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Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells
Placenta 35 (2014) 932e936
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Placenta journal homepage: www.elsevier.com/locate/placenta
Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells* J.C. Ehrig a, D. Horvat a, S.R. Allen a, R.O. Jones a, T.J. Kuehl a, b, c, M.N. Uddin a, b, * a From the Departments of Obstetrics & Gynecology, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX 76508, USA b Pediatrics, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX 76508, USA c Molecular & Cellular Medicine, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX 76508, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 28 July 2014
Objective: Preeclampsia (preE), is characterized by abnormal placental invasion and function. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits cytotrophoblast (CTB) cell functions that are critical for normal placental development. This study tests the hypothesis that CTSs induce antiangiogenic and anti-proliferative effects in CTB cells. Methods: Human extravillous CTB cells of the line Sw-71, derived from first trimester chorionic villus tissue, were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Thereafter, levels of pro-angiogenic (vascular endothelial growth factor (VEGF165), placental growth factor (PlGF)) and anti-angiogenic (soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng)) factors were measured in culture media using ELISA kits. Expression of three receptors (VEGF receptor 1 (VEGFR1), angiogenic angiotensin type 1 receptor (AT1) and anti-angiogenic angiotensin type 2 receptor (AT2)) were assayed using immunoblotting (western blots) in cell lysates. Results: sFlt-1 and sEng secretion were increased while VEGF165 and PIGF were decreased in the culture media of CTB cells treated with 1 nM or more of each CTSs (p < 0.01 for each). The AT2 receptor expression was up-regulated (p < 0.05) in CTB cells treated with 1 nM or more of MBG and CINO and with 100 nM OUB, while AT1 and VEGFR1 expressions decreased (p < 0.05) with 1 nM or more of MBG and 10 nM or more of CINO and OUB. Conclusions: CTSs influence extravillous CTB cells to induce an anti-angiogenic and anti-proliferative profile. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Cardiac steroids Cytotrophoblast cells Angiogenesis Cell proliferation
1. Introduction Preeclampsia (preE) is a disorder of pregnancy characterized by de novo development of hypertension and proteinuria after 20 weeks gestation. This disorder affects about 1 in 20 pregnancies and is a leading cause of maternal and fetal morbidity and mortality [1e5]. Potential multi-system impacts and sequelae of preE include eclampsia, pulmonary edema, thrombocytopenia and fetal growth restriction [6e9]. Early diagnosis and treatment are not yet available and strategies to prevent the symptom cascade have only
* Presented at the 33rd annual meeting of the Society of Maternal-Fetal Medicine, Hilton San Francisco Union Square, San Francisco, CA, Feb. 11e16, 2013. * Corresponding author. Scott & White Hospital (MS-01-E316A), 2401 South 31st Street, Temple, TX 76508, USA. Tel.: þ1 254 724 3624; fax: þ1 254 724 1046. E-mail address: [email protected] (M.N. Uddin).
http://dx.doi.org/10.1016/j.placenta.2014.07.014 0143-4004/© 2014 Elsevier Ltd. All rights reserved.
minimal success. The exact mechanism(s) leading to preE continues to elude investigators, but several theories have been proposed. One theory invokes a role for bufadienolides, a subgroup of cardiotonic steroids (CTSs). The most studied of this group, marinobufagenin (MBG), is elevated in both animal models of volume expansion and patients with preE [2,10e12]. Evaluation of MBG serum levels prior to the onset of hypertension and proteinuria in women implicates MBG in the pathogenesis of preE [1,2,4,11,12]. MBG also has been shown to interfere with proliferation, migration and invasion of CTB cells [13e15], and specifically to disrupt endothelial cell junctional complexes [9,16]. MBG is increased in a rat model of preE and pregnant rats treated with MBG develop preE symptoms [17]. Angiotensin II (Ang II), a bioactive effector component of the renin-angiotensin system (RAS), influences cell growth, cell contraction, hypertrophy, inflammation, fibrosis, and progression to cellular damage in certain cell types [18,19]. The biological
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actions of Ang II are mediated via two G-protein coupled receptors, Angiotensin receptor type 1 (AT1) and Angiotensin receptor type 2 (AT2) [20]. Changes in AT1 and AT2 expression, in the presence of Ang II, can result in vasoconstriction, cell proliferation, and tissue-
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remodeling through stimulation of AT1 [21] or vasodilatation, cell growth inhibition and activation of apoptosis through AT2 [22] depending on the balance between expression of these two receptors. Angiotensin (1e7) (Ang (1e7)) is a heptapeptide hormone
Fig. 1. A and B. Graph presents means with SE of three experiments for the secretion of 2 pro-angiogenic factors (VEGF165 and PIGF) (A) and 2 anti-angiogenic factors (sFlt-1 and sEng) (B) by CTB cells treated with 5 doses (0, 0.1 nM, 1 nM, 10 nM, and 100 nM) of 3 CTSs (MBG, CINO, and OUB) for 48 h. Comparisons with basal (0 nM) doses were performed using repeated measures analysis of variance with Dunnett's post-hoc test. All of 3 CTSs at levels of 1 nM or greater decreased the secretion of VEGF165 and PIGF and increased the secretion of sFlt-1 by CTB cells. CINO and OUB at levels of 0.1 nM altered the secretion of sEng. The sEng dose response curves varied between the 3 CTSs such that MBG differed (p < 0.01) from those of CINO and OUB, while CINO and OUB are similar (p ¼ 0.88). For the other 3 secreted factors (VEGF165, PIGF, and sFlt-1), all 3 CTSs (MBG, CINO and OUB) produce similar response curves (p > 0.4).
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generated by the cleavage of Ang I by prolyl endopeptidase and neutral endopeptidase. Ang-(1e7) is also generated by cleavage of Ang II by several enzymes, in particular the angiotensin-converting enzyme homolog (ACE2) [23,24]. Ang 1e7, acting through the G protein-coupled receptor Mas (MasR), counteracts many of the actions of Ang II inducing vasodilatation and inhibiting cell growth [25]. Action of CTSs on Ang II receptors has not been described in CTB cells. CTSs are secreted by the adrenal gland in humans and act by inhibiting Na/K-ATPase in target cells [26]. Angiogenic balance is created by the inter-play of antiangiogenic factors, including soluble fms-like tyrosine kinase (sFlt-1) and soluble endoglin (sEng), and pro-angiogenic factors, including vascular endothelial growth factor (VEGF165) and placental growth factor (PlGF) [7,12,27e32]. Angiogenic imbalance during pregnancy may contribute to preE [33]. The action of CTSs on the angiogenic balance in CTB cells is unknown. We thus designed this study to examine the effects of three CTSs on the angiogenic milieu produced by first trimester CTBs in vitro and the potential for RAS involvement based on alterations in receptor expression of Ang II. 2. Methods 2.1. Cell culture The human extravillous CTB cell line Sw-71 utilized in these studies was derived from first trimester chorionic villous tissue and was kindly provided by Dr. Gil G. Mor at Yale University School of Medicine, New Haven, CT, USA. These cells are well characterized and share many characteristics with isolated primary cells, including the expression of cytokeratin-7, HLA class I antigen, HLA-G, BC-1, CD9, human chorionic gonadotropin, and human placental lactogen. Sw-71 CTB cells were cultured in Ham's F-10 nutrient mix (Invitrogen) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/ml), streptomycin (100 mg/ml), and 2 mM L-glutamine (Sigma Chemical, St Louis, MO). Cells were incubated at 37 C, 5% CO2, 10% O2 tension in humidified air.
2.2. Treatments The extravillous CTB cells were serum-starved in medium containing 0.5% FBS for 24 h, washed twice with 1 phosphate buffered saline (PBS) and replicates were subsequently treated with 10% FBS/Ham F10 containing DMSO (vehicle) or 0.1, 1, 10 or 100 nM of each of three types of CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. The cell culture media were collected for the analysis of pro-angiogenic and anti-angiogenic factors. The cell lysates were utilized for Western Blot analyses. The anti-proliferative and anti-angiogenic capacity of MBG, CINO and OUB on the cells were not due to a cytotoxic effect of the steroids, as evaluated by a cell viability assay where fewer than 3% were non-viable in all conditions. The cell viability was measured using a CellTiter Assay (Promega) as described previously [4,13]. Measurement of pro-angiogenic and anti-angiogenic factors: The levels of proangiogenic factors, VEGF165 and PlGF, and anti-angiogenic factors, sFlt-1 and sEng, were measured by commercially available kits (Human VEGF Quantikine ELISA Kit (DVE00); Human PlGF Quantikine ELISA Kit (DPG00); Human sVEGF R1/Flt-1 Quantikine ELISA Kit (DVR100B); Human Endoglin/CD105 Quantikine ELISA Kit (DNDG00); R&D Systems, Minneapolis, MN) according to the instruction provided by the manufacturer as described previously [22]. Western Blots of VEGFR1, angiogenic angiotensin type 1 receptor (AT1) and antiangiogenic angiotensin type 2 receptor (AT2): The expression of three receptors in the CTB cells was measured by gel electrophoresis of the CTS-treated cell lysates followed by detection with immunoblotting (Western blotting) using antiVEGFR1(Polyclonal rabbit antibody specific for soluble human VEGFR1 which recognizes the expressed product of FLT1 gene, Catalog number 361100, Life Science), anti-AT1 (AT1 (1E10-1A9) mouse monoclonal, Catalog number sc-81671, Santa Cruz) or anti-AT2 antibodies (ab19134 rabbit polyclonal, Abcam) [17,34]. While these antibodies are reported by the commercial sources to be validated for western blots, independent specificity has not been reported. Proteins were visualized with chemiluminescence detection system. The intensity of the bands was determined by scanning video densitometry using the phospho-imager, Storm 860 with ImageQuant TL software version 2003.2 (GE Healthcare, Little, Chalfont, Buckinghamshire, UK). Antibodies to beta-actin (Santa Cruz) were used to calculate the ratio of receptors to beta-actin as a control for loading variation. 2.3. Statistical method Data are presented as mean ± SE. Data from CTSs-treated replicates were compared to basal (DMSO)-treated replicates in within each experimental trial using analysis of variance for repeated measures with Dunnett's post-hoc test (Statistica,
Fig. 2. Graph presents means with SE of five experiments for the expression of AT1, AT2, and VEGFR1 receptors relative to beta-actin in the cell lysates by Western Blot following treatment with CTS. Comparisons with basal (0 nM) doses were performed using repeated measures analysis of variance with Dunnett's post-hoc test for differences with p < 0.05 shown as filled symbols. All three CTSs downregulated the expression of AT1 and VEGFR1 receptors and upregulated expression of AT2 receptors in CTB cells compared to basal (0 nM) levels. The data are presented as means with SE for five experiments. The CTS dose responses were similar for the three CTS effects on AT1 (p ¼ 0.33) and VEGFR1 (p ¼ 0.25), but differed (p ¼ 0.03) for AT2.
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StatSoft, Tulsa, OK). P < 0.05 was considered significant. Levene's test for homogeneity of variance was used to verify that variance terms were homogeneous and appropriate for ANOVA analyses.
3. Results Extravillous CTB cells were treated with 5 doses (0, 0.1 nM, 1 nM, 10 nM, and 100 nM) of 3 CTSs (MBG, CINO, and OUB) for 48 h and the concentrations of 2 pro-angiogenic factors (VEGF165 and PIGF) and 2 anti-angiogenic factors (sFlt-1 and sEng) were measured in the cell culture media by ELISA (Fig. 1). Comparisons with basal (0 nM) doses were performed and all three CTSs at levels of 1 nM or greater demonstrated decreases (p < 0.05) in the secretion of VEGF165 and PIGF. The secretion of sFlt-1 by CTB cells was increased (p < 0.05) by exposure to all three CTSs at levels of 1 nM or greater. All 3 CTSs at levels of 0.1 nM altered (p < 0.05) the secretion of sEng. The sEng dose response curves varied between the 3 CTSs such that MBG. For the other 3 secreted factors (VEGF165, PIGF, and sFlt-1), all 3 CTSs (MBG, CINO and OUB) produced similar response curves (p > 0.4). The receptor expressions of AT1, AT2, and VEGFR1 in response to 4 doses of three CTSs are shown in Fig. 2. AT1, AT2, and VEGFR1 receptor expressions were measured relative to beta-actin in the cell lysates by Western Blot. All three CTSs significantly (p < 0.05) downregulated the expression of AT1 and VEGFR1 receptors in CTB cells compared to basal (0 nM) levels. However, AT2 receptor levels were increased (p < 0.05) by the CTSs. The CTS dose response curves were similar for the three CTS effects on AT1 (p ¼ 0.33) and VEGFR1 (p ¼ 0.25). The dose response curves to the three CTSs differed (p ¼ 0.03) for AT2. A representative blot of each of three receptors has been shown in Fig. 3 (A. AT1; B. AT2; and C. VEGFR1) and b-actin in CTBs that were treated with MBG, CINO, and OUB at 5 concentrations from 0 nM to 100 nM. 4. Discussion These data demonstrate that CTSs altered the angiogenic balance of a cell line of extravillous CTBs cultured in vitro. Exposure to any of the three CTSs result in decreased expression of the proangiogenic factors (VEGF165, PIGF) and receptors (VEGFR1, AT1) while upregulating expression of the anti-angiogenic factors (sFlt-1, sEng) and receptor (AT2). The dose response curves show the greatest increase in sFlt-1 and sEng expression between 01 nM and 1 nM for all three CTS. This is the same interval that the largest decrease in VEGF165 and PlGF secretion are seen. One explanation for the lack of a dose dependent effect on any of the factors at concentrations greater than 1 nM is receptor saturation. The overall result is to favor an anti-angiogenic environment at the interface between fetal placentation and maternal uterine vasculature. The shift to predominantly AT2 expression also may inhibit CTB growth and activate apoptosis. If these responses are triggered when cytotrophoblasts are the predominant placental cell type, placental volume and future perfusion capacity may be restricted. Such restriction of perfusion capacity may set the stage for future onset of the preE syndrome as the placenta releases agents into the maternal circulation to enhance profusion later in pregnancy. These same pathways may be involved in the progression to preE in response to CTS elevations in maternal circulation later in pregnancy. For example, CTS concentrations in human urine and plasma increase during pregnancy. Non-pregnant plasma levels of MBG and a OUB-like compound (OLC) in healthy humans rise from 0.190 ± 0.04 nmol/l and 0.297 ± 0.037 nmol/l, respectively, to 0.625 ± 0.067 nmol/l (MBG) and 0.32 ± 0.07 nmol/l (OLC). In patients with preE, plasma levels of both MBG and OLC increase further to 2.63 ± 0.10 nmol/l and 0.697 ± 0.16 nmol/l, respectively [11,28].
Fig. 3. Representative blots of each of three receptors (AT1 in panel A, AT2 in panel B, and VEGFR1 in panel C) and beta-actin in CTBs that were treated with the three CTSs (MBG, CINO, and OUB) at 5 concentrations from 0 nM to 100 nM.
Urinary MBG also is higher in women with preE (2341.8 ± 244.9 pg/ mg creatinine) than in normal pregnant patients (584.1 ± 34.3 pg/mg creatinine). The concentration of CTSs at which the anti-angiogenic impact occurs in vitro in cultured CTB cells, 1 nM, occurs at a concentration of MBG seen in the circulation of women with preE. CTSs are secreted by the adrenal gland and inhibit Na/K ATPase [26]. CTS action through the Na/K ATPase pathway raises the
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question of potential for treatment strategies. In a DOCA/saline preE rat model, resibufogenin (RBG), a CTS antagonist of MBG, normalizes Na/K-ATPase activity, reverses vasoconstriction, and lowers blood pressure and proteinuria [35]. MBG to cause natriuresis both by the inhibition of Naþ/K þ ATPase activity and by its ability to hinder mineralocorticoid receptor-dependent expression of the Na/K/H exchanger in the late distal nephron [36]. It is not known whether RBG can only act as an antagonist to MBG, or whether it can act independently. It is well established that an anti-angiogenic milieu exists in women with preE [7,8,12,13,27e33,35e38]. This study shows that CTSs, which circulate in higher concentrations during pregnancy, can induce such changes in extravillous cytotrophoblast cell protein expression, including inhibited production of VEGF165, PlGF, VEGFR1, AT1 and increased production of sFlt-1, sEng, AT2. With further study and confirmation of our observations, CTSs may be a target for preE screening and therapeutics.
Conflict of interest The authors have no conflicts of interest to disclose. Financial support Funding for this work was provided by Scott, Sherwood and Brindley Foundation and Department of Obstetrics and Gynecology (MNU) and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (TJK), Baylor Scott & White Health, Temple, Texas.
Condensation CTSs influenced CTB cells to induce an anti-angiogenic and antiproliferative profile. Acknowledgment Funding for this work was provided by Scott, Sherwood and Brindley Foundation and Department of Obstetrics and Gynecology (MNU) and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (TJK), Scott & White Healthcare, Temple, Texas. The CTB cell line Sw-71 was kindly provided by Dr. Gil G. Mor at Yale University School of Medicine, New Haven, CT, USA. MBG was a kind gift from Drs. Alexei Y. Bagrov, Edward G. Lakatta, and Olga V. Fedorova at the National Institute on Aging (NIA), Baltimore, Maryland. References [1] Leung DN, Smith SC, To KF, Sahota DS, Baker PN. Increased placental apoptosis in pregnancies complicated by preeclampsia. Am J Obstet Gynecol 2001;184: 1249e50. [2] Pridjian G, Puschett JB. Preeclampsia, part II: experimental and genetic considerations. Obstet Gynecol Surv 2002;57:619e40. [3] Puschett JB, Agunanne E, Uddin MN. Emerging role of the bufadienolides in cardiovascular and kidney disease. Am J Kidney Dis 2010;56:359e70. [4] Uddin MN, Allen S, Jones R, Zawieja D, Kuehl T. Pathogenesis of preeclampsia: marinobufagenin and angiogenic imbalance as biomarkers of the syndrome. Transl Res 2012;160:99e113. [5] Vu H, Ianosi Irimie M, Pridjian CA, Whitbred JM, Durst JM, Bagrov AY, et al. Involvement of marinobufagenin in a rat model of human preeclampsia. Am J Nephrol 2005;25:520e8. [6] Campbell DM, Campbell AJ. Evans blue disappearance rate in normal and preeclamptic pregnancy. Clin Exp Hypertens B 1983;2:163e9. [7] Maynard SE, Min J, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1(sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649e58.
[8] Sharkey AM, Cooper JC, Balmforth JR, McLaren J, Clark DE, Charnock-Jones DS, et al. Maternal plasma levels of vascular endothelial growth factor in normotensive pregnancies and in pregnancies complicated by pre-eclampsia. Eur J Clin Invest 1996;26:1182e5. [9] Uddin MN, McLean LB, Hunter FA, Horvat D, Severson J, Tharakan B, et al. Vascular leak in a rat model of preeclampsia. Am J Nephrol 2009;30:26e33. [10] Cunningham FG, Leveno KJ, Bloom SL, Hauth JC, Rouse DJ, Spong CY. Williams obstetrics. Chapter 3 implantation, embryogenesis and placentation. 23rd ed. The McGraw-Hill Companies; 2010. [11] Gonick HC, Ding Y, Vaziri ND, Bagrov AY, Fedorova OV. Simultaneous measurement of marinobufagenin, ouabain, and hypertension-associated protein in various disease states. Clin Exp Hypertens 1998;20:617e27. [12] Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, et al. Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat Med 2006;12: 642e9. [13] Uddin MN, Horvat D, Glaser SS, Danchuk S, Mitchell BM, Sullivan DE, et al. Marinobufagenin inhibits proliferation and migration of cytotrophoblast and CHO cells. Placenta 2008;29:266e73. [14] Uddin MN, Horvat D, Glaser SS, Mitchell BM, Puschett JB. Examination of the cellular mechanisms by which marinobufagenin inhibits cytotrophoblast function. J Biol Chem 2008;283:17946e53. [15] LaMarca HL, Morris CA, Pettit GR, Nagowa T, Puschett JB. Placenta 2006;27: 984e8. [16] Uddin MN, Horvat D, Childs EW, Puschett JB. Marinobufagenin causes endothelial cell monolayer hyperpermeability by altering apoptotic signaling. Am J Physiol Regul Integr Comp Physiol 2009;296:1726e34. [17] Uddin MN, Agunanne E, Horvat D, Puschett JB. Alterations in the reninangiotensin system in a rat model of human preeclampsia. Am J Nephrol 2010;31:171e7. [18] Ino K, Uehara C, Kikkawa F, Shibata K, Suzuki T, Khin EE, et al. Enhancement of aminopeptidase A expression during angiotensin II-induced choriocarcinoma cell proliferation through AT1 receptor involving protein kinase C- and mitogen-activated protein kinase-dependent signaling pathway. J Clin Endocrinol Metab 2003;88:3973e82. [19] Ruperez M, Lorenzo O, Blanco-Colio LM, Esteban V, Egido J, Ruiz-Ortega M. Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis. Circulation 2003;108:1499e505. [20] Norwitz ER, Schust DJ, Fisher SJ. Implantation and the survival of early pregnancy. N Engl J Med 2001;345:1400e8. [21] Mezzano SA, Ruiz-Ortega M, Egido J. Angiotensin II and renal fibrosis. Hypertension 2001;38:635e8. [22] Sadoshima J. Cytokine actions of angiotensin II. Circ Res 2000;86:1187e9. [23] Zisman LS, Meixell GE, Bristow MR, Canver CC. Angiotensin (1-7) formation in the intact human heart: in vivo dependence on angiotensin II as substrate. Circulation 2003;108:679e81. [24] Li N, Zimpelmann J, Cheng K, Wilkins JA, Burns KD. The role of angiotensin converting enzyme 2 in the generation of angiotensin 1e7 by rat proximal tubules. Am J Physiol Renal Physiol 2005;288:353e62. [25] Iwai M, Horiuchi M. Devil and angel in the renin-angiotensin system: ACEangiotensin II-AT1 receptor axis vs. ACE2-angiotensin-(1-7)-Mas receptor axis. Hypertens Res 2009;32:533e6. [26] Fedorova OV, Simbirtsev AS, Kolodkin NI, Kotov AY, Agalakova NI, Kashkin VA, et al. Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension. J Hypertens 2008;26:2414e25. [27] Agarwal I, Karumanchi SA. Preeclampsia and the anti-angiogenic state. Pregnancy Hypertens 2011;1:17e21. [28] Berg CJ, Atrash HK, Koonin LM, Tucker M. Pregnancy-related mortality in the United States, 1987-1990. Obstet Gynecol 1996;88:161e7. [29] Levine R, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med 2006;355:992e1005. [30] Levine R, Maynard S, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672e83. [31] Livingston JC, Chin R, Haddad B, McKinney ET, Ahokas R, Sibai BM. Reductions of vascular endothelial growth factor and placental growth factor concentrations in severe preeclampsia. Am J Obstet Gynecol 2000;183:1554e7. [32] Maynard S, Karumanchi S. Angiogenic factors and preeclampsia. Semin Nephrol 2001;31:33e46. [33] Ahmed A. Heparin-binding angiogenic growth factors in pregnancy. Trophobl Res 1997;10:215e58. [34] Goyal R, Wong C, Van Wickle J, Longo LD. Antenatal maternal protein deprivation: sexually dimorphic programming of the pancreatic renin-angiotensin system. J Renin Angiotensin Aldosterone Syst 2013;14:137e45. [35] Horvat D, Severson J, Uddin MN, Mitchell B, Puschett JB. Resibufogenin prevents the manifestations of pre-eclampsia in an animal model of the syndrome. Hypertens Pregnancy 2010;29:1e9. [36] Smith CL, He Q, Huang L, Foster E, Puschett JB. Marinobufagenin interferes with the function of the mineralocorticoid receptor. Biochem Biophys Res Commun 2007;356:930e4. [37] Lopatin DA, Ailamazian EK, Dmitrieva RI, Shpen VM, Fedorova OV, Doris PA, et al. Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia. J Hypertens 1999;17:1179e87. [38] Levine RJ, Thadhani R, Qian C, Lam C, Lim KH, Yu KF, et al. Urinary placental growth factor and the risk of preeclampsia. J Am Med Assoc 2005;293:77e85.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells* J.C. Ehrig a, D. Horvat a, S.R. Allen a, R.O. Jones a, T.J. Kuehl a, b, c, M.N. Uddin a, b, * a From the Departments of Obstetrics & Gynecology, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX 76508, USA b Pediatrics, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX 76508, USA c Molecular & Cellular Medicine, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, TX 76508, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 28 July 2014
Objective: Preeclampsia (preE), is characterized by abnormal placental invasion and function. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits cytotrophoblast (CTB) cell functions that are critical for normal placental development. This study tests the hypothesis that CTSs induce antiangiogenic and anti-proliferative effects in CTB cells. Methods: Human extravillous CTB cells of the line Sw-71, derived from first trimester chorionic villus tissue, were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Thereafter, levels of pro-angiogenic (vascular endothelial growth factor (VEGF165), placental growth factor (PlGF)) and anti-angiogenic (soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng)) factors were measured in culture media using ELISA kits. Expression of three receptors (VEGF receptor 1 (VEGFR1), angiogenic angiotensin type 1 receptor (AT1) and anti-angiogenic angiotensin type 2 receptor (AT2)) were assayed using immunoblotting (western blots) in cell lysates. Results: sFlt-1 and sEng secretion were increased while VEGF165 and PIGF were decreased in the culture media of CTB cells treated with 1 nM or more of each CTSs (p < 0.01 for each). The AT2 receptor expression was up-regulated (p < 0.05) in CTB cells treated with 1 nM or more of MBG and CINO and with 100 nM OUB, while AT1 and VEGFR1 expressions decreased (p < 0.05) with 1 nM or more of MBG and 10 nM or more of CINO and OUB. Conclusions: CTSs influence extravillous CTB cells to induce an anti-angiogenic and anti-proliferative profile. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Cardiac steroids Cytotrophoblast cells Angiogenesis Cell proliferation
1. Introduction Preeclampsia (preE) is a disorder of pregnancy characterized by de novo development of hypertension and proteinuria after 20 weeks gestation. This disorder affects about 1 in 20 pregnancies and is a leading cause of maternal and fetal morbidity and mortality [1e5]. Potential multi-system impacts and sequelae of preE include eclampsia, pulmonary edema, thrombocytopenia and fetal growth restriction [6e9]. Early diagnosis and treatment are not yet available and strategies to prevent the symptom cascade have only
* Presented at the 33rd annual meeting of the Society of Maternal-Fetal Medicine, Hilton San Francisco Union Square, San Francisco, CA, Feb. 11e16, 2013. * Corresponding author. Scott & White Hospital (MS-01-E316A), 2401 South 31st Street, Temple, TX 76508, USA. Tel.: þ1 254 724 3624; fax: þ1 254 724 1046. E-mail address: [email protected] (M.N. Uddin).
http://dx.doi.org/10.1016/j.placenta.2014.07.014 0143-4004/© 2014 Elsevier Ltd. All rights reserved.
minimal success. The exact mechanism(s) leading to preE continues to elude investigators, but several theories have been proposed. One theory invokes a role for bufadienolides, a subgroup of cardiotonic steroids (CTSs). The most studied of this group, marinobufagenin (MBG), is elevated in both animal models of volume expansion and patients with preE [2,10e12]. Evaluation of MBG serum levels prior to the onset of hypertension and proteinuria in women implicates MBG in the pathogenesis of preE [1,2,4,11,12]. MBG also has been shown to interfere with proliferation, migration and invasion of CTB cells [13e15], and specifically to disrupt endothelial cell junctional complexes [9,16]. MBG is increased in a rat model of preE and pregnant rats treated with MBG develop preE symptoms [17]. Angiotensin II (Ang II), a bioactive effector component of the renin-angiotensin system (RAS), influences cell growth, cell contraction, hypertrophy, inflammation, fibrosis, and progression to cellular damage in certain cell types [18,19]. The biological
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actions of Ang II are mediated via two G-protein coupled receptors, Angiotensin receptor type 1 (AT1) and Angiotensin receptor type 2 (AT2) [20]. Changes in AT1 and AT2 expression, in the presence of Ang II, can result in vasoconstriction, cell proliferation, and tissue-
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remodeling through stimulation of AT1 [21] or vasodilatation, cell growth inhibition and activation of apoptosis through AT2 [22] depending on the balance between expression of these two receptors. Angiotensin (1e7) (Ang (1e7)) is a heptapeptide hormone
Fig. 1. A and B. Graph presents means with SE of three experiments for the secretion of 2 pro-angiogenic factors (VEGF165 and PIGF) (A) and 2 anti-angiogenic factors (sFlt-1 and sEng) (B) by CTB cells treated with 5 doses (0, 0.1 nM, 1 nM, 10 nM, and 100 nM) of 3 CTSs (MBG, CINO, and OUB) for 48 h. Comparisons with basal (0 nM) doses were performed using repeated measures analysis of variance with Dunnett's post-hoc test. All of 3 CTSs at levels of 1 nM or greater decreased the secretion of VEGF165 and PIGF and increased the secretion of sFlt-1 by CTB cells. CINO and OUB at levels of 0.1 nM altered the secretion of sEng. The sEng dose response curves varied between the 3 CTSs such that MBG differed (p < 0.01) from those of CINO and OUB, while CINO and OUB are similar (p ¼ 0.88). For the other 3 secreted factors (VEGF165, PIGF, and sFlt-1), all 3 CTSs (MBG, CINO and OUB) produce similar response curves (p > 0.4).
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generated by the cleavage of Ang I by prolyl endopeptidase and neutral endopeptidase. Ang-(1e7) is also generated by cleavage of Ang II by several enzymes, in particular the angiotensin-converting enzyme homolog (ACE2) [23,24]. Ang 1e7, acting through the G protein-coupled receptor Mas (MasR), counteracts many of the actions of Ang II inducing vasodilatation and inhibiting cell growth [25]. Action of CTSs on Ang II receptors has not been described in CTB cells. CTSs are secreted by the adrenal gland in humans and act by inhibiting Na/K-ATPase in target cells [26]. Angiogenic balance is created by the inter-play of antiangiogenic factors, including soluble fms-like tyrosine kinase (sFlt-1) and soluble endoglin (sEng), and pro-angiogenic factors, including vascular endothelial growth factor (VEGF165) and placental growth factor (PlGF) [7,12,27e32]. Angiogenic imbalance during pregnancy may contribute to preE [33]. The action of CTSs on the angiogenic balance in CTB cells is unknown. We thus designed this study to examine the effects of three CTSs on the angiogenic milieu produced by first trimester CTBs in vitro and the potential for RAS involvement based on alterations in receptor expression of Ang II. 2. Methods 2.1. Cell culture The human extravillous CTB cell line Sw-71 utilized in these studies was derived from first trimester chorionic villous tissue and was kindly provided by Dr. Gil G. Mor at Yale University School of Medicine, New Haven, CT, USA. These cells are well characterized and share many characteristics with isolated primary cells, including the expression of cytokeratin-7, HLA class I antigen, HLA-G, BC-1, CD9, human chorionic gonadotropin, and human placental lactogen. Sw-71 CTB cells were cultured in Ham's F-10 nutrient mix (Invitrogen) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/ml), streptomycin (100 mg/ml), and 2 mM L-glutamine (Sigma Chemical, St Louis, MO). Cells were incubated at 37 C, 5% CO2, 10% O2 tension in humidified air.
2.2. Treatments The extravillous CTB cells were serum-starved in medium containing 0.5% FBS for 24 h, washed twice with 1 phosphate buffered saline (PBS) and replicates were subsequently treated with 10% FBS/Ham F10 containing DMSO (vehicle) or 0.1, 1, 10 or 100 nM of each of three types of CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. The cell culture media were collected for the analysis of pro-angiogenic and anti-angiogenic factors. The cell lysates were utilized for Western Blot analyses. The anti-proliferative and anti-angiogenic capacity of MBG, CINO and OUB on the cells were not due to a cytotoxic effect of the steroids, as evaluated by a cell viability assay where fewer than 3% were non-viable in all conditions. The cell viability was measured using a CellTiter Assay (Promega) as described previously [4,13]. Measurement of pro-angiogenic and anti-angiogenic factors: The levels of proangiogenic factors, VEGF165 and PlGF, and anti-angiogenic factors, sFlt-1 and sEng, were measured by commercially available kits (Human VEGF Quantikine ELISA Kit (DVE00); Human PlGF Quantikine ELISA Kit (DPG00); Human sVEGF R1/Flt-1 Quantikine ELISA Kit (DVR100B); Human Endoglin/CD105 Quantikine ELISA Kit (DNDG00); R&D Systems, Minneapolis, MN) according to the instruction provided by the manufacturer as described previously [22]. Western Blots of VEGFR1, angiogenic angiotensin type 1 receptor (AT1) and antiangiogenic angiotensin type 2 receptor (AT2): The expression of three receptors in the CTB cells was measured by gel electrophoresis of the CTS-treated cell lysates followed by detection with immunoblotting (Western blotting) using antiVEGFR1(Polyclonal rabbit antibody specific for soluble human VEGFR1 which recognizes the expressed product of FLT1 gene, Catalog number 361100, Life Science), anti-AT1 (AT1 (1E10-1A9) mouse monoclonal, Catalog number sc-81671, Santa Cruz) or anti-AT2 antibodies (ab19134 rabbit polyclonal, Abcam) [17,34]. While these antibodies are reported by the commercial sources to be validated for western blots, independent specificity has not been reported. Proteins were visualized with chemiluminescence detection system. The intensity of the bands was determined by scanning video densitometry using the phospho-imager, Storm 860 with ImageQuant TL software version 2003.2 (GE Healthcare, Little, Chalfont, Buckinghamshire, UK). Antibodies to beta-actin (Santa Cruz) were used to calculate the ratio of receptors to beta-actin as a control for loading variation. 2.3. Statistical method Data are presented as mean ± SE. Data from CTSs-treated replicates were compared to basal (DMSO)-treated replicates in within each experimental trial using analysis of variance for repeated measures with Dunnett's post-hoc test (Statistica,
Fig. 2. Graph presents means with SE of five experiments for the expression of AT1, AT2, and VEGFR1 receptors relative to beta-actin in the cell lysates by Western Blot following treatment with CTS. Comparisons with basal (0 nM) doses were performed using repeated measures analysis of variance with Dunnett's post-hoc test for differences with p < 0.05 shown as filled symbols. All three CTSs downregulated the expression of AT1 and VEGFR1 receptors and upregulated expression of AT2 receptors in CTB cells compared to basal (0 nM) levels. The data are presented as means with SE for five experiments. The CTS dose responses were similar for the three CTS effects on AT1 (p ¼ 0.33) and VEGFR1 (p ¼ 0.25), but differed (p ¼ 0.03) for AT2.
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StatSoft, Tulsa, OK). P < 0.05 was considered significant. Levene's test for homogeneity of variance was used to verify that variance terms were homogeneous and appropriate for ANOVA analyses.
3. Results Extravillous CTB cells were treated with 5 doses (0, 0.1 nM, 1 nM, 10 nM, and 100 nM) of 3 CTSs (MBG, CINO, and OUB) for 48 h and the concentrations of 2 pro-angiogenic factors (VEGF165 and PIGF) and 2 anti-angiogenic factors (sFlt-1 and sEng) were measured in the cell culture media by ELISA (Fig. 1). Comparisons with basal (0 nM) doses were performed and all three CTSs at levels of 1 nM or greater demonstrated decreases (p < 0.05) in the secretion of VEGF165 and PIGF. The secretion of sFlt-1 by CTB cells was increased (p < 0.05) by exposure to all three CTSs at levels of 1 nM or greater. All 3 CTSs at levels of 0.1 nM altered (p < 0.05) the secretion of sEng. The sEng dose response curves varied between the 3 CTSs such that MBG. For the other 3 secreted factors (VEGF165, PIGF, and sFlt-1), all 3 CTSs (MBG, CINO and OUB) produced similar response curves (p > 0.4). The receptor expressions of AT1, AT2, and VEGFR1 in response to 4 doses of three CTSs are shown in Fig. 2. AT1, AT2, and VEGFR1 receptor expressions were measured relative to beta-actin in the cell lysates by Western Blot. All three CTSs significantly (p < 0.05) downregulated the expression of AT1 and VEGFR1 receptors in CTB cells compared to basal (0 nM) levels. However, AT2 receptor levels were increased (p < 0.05) by the CTSs. The CTS dose response curves were similar for the three CTS effects on AT1 (p ¼ 0.33) and VEGFR1 (p ¼ 0.25). The dose response curves to the three CTSs differed (p ¼ 0.03) for AT2. A representative blot of each of three receptors has been shown in Fig. 3 (A. AT1; B. AT2; and C. VEGFR1) and b-actin in CTBs that were treated with MBG, CINO, and OUB at 5 concentrations from 0 nM to 100 nM. 4. Discussion These data demonstrate that CTSs altered the angiogenic balance of a cell line of extravillous CTBs cultured in vitro. Exposure to any of the three CTSs result in decreased expression of the proangiogenic factors (VEGF165, PIGF) and receptors (VEGFR1, AT1) while upregulating expression of the anti-angiogenic factors (sFlt-1, sEng) and receptor (AT2). The dose response curves show the greatest increase in sFlt-1 and sEng expression between 01 nM and 1 nM for all three CTS. This is the same interval that the largest decrease in VEGF165 and PlGF secretion are seen. One explanation for the lack of a dose dependent effect on any of the factors at concentrations greater than 1 nM is receptor saturation. The overall result is to favor an anti-angiogenic environment at the interface between fetal placentation and maternal uterine vasculature. The shift to predominantly AT2 expression also may inhibit CTB growth and activate apoptosis. If these responses are triggered when cytotrophoblasts are the predominant placental cell type, placental volume and future perfusion capacity may be restricted. Such restriction of perfusion capacity may set the stage for future onset of the preE syndrome as the placenta releases agents into the maternal circulation to enhance profusion later in pregnancy. These same pathways may be involved in the progression to preE in response to CTS elevations in maternal circulation later in pregnancy. For example, CTS concentrations in human urine and plasma increase during pregnancy. Non-pregnant plasma levels of MBG and a OUB-like compound (OLC) in healthy humans rise from 0.190 ± 0.04 nmol/l and 0.297 ± 0.037 nmol/l, respectively, to 0.625 ± 0.067 nmol/l (MBG) and 0.32 ± 0.07 nmol/l (OLC). In patients with preE, plasma levels of both MBG and OLC increase further to 2.63 ± 0.10 nmol/l and 0.697 ± 0.16 nmol/l, respectively [11,28].
Fig. 3. Representative blots of each of three receptors (AT1 in panel A, AT2 in panel B, and VEGFR1 in panel C) and beta-actin in CTBs that were treated with the three CTSs (MBG, CINO, and OUB) at 5 concentrations from 0 nM to 100 nM.
Urinary MBG also is higher in women with preE (2341.8 ± 244.9 pg/ mg creatinine) than in normal pregnant patients (584.1 ± 34.3 pg/mg creatinine). The concentration of CTSs at which the anti-angiogenic impact occurs in vitro in cultured CTB cells, 1 nM, occurs at a concentration of MBG seen in the circulation of women with preE. CTSs are secreted by the adrenal gland and inhibit Na/K ATPase [26]. CTS action through the Na/K ATPase pathway raises the
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question of potential for treatment strategies. In a DOCA/saline preE rat model, resibufogenin (RBG), a CTS antagonist of MBG, normalizes Na/K-ATPase activity, reverses vasoconstriction, and lowers blood pressure and proteinuria [35]. MBG to cause natriuresis both by the inhibition of Naþ/K þ ATPase activity and by its ability to hinder mineralocorticoid receptor-dependent expression of the Na/K/H exchanger in the late distal nephron [36]. It is not known whether RBG can only act as an antagonist to MBG, or whether it can act independently. It is well established that an anti-angiogenic milieu exists in women with preE [7,8,12,13,27e33,35e38]. This study shows that CTSs, which circulate in higher concentrations during pregnancy, can induce such changes in extravillous cytotrophoblast cell protein expression, including inhibited production of VEGF165, PlGF, VEGFR1, AT1 and increased production of sFlt-1, sEng, AT2. With further study and confirmation of our observations, CTSs may be a target for preE screening and therapeutics.
Conflict of interest The authors have no conflicts of interest to disclose. Financial support Funding for this work was provided by Scott, Sherwood and Brindley Foundation and Department of Obstetrics and Gynecology (MNU) and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (TJK), Baylor Scott & White Health, Temple, Texas.
Condensation CTSs influenced CTB cells to induce an anti-angiogenic and antiproliferative profile. Acknowledgment Funding for this work was provided by Scott, Sherwood and Brindley Foundation and Department of Obstetrics and Gynecology (MNU) and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (TJK), Scott & White Healthcare, Temple, Texas. The CTB cell line Sw-71 was kindly provided by Dr. Gil G. Mor at Yale University School of Medicine, New Haven, CT, USA. MBG was a kind gift from Drs. Alexei Y. Bagrov, Edward G. Lakatta, and Olga V. Fedorova at the National Institute on Aging (NIA), Baltimore, Maryland. References [1] Leung DN, Smith SC, To KF, Sahota DS, Baker PN. Increased placental apoptosis in pregnancies complicated by preeclampsia. Am J Obstet Gynecol 2001;184: 1249e50. [2] Pridjian G, Puschett JB. Preeclampsia, part II: experimental and genetic considerations. Obstet Gynecol Surv 2002;57:619e40. [3] Puschett JB, Agunanne E, Uddin MN. Emerging role of the bufadienolides in cardiovascular and kidney disease. Am J Kidney Dis 2010;56:359e70. [4] Uddin MN, Allen S, Jones R, Zawieja D, Kuehl T. Pathogenesis of preeclampsia: marinobufagenin and angiogenic imbalance as biomarkers of the syndrome. Transl Res 2012;160:99e113. [5] Vu H, Ianosi Irimie M, Pridjian CA, Whitbred JM, Durst JM, Bagrov AY, et al. Involvement of marinobufagenin in a rat model of human preeclampsia. Am J Nephrol 2005;25:520e8. [6] Campbell DM, Campbell AJ. Evans blue disappearance rate in normal and preeclamptic pregnancy. Clin Exp Hypertens B 1983;2:163e9. [7] Maynard SE, Min J, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1(sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649e58.
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