Abstract
Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.
Similar content being viewed by others
References
Meekins JW, Pijnenborg R, Hanssens M, McFadyen IR, van Asshe A (1994) A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies. Br J Obstet Gynaecol 101(8):669–674
Jauniaux E, Watson AL, Hempstock J, Bao YP, Skepper JN, Burton GJ (2000) Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am J Pathol 157(6):2111–2122
Huppertz B, Frank HG, Reister F, Kingdom J, Korr H, Kaufmann P (1999) Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous cytotrophoblast and syncytial fragments in vitro. Lab Invest 79(12):1687–1702
Allaire AD, Ballenger KA, Wells SR, McMahon MJ, Lessey BA (2000) Placental apoptosis in preeclampsia. Obstet Gynecol 96(2):271–276
Leung DN, Smith SC, To KF, Sahota DS, Baker PN (2001) Increased placental apoptosis in pregnancies complicated by preeclampsia. Am J Obstet Gynecol 184(6):1249–1250
Heazell AE, Moll SJ, Jones CJ, Baker PN, Crocker IP (2006) Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species. Placenta 28(Supplement 1):S33–S40
Smith SC, Baker PN, Symonds EM (1997) Increased placental apoptosis in intrauterine growth restriction. Am J Obstet Gynecol 177(6):1395–1401
Levy R, Smith SD, Yusuf K, Huettner PC, Kraus FT, Sadovsky Y, Nelson DM (2002) Trophoblast apoptosis from pregnancies complicated by fetal growth restriction is associated with enhanced p53 expression. Am J Obstet Gynecol 186(5):1056–1061
Ishihara N, Matsuo H, Murakoshi H, Laoag-Fernandez JB, Samoto T, Maruo T (2002) Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. Am J Obstet Gynecol 186(1):158–166
Johansen M, Redman CW, Wilkins T, Sargent IL (1999) Trophoblast deportation in human pregnancy—its relevance for pre-eclampsia. Placenta 20(7):531–539
Levy R, Smith SD, Chandler K, Sadovsky Y, Nelson DM (2000) Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor. Am J Physiol Cell Physiol 278(5):C982–C988
Hung TH, Skepper JN, Charnock-Jones DS, Burton GJ (2002) Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia. Circ Res 90(12):1274–1281
Fadeel B, Orrenius S (2005) Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med 258(6):479–517
Levy R, Nelson DM (2000) To be, or not to be, that is the question. Apoptosis in human trophoblast. Placenta 21(1):1–13
Yui J, Garcia-Lloret M, Wegmann TG, Guilbert LJ (1994) Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts. Placenta 15(8):819–835
Crocker IP, Tansinda DM, Jones CJ, Baker PN (2004) The influence of oxygen and tumor necrosis factor-alpha on the cellular kinetics of term placental villous explants in culture. J Histochem Cytochem 52(6):749–757
Barres BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, Raff MC (1992) Cell death and control of cell survival in the oligodendrocyte lineage. Cell, 70(1):31–46
Raff MC (1992) Social controls on cell survival and cell death. Nature 356(6368):397–400
Smith S, Francis R, Guilbert L, Baker PN (2002) Growth factor rescue of cytokine mediated trophoblast apoptosis. Placenta 23(4):322–330
Garcia-Lloret MI, Yui J, Winkler-Lowen B, Guilbert LJ (1996) Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts. J Cell Physiol 167(2):324–332
Payne SG, Brindley DN, Guilbert LJ (1999) Epidermal growth factor inhibits ceramide-induced apoptosis and lowers ceramide levels in primary placental trophoblasts. J Cell Physiol 180(2):263–270
Morrish DW, Bhardwaj D, Dabbagh LK, Marusyk H, Siy O (1987) Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J Clin Endocrinol Metab 65(6):1282–1290
Anteby EY, Greenfield C, Natanson-Yaron S, Goldman-Wohl D, Hamani Y, Khudyak V, Ariel I, Yagel S (2004) Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase-9. Mol Hum Reprod 10(4):229–235
Muhlhauser J, Crescimanno C, Kaufmann P, Hofler H, Zaccheo D, Castellucci M (1993) Differentiation and proliferation patterns in human trophoblast revealed by c-erbB-2 oncogene product and EGF-R. J Histochem Cytochem 41(2):165–173
Bulmer JN, Thrower S, Wells M (1989) Expression of epidermal growth factor receptor and transferrin receptor by human trophoblast populations. Am J Reprod Immunol 21(3–4):87–93
Hofmann GE, Drews MR, Scott RT Jr, Navot D, Heller D, Deligdisch L (1992) Epidermal growth factor and its receptor in human implantation trophoblast: immunohistochemical evidence for autocrine/paracrine function. J Clin Endocrinol Metab 74(5):981–988
Ladines-Llave CA, Maruo T, Manalo AS, Mochizuki M (1991) Cytologic localization of epidermal growth factor and its receptor in developing human placenta varies over the course of pregnancy. Am J Obstet Gynecol 165(5 Pt 1):1377–1382
Toth FD, Aboagye-Mathiesen G, Nemes J, Liu X, Andirko I, Hager H, Zdravkovic M, Szabo J, Kiss J, Aranyosi J, Ebbesen P (1997) Epstein-Barr virus permissively infects human syncytiotrophoblasts in vitro and induces replication of human T cell leukemia-lymphoma virus type I in dually infected cells. Virology 229(2):400–414
Tavare JM, Holmes CH (1989) Differential expression of the receptors for epidermal growth factor and insulin in the developing human placenta. Cell Signal 1(1):55–64
Filla MS, Zhang CX, Kaul KL (1993) A potential transforming growth factor alpha/epidermal growth factor receptor autocrine circuit in placental cytotrophoblasts. Cell Growth Differ 4(5):387–393
Lindqvist P, Grennert L, Marsal K (1999) Epidermal growth factor in maternal urine–a predictor of intrauterine growth restriction? Early Hum Dev 56(2–3):143–150
Kamei Y, Tsutsumi O, Yamakawa A, Oka Y, Taketani Y, Imaki J (1999) Maternal epidermal growth factor deficiency causes fetal hypoglycemia and intrauterine growth retardation in mice: possible involvement of placental glucose transporter GLUT3 expression. Endocrinology 140(9):4236–4243
Maruo T, Matsuo H, Murata K, Mochizuki M (1992) Gestational age-dependent dual action of epidermal growth factor on human placenta early in gestation. J Clin Endocrinol Metab 75(5):1362–1367
Soleymanlou N, Jurisica I, Nevo O, Ietta F, Zhang X, Zamudio S, Post M, Caniggia I (2005) Molecular evidence of placental hypoxia in preeclampsia. J Clin Endocrinol Metab 90(7):4299–4308. Epub 2005 Apr 19
Halliwell B, Clement MV, Long LH (2000) Hydrogen peroxide in the human body. FEBS Lett 486(1):10–13
Crocker IP, Daayana SL, Baker PN (2004) An image analysis technique for the investigation of human placental morphology in pre-eclampsia and intrauterine growth restriction. J Soc Gynecol Invest 11(8):545–552
Siman CM, Sibley CP, Jones CJ, Turner MA, Greenwood SL (2001) The functional regeneration of syncytiotrophoblast in cultured explants of term placenta. Am J Physiol Regul Integr Comp Physiol 280(4):R1116–R1122
Crocker IP, Tansinda DM, Baker PN (2004) Altered cell kinetics in cultured placental villous explants in pregnancies complicated by pre-eclampsia and intrauterine growth restriction. J Pathol 204(1):11–18
Maddox PH, Jenkins D (1987) 3-Aminopropyltriethoxysilane (APES): a new advance in section adhesion. J Clin Pathol 40(10):1256–1257
Jones CJ, Mosley SM, Jeffrey IJ, Stoddart RW (1987) Elimination of the non-specific binding of avidin to tissue sections. Histochem J 19(5):264–268
Cheung AN, Ngan HY, Collins RJ, Wong YL (1994) Assessment of cell proliferation in hydatidiform mole using monoclonal antibody MIB1 to Ki-67 antigen. J Clin Pathol 47(7):601–604
Jinno Y, Merlino GT, Pastan I (1988) A novel effect of EGF on mRNA stability. Nucleic Acids Res 16(11):4957–4966
Burdon RH (1995) Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 18(4):775–794
Castellucci M, Kaufmann P (2006) Basic structure of the villous trees. In: Benirschke K, Kaufmann P (eds) Pathology of the placenta. Springer, New York, pp 50–120
Levy R, Smith SD, Chandler K, Sadovsky Y, Nelson DM (2000) Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor. Am J Physiol Cell Physiol 278(5):C982–C988
Ka H, Hunt JS (2003) Temporal and spatial patterns of expression of inhibitors of apoptosis in human placentas. Am J Pathol 163(2):413–422
Fulop V, Mok SC, Genest DR, Gati I, Doszpod J, Berkowitz RS (1998) p53, p21, Rb and mdm2 oncoproteins. Expression in normal placenta, partial and complete mole, and choriocarcinoma. J Reprod Med 43(2):119–127
Toki T, Horiuchi A, Ichikawa N, Mori A, Nikaido T, Fujii S (1999) Inverse relationship between apoptosis and Bcl-2 expression in syncytiotrophoblast and fibrin-type fibrinoid in early gestation. Mol Hum Reprod 5(3):246–251
Yui J, Garcia-Lloret M, Brown AJ, Berdan RC, Morrish DW, Wegmann TG, Guilbert LJ (1994) Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells. Placenta 15(3):231–246
Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF 3rd (1986) Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118(4):1567–1582
Crocker IP, Barratt S, Kaur M, Baker PN (2001) The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts. Placenta 22(10):822–830
Sakuragi N, Matsuo H, Coukos G, Furth EE, Bronner MP, VanArsdale CM, Krajewsky S, Reed JC, Strauss JF 3rd (1994) Differentiation-dependent expression of the BCL-2 proto-oncogene in the human trophoblast lineage. J Soc Gynecol Investig 1(2):164–172
Kawamata T, Yamaguchi T, Shin-ya K, Hori T (2003) Divergence in signaling pathways involved in promotion of cell viability mediated by bFGF, NGF, and EGF in PC12 cells. Neurochem Res 28(8):1221–1225
Qiu Q, Yang M, Tsang BK, Gruslin A (2004) EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways. Reproduction 128(3):355–363
Johnstone ED, Sibley CP, Lowen B, Guilbert LJ (2005) Epidermal growth factor stimulation of trophoblast differentiation requires MAPK11/14 (p38 MAP kinase) activation. Biol Reprod 73(6):1282–1288
Perkins J, St John J, Ahmed A (2002) Modulation of trophoblast cell death by oxygen and EGF. Mol Med 8(12):847–856
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91(2):231–241
Ho S, Winkler-Lowen B, Morrish DW, Dakour J, Li H, Guilbert LJ (1999) The role of Bcl-2 expression in EGF inhibition of TNF-alpha/IFN-gamma-induced villous trophoblast apoptosis. Placenta 20(5–6):423–430
Acknowledgements
The authors would like to thank the midwives and clinical research fellows at St Mary’s Hospital, Manchester for their assistance in obtaining term placental tissue. This study was supported by Tommy’s—the baby charity, the Department of Health, UK, The Castang Foundation and a student stipend from the Society for Gynecological Investigation.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Moll, S.J., Jones, C.J.P., Crocker, I.P. et al. Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species. Apoptosis 12, 1611–1622 (2007). https://doi.org/10.1007/s10495-007-0092-6
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10495-007-0092-6