Lim K.H. Yuan H.T. Libermann T.A. Stillman I.E. Roberts D. D'Amore P.A. Epstein F.H. Sellke F.W. Romero R. Sukhatme V.P. Letarte M. Karumanchi S.A. Soluble endoglin contributes to the pathogenesis of preeclampsia.Nat Med. 2006; 12: 642-649Crossref PubMed Scopus (1438) Google Scholar, 11Masuyama H. Nakatsukasa H. Takamoto N. Hiramatsu Y. Correlation between soluble endoglin, vascular endothelial growth factor receptor-1, and adipocytokines in preeclampsia.J Clin Endocrinol Metab. 2007; 92: 2672-2679Crossref PubMed Scopus (82) Google Scholar Circulating sEng levels were markedly increased 2 to 3 months before the clinical onset of preeclampsia.12Levine R.J. Lam C. Qian C. Yu K.F. Maynard S.E. Sachs B.P. Sibai B.M. Epstein F.H. Romero R. Thadhani R. Karumanchi S.A. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia.N Engl J Med. 2006; 355: 992-1005Crossref PubMed Scopus (1456) Google ScholarPlacental hypoxia may play a major role in the pathophysiological characteristics of preeclampsia9Makris A. Thornton C. Thompson J. Thomson S. Martin R. Ogle R. Waugh R. McKenzie P. Kirwan P. Hennessy A. Uteroplacental ischemia results in proteinuric hypertension and elevated sFlt-1.Kidney Int. 2007; 71: 977-984Crossref PubMed Scopus (259) Google Scholar, 13Sandau K.B. Zhou J. Kietzmann T. Brune B. Regulation of the hypoxia-inducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis factor-alpha in contrast to desferroxamine and phenylarsine oxide.J Biol Chem. 2001; 276: 39805-39811Crossref PubMed Scopus (192) Google Scholar, 14Soleymanlou N. Jurisica I. Nevo O. Ietta F. Zhang X. Zamudio S. Post M. Caniggia I. Molecular evidence of placental hypoxia in preeclampsia.J Clin Endocrinol Metab. 2005; 90: 4299-4308Crossref PubMed Scopus (294) Google Scholar; hypoxia increases the secretion of sFlt-1 and sEng from first- and second-trimester chorionic villi explants.9Makris A. Thornton C. Thompson J. Thomson S. Martin R. Ogle R. Waugh R. McKenzie P. Kirwan P. Hennessy A. Uteroplacental ischemia results in proteinuric hypertension and elevated sFlt-1.Kidney Int. 2007; 71: 977-984Crossref PubMed Scopus (259) Google Scholar, 15Yinon Y. Nevo O. Xu J. Many A. Rolfo A. Todros T. Post M. Caniggia I. Severe intrauterine growth restriction pregnancies have increased placental endoglin levels: hypoxic regulation via transforming growth factor-beta 3.Am J Pathol. 2008; 172: 77-85Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 16Zamudio S. Wu Y. Ietta F. Rolfo A. Cross A. Wheeler T. Post M. Illsley N.P. Caniggia I. Human placental hypoxia-inducible factor-1α expression correlates with clinical outcomes in chronic hypoxia in vivo.Am J Pathol. 2007; 170: 2171-2179Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Nevo O. Soleymanlou N. Wu Y. Increased expression of sFlt-1 in in vivo and in vitro models of human placental hypoxia is mediated by HIF-1.Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1085Crossref PubMed Scopus (254) Google Scholar Although hypoxia also increases the secretion of sFlt-1 by third-trimester explants,18Rajakumar A. Powers R. Hubel C. Shibata E. von Versen-Hoynck F. Plymire D. Jeyabalan A. Novel soluble Flt-1 isoforms in plasma and cultured placental explants from normotensive pregnant and preeclamptic women.Placenta. 2009; 30: 25-34Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar its effect on sEng secretion by third-trimester explants has not been determined. Because the clinical signs of preeclampsia only appear in the second half of pregnancy, it is possible that a hypoxia-induced increase in sFlt-1 and sEng secretion in the second and third trimesters of gestation contributes to the pathophysiological characteristics of preeclampsia.The hypoxia response pathway represents a potential therapeutic target for inhibiting placental secretion of sFlt-1 and sEng. A key player that mediates many adaptive responses to hypoxia is the transcriptional activator hypoxia-inducible factor (HIF)-1. This transcription factor is a basic helix-loop-helix-PAS domain protein composed of α and β subunits. Although the HIF-1β subunit is constitutively expressed, the levels of HIF-1α increase in response to hypoxia.19Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci U S A. 1995; 92: 5510-5514Crossref PubMed Scopus (4975) Google Scholar The stability of HIF-1α is regulated by hydroxylation of proline residues 402, 564, or both in an oxygen-dependent reaction.20Semenza G.L. Hypoxia-inducible factor 1 (HIF-1) pathway.Sci STKE. 2007; 2007: cm8Crossref PubMed Scopus (708) Google Scholar Proline hydroxylation of HIF-1α promotes the binding of the von Hippel–Lindau protein, leading to ubiquitination and proteasomal degradation of HIF-1α.20Semenza G.L. Hypoxia-inducible factor 1 (HIF-1) pathway.Sci STKE. 2007; 2007: cm8Crossref PubMed Scopus (708) Google Scholar, 21Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4369) Google ScholarThere is evidence that relatively low concentrations of nitric oxide (NO) inhibit the accumulation of HIF-1α under hypoxia, thereby interfering with HIF-1–mediated adaptive responses.22Brune B. Zhou J. Nitric oxide and superoxide: interference with hypoxic signaling.Cardiovasc Res. 2007; 75: 275-282Crossref PubMed Scopus (106) Google Scholar, 23Hagen T. Taylor C.T. Lam F. Moncada S. Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha.Science. 2003; 302: 1975-1978Crossref PubMed Scopus (629) Google Scholar Low concentrations (lower than micromolar) of NO mimetics [eg, glyceryl trinitrate (GTN)] inhibit the hypoxia-induced acquisition of malignant phenotypes in cancer cells, such as invasiveness, metastasis, and drug resistance.24Frederiksen L.J. Sullivan R. Maxwell L.R. Macdonald-Goodfellow S.K. Adams M.A. Bennett B.M. Siemens D.R. Graham C.H. Chemosensitization of cancer in vitro and in vivo by nitric oxide signaling.Clin Cancer Res. 2007; 13: 2199-2206Crossref PubMed Scopus (113) Google Scholar, 25Frederiksen L.J. Siemens D.R. Heaton J.P. Maxwell L.R. Adams M.A. Graham C.H. Hypoxia induced resistance to doxorubicin in prostate cancer cells is inhibited by low concentrations of glyceryl trinitrate.J Urol. 2003; 170: 1003-1007Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Although the precise role of endogenous NO in normal and pathological pregnancy is unclear, there is evidence that decreased endogenous NO availability contributes to the pathophysiological characteristics of preeclampsia.26Lowe D.T. Nitric oxide dysfunction in the pathophysiology of preeclampsia.Nitric Oxide. 2000; 4: 441-458Crossref PubMed Scopus (145) Google Scholar A previous study27Belkacemi L. Bainbridge S.A. Dickinson M.A. Smith G.N. Graham C.H. Glyceryl trinitrate inhibits hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of the human placenta: therapeutic implications for preeclampsia.Am J Pathol. 2007; 170: 909-920Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar demonstrated that low concentrations of GTN inhibit apoptosis of the syncytiotrophoblast in chorionic villi explants exposed to hypoxia/reoxygenation (H/R). In the present study, we determined the effect of administration of low concentrations of GTN on the secretion of sFlt-1 and sEng from term chorionic villi explants exposed to hypoxia.Materials and MethodsCollection and Culture of Chorionic Villi ExplantsHuman term placentas (n = 22) were obtained from uncomplicated pregnancies immediately after caesarean deliveries at Kingston General Hospital, Kingston, ON. Placentas were collected with the approval of the Queen's University Research Ethics Board. Explants of chorionic villi were prepared as previously described27Belkacemi L. Bainbridge S.A. Dickinson M.A. Smith G.N. Graham C.H. Glyceryl trinitrate inhibits hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of the human placenta: therapeutic implications for preeclampsia.Am J Pathol. 2007; 170: 909-920Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar and incubated in 1.3 mL of serum-free RPMI 1640 medium (Invitrogen, Burlington, ON). For exposures to hypoxia, explants (approximately 60 per experiment) treated with or without GTN (10 nmol/L or 1 μmol/L; Sabex, Boucherville, QC) were incubated at 37°C for 24 hours in a humidified Plexiglas chamber flushed with a gas mixture of 5% CO2 and 95% N2. Oxygen concentrations within the chamber were maintained at 0.5% (PO2 = 3.8 mm Hg) by oxygen regulators (ProOx 110; Biospherix Inc., Lacona, NY). Controls consisted of explants incubated in either 8% O2 (PO2 = 60 mm Hg, physiological concentrations) or 20% O2 (PO2 = 152 mm Hg) and 5% CO2 for 24 hours. Explants were then collected or exposed to conditions of reoxygenation (20% O2) for 3 additional hours. At the end of all incubations, placental tissues were either flash frozen for molecular analysis or fixed in 4% paraformaldehyde for histological analysis. Media of cultured explants were collected, centrifuged for 10 minutes at 8000 × g, divided into small tubes, and stored at -80°C until further analysis.ImmunohistochemistryRandomly selected paraffin-embedded sections of chorionic villi were deparaffinized and subjected to antigen retrieval by heating them in a microwave oven for 22 minutes in citrate buffer (0.1 mol/L sodium citrate, pH 6.0). Primary antibodies used were goat polyclonal anti-human Eng or Flt-1 antibody (1 μg/mL; R&D Systems, Minneapolis, MN), mixed with mouse anti–human pan-cytokeratin antibody (1:100; Sigma-Aldrich, St Louis, MO) and incubated at 4°C overnight. Secondary antibodies were a mix of donkey anti-mouse (Alexa Fluor 568) and donkey anti-goat (Alexa Fluor 488) antibodies (1:500 each; Invitrogen, Carlsbad, CA). Slides were mounted with medium (VECTASHIELD Mounting Medium) containing DAPI (Vector Laboratories, Inc., Burlingame, CA). Micrographs were taken using a microscope (Zeiss Imager A1 Microscope; Carl Zeiss Canada Ltd, Toronto, ON) and software (Axiovision v 4.7.1.0; Carl Zeiss Canada Ltd).Western Blot Analysis of HIF-1αTo assess the ability of chorionic villi to respond to hypoxia, the levels of HIF-1α were determined by Western blot analysis of flash-frozen chorionic villi preexposed to 0.5%, 8%, or 20% O2 for 24 hours (n = 3 to 6 placentas). Primary antibodies used were mouse monoclonal antibody against HIF-1α (1:250; BD Biosciences, San Diego, CA), goat polyclonal anti–TGF-β1 (1:1000, R&D Systems), and monoclonal anti–β-actin antibody (clone AC-15, 1:8000; Bio-Rad Laboratories Ltd, Mississauga, ON, Canada). Goat anti-mouse antibody (1:5000, Vector Laboratories Inc.) labeled with horseradish peroxidase was used as a secondary antibody. The relative intensities of bands were determined by densitometry using software (AlphaErase; α Innotech Corp, San Leandro, CA).Knockdown of HIF-1α in Chorionic Villi ExplantsTo down-regulate HIF-1α expression in chorionic villi, HIF-1α small-interfering RNA (siRNA) was introduced into explants using a transfection reagent (siPORT NeoFX; Ambion Inc., Austin, TX). This HIF-1α siRNA was validated and targets exon 5 of the human HIF-1α gene (number 42840; Ambion Inc.). Explants were transfected with 200 nmol/L HIF-1α siRNA or negative control siRNA 2 (Silencer; Ambion Inc.) and then incubated in a standard incubator at 5% CO2 and 20% O2 (151 mm Hg) for 24 hours. After this incubation, the culture medium was replaced with fresh RPMI 1640 medium (Invitrogen, Burlington, ON, Canada) and explants were further incubated for 24 hours in either 20% or 0.5% O2, as previously described.Determination of sFlt-1 and sEng Levels in the Culture MediaTo determine the levels of sFlt-1 or sEng in the culture media, kits (DuoSet ELISA Development System kits; R&D Systems) were used according to the manufacturer's instructions.Real-Time PCRTotal RNA was isolated using a kit (High Pure RNA Isolation Kit; Qiagen, Valencia, CA), according to the manufacturer's protocol. Total RNA, 1 μg, was reverse transcribed with a kit (Omniscript RT Kit; Qiagen) using a random hexamer (Cortex, Kingston). Real-time PCR was performed with a system (LightCycler 480 Real-Time PCR System; Roche, Mississauga, ON) using a mix (SYBR Green PCR Master Mix; Roche) and primers, as follows: β-actin, 5′-TGGGACGACATGGAGAAAAT-3′ (sense) and 5′-GAGGCGTACAGGGATAGCAC-3′ (antisense); Flt-1, 5′-GCACCTTGGTTGTGGCTGAC-3′ (sense) and 5′-TGGAATTCGTGCTGCTTCCTGGTCC-3′ (antisense); sFlt-1, 5′-GCACTGCAACAAAAAGGC-3′ (sense) and 5′-CCAGGAATGTATACACAGG-3′ (antisense); Eng, 5′-GCTGGATGAGCCGGGAGCTCCCTGCTG-3′ (sense) and 5′-CACAGGCTGAAGGTCACAATGGACTG-3′ (antisense); and HIF-1α, 5′-TGCTTGGTGCTGATTTGTGA-3′ (sense) and 5′-GGTCAGATGATCAGAGTCCA-3′ (antisense). Real-time reaction conditions for all sets of primers were as follows: 94°C for 5 minutes, followed by 60 cycles at 94°C for 20 seconds, 55°C for 20 seconds, and 72°C for 30 seconds. The intensity of dye (FastStart SYBR Green) was analyzed using software (LightCycler 480; Roche). Transcript levels were normalized against β-actin mRNA levels.Statistical AnalysisFor statistical analysis of enzyme-linked immunosorbent assay and quantitative PCR results, one-way analysis of variance was performed and significant differences between groups were determined using Tukey's multiple comparisons post hoc test. All statistical tests were two sided, and differences were considered significant at P < 0.05.ResultsImmunolocalization of Flt-1 and Eng in Chorionic Villi ExplantsTo determine the potential source of secreted Flt-1 and Eng, immunofluorescence using polyclonal antibodies against Flt-1 and Eng was performed on sections of chorionic villi explants incubated in 20% O2 (see Supplemental Figure S1, A and E, at http://ajp.amjpathol.org). Cytokeratin was used as a marker for trophoblast (see Supplemental Figure S1, B and F, at http://ajp.amjpathol.org). Both Flt-1 and Eng colocalized with cytokeratin primarily to the syncytiotrophoblast layer (see Supplemental Figure S1, D and H, at http://ajp.amjpathol.org), indicating that this tissue is a potential source of the secreted molecules. We also observed that Flt-1, but not Eng, is expressed in the fetal vessels within the villous stroma, indicating that fetal vessels are another potential source of sFlt-1 but not of sEng (see Supplemental Figure S1, D versus H, respectively, at http://ajp.amjpathol.org).Effect of Hypoxia on the Levels of sFlt-1 and Eng mRNA and Secreted ProteinPrevious studies14Soleymanlou N. Jurisica I. Nevo O. Ietta F. Zhang X. Zamudio S. Post M. Caniggia I. Molecular evidence of placental hypoxia in preeclampsia.J Clin Endocrinol Metab. 2005; 90: 4299-4308Crossref PubMed Scopus (294) Google Scholar, 15Yinon Y. Nevo O. Xu J. Many A. Rolfo A. Todros T. Post M. Caniggia I. Severe intrauterine growth restriction pregnancies have increased placental endoglin levels: hypoxic regulation via transforming growth factor-beta 3.Am J Pathol. 2008; 172: 77-85Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Nevo O. Soleymanlou N. Wu Y. Increased expression of sFlt-1 in in vivo and in vitro models of human placental hypoxia is mediated by HIF-1.Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1085Crossref PubMed Scopus (254) Google Scholar, 28Munaut C. Lorquet S. Pequeux C. Blacher S. Berndt S. Frankenne F. Foidart J.-M. Hypoxia is responsible for soluble vascular endothelial growth factor receptor-1 (VEGFR-1) but not for soluble endoglin induction in villous trophoblast.Hum Reprod. 2008; 23: 1407-1415Crossref PubMed Scopus (104) Google Scholar reported hypoxia-induced sFlt-1 and sEng secretion by trophoblast cell lines and first-trimester chorionic villi explants. Given that preeclampsia is mostly a disease of the second half of pregnancy, we examined the effect of hypoxia on the mRNA levels of Flt-1 variants and Eng by term chorionic villi explants. The secreted form of Flt-1 either can be encoded by a spliced variant of the Flt-1 (spliced Flt-1) gene or, less frequently, is the product of the cleaved ectodomain of the Flt-1 receptor (receptor Flt-1).29Kishuku M. Nishioka Y. Abe S. Kishi J. Ogino H. Aono Y. Azuma M. Kinoshita K. Rentsenhand B. Makino H. Ranjan P. Minakuchi K. Sone S. Expression of soluble vascular endothelial growth factor receptor-1 in human monocyte-derived mature dendritic cells contributes to their antiangiogenic property.J Immunol. 2009; 183: 8176-8185Crossref PubMed Scopus (24) Google Scholar, 30Kendall R.L. Thomas K.A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.Proc Natl Acad Sci U S A. 1993; 90: 10705-10709Crossref PubMed Scopus (1177) Google Scholar To determine whether a specific variant of Flt-1 is responsive to hypoxia in term villous explants, we examined both species of Flt-1 mRNA using real-time PCR. Exposure to 0.5% O2 increased the expression of both the spliced Flt-1 and receptor Flt-1 mRNA species compared with exposure to either 20% or 8% O2 (Figure 1A). However, although the expression of the spliced Flt-1 variant increased several thousandfold, the levels of transcript encoding the receptor Flt-1 variant increased by only approximately twofold. Consequently, the ratio of spliced Flt-1/receptor Flt-1 significantly increased in chorionic villi exposed to hypoxia compared with chorionic villi exposed to either 20% or 8% O2 (Figure 1A: P < 0.001 for both). This ratio is an indication of increased bioavailability of the spliced (secreted) Flt-1 variant compared with nonspliced Flt-1 receptor in chorionic villi exposed to hypoxia. Exposure of chorionic villi to hypoxia (0.5% O2) resulted in a fivefold and a threefold increase in Eng mRNA levels compared with exposure to 20% and 8% O2, respectively (Figure 1B: P < 0.001 for both). Compared with exposure to 20% O2, exposure to 8% O2 did not result in statistically significant increases in the levels of sFlt-1 and sEng mRNA.To further characterize the effect of hypoxia on the secretion of sFlt-1 and sEng, we performed an enzyme-linked immunosorbent assay on the media of explant cultures. Explants exposed to 0.5% O2 for 24 hours secreted significantly higher levels of sFlt-1 and sEng than explants incubated in 20% or 8% O2 (Figure 2, A and B). Although the data shown in Figure 2 are expressed as pictograms of sFlt-1 or sEng per microgram of protein in the media, differences in the reported concentrations of these molecules were not the result of differences in the total amount of protein secreted by chorionic villi; the latter was similar across the different treatment groups (see Supplemental Figure S2 at http://ajp.amjpathol.org). Compared with exposure to 20% O2 alone, exposure of chorionic villi to H/R (24 hours in 0.5% O2, followed by 3 hours in 20% O2) did not significantly increase the detectable levels of sFlt-1 and sEng in the culture medium (see Supplemental Figure S3, A and B, at http://ajp.amjpathol.org). Accordingly, compared with exposure to hypoxia for 24 hours, subsequent reoxygenation of chorionic villi explants (3 hours in 20% O2) resulted in a significant decrease in the levels of detectable sFlt-1 and sEng (see Supplemental Figure S4, A and B, at http://ajp.amjpathol.org; P < 0.05 for both).Figure 2Effect of GTN on the secretion and expression of sFlt-1 and sEng by chorionic villi explants exposed to hypoxia. A and B: Relative sFlt-1 and sEng levels, respectively, in the medium of explant cultures (six explants per placenta, with a minimum of three placentas per treatment). Concentrations (in ng/mL) were divided by the total amount of protein (in μg/mL) i

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