Placenta (2004), 25, Supplement A, Trophoblast Research, Vol. 18, S109–S111 doi:10.1016/j.placenta.2004.01.021
From Basic Research to Clinical Application of Placental Proteins—A Workshop Report N. G. Than a,b, H. Bohn c, P. Toth d, N. Bersinger e, J. G. Grudzinskas f and P. Bischof g,* a
First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary; b Department of Biochemistry and Medical Chemistry, University of Pe´cs, Hungary; c Behringwerke AG, Marburg/Lahn, Germany; d Second Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary; e Department of Obstetrics and Gynecology, University of Berne, Switzerland; f The Bridge Centre/Department of Obstetrics and Gynecology, St Bartholomew’s Hospital, London, UK; g Department of Obstetrics and Gynecology, Maternity, Laboratory of Hormonology, University of Geneva, Boulevard de la Cluse, 1211 Geneva 14, Switzerland
INTRODUCTION The workshop focused on proteins synthesized by human placenta, which have not only local, but also general developmental and regulatory functions, and essential effects on the outcome of pregnancy. After their isolation these proteins were clinically characterized. Data on their placental expression and levels in maternal blood, urine, or amniotic fluid showed that some of them might have clinical importance in the early diagnosis of pregnancy, or have diagnostic value in the assessment of placental functions, fetal well-being or pregnancyrelated disorders. The intention of the workshop was to give insight into the most important advances in basic research and clinical application of these placental proteins.
PREFACE H. Bohn reviewed the history of placental protein research and reported on his investigations at Behringwerke AG. Though hCG was detected in pregnant urine by Aschheim in 1927, it is only in the early 1970s that a systematic search for placental proteins was started in collaborating laboratories. At Behringwerke, more than fifty proteins were discovered, isolated and named sequentially [1,2]. During this pioneering era, different research groups tried to isolate and characterize proteins from placenta, maternal and fetal sera and amniotic fluid, as well as to establish their nomenclature. Efforts were undertaken to improve their basic and applied methodology [3], biological, regulational and immunological aspects, and their role in human reproduction were widely studied [4,5]. From the 1980s, recombinant techniques allowed for molecular biological analyses. Function and clinical importance of many of these proteins still remains unknown. Our limited knowledge will probably be widened by the efforts made by the human genome and proteome projects, and by well-established clinical studies. In the near future new light will be shed on our fragmentary knowledge that will lead to the discovery of new *
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placental genes and proteins, and exciting diagnostic and therapeutic applications.
LEPTIN, A PLACENTAL PROTEIN WITH REGULATORY FUNCTIONS ON HCG SECRETION P. Bischof reported studies on the obese-gene product leptin conducted with D. Islami and D. Chardonnens. Leptin, a 167 amino acid peptide, has provided a fundamental link between food intake, energy balance and reproductive events. It has been demonstrated that circulating leptin levels are elevated during pregnancy, reaching a peak during the second trimester. The Geneva group has shown that human trophoblast was another important source of leptin, and that interleukin-6 and oestradiol stimulate its secretion. In one study, the expression of leptin and leptin-R was investigated in the human placenta with particular emphasis on extravillous cytotrophoblastic cell (evCTB) islands and cell columns, which play a pivotal role in trophoblast invasion [6]. It was demonstrated that leptin-R immunoreactivity showed a strong expression in the distal evCTB of cell columns invading the basal plate, whereas leptin expression was homogeneously expressed in all the cellular components of cell columns. Since the invasive ability of the distally located evCTB cell columns was known to be regulated by a variety of proteases and some extracellular matrix molecules, the influence of leptin on the in-vitro production of matrix metalloproteinase (MMP)-2, MMP-9 and fetal fibronectin (fFN) by CTB was tested. It was demonstrated that leptin increased, in a dose-dependent manner, the secretion of immunoreactive MMP-2 and fFN, and enhanced the activity of MMP-9 in cultured CTB. These results suggested, that leptin and leptin-R could have a role in regulating the invasive behavior of evCTB by modulating their expression of MMPs. This research group assessed also the potential effects of leptin on placental function by measuring hCG and vascular endothelial growth factor (VEGF) secretion in cultured first trimester CTB in presence or absence of human recombinant 2004 IFPA and Elsevier Ltd. All rights reserved.
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leptin, GnRH and GnRH antagonists [7]. A concentrationdependent leptin-induced hCG secretion was observed, reaching a 5-fold increase over baseline values with a leptin concentration of 1 µg/ml. GnRH (8.5108 ) also increased hCG secretion. Both GnRH antagonists, Cetrorelix and Azaline (0.1 µg/ml) inhibited leptin-induced hCG secretion. Higher concentrations of GnRH antagonists did not show such an inhibition. These data represent the first in vitro evidence of leptin-induced hCG secretion that seems to be mediated via the GnRH pathway. In conclusion, these novel data strongly suggest that leptin is a key regulator of trophoblastic invasion and placental endocrine function. FUNCTIONAL RELEVANCE OF HCG/LH RECEPTORS IN ENDOTHELIAL CELLS P. Toth reported studies on hCG/LH receptors in endothelial cells. The presence of hCG/LH receptors in human uterine blood vessels was detected a decade ago [8]. Their functional relevance was investigated by in vivo and in vitro studies, which showed that activation of the vascular receptors could lead to vasorelaxation [9]. The underlying mechanism is the effect of hCG on the eicosanoid synthesis in vessel walls. Human Doppler blood flow measurements showed a decrease of vascular resistance indices after administration of hCG to infertile patients and during the first trimester of pregnancy in patients with threatened abortion. HCG administration to these patients significantly decreased the rate of spontaneous abortions [10]. Based on these results, further studies were carried out. One study investigated whether there was any long-term effect of hCG administration to patients with threatened abortion. Pregnancies treated by either magnesium or hCG in the first trimester because of signs of threatened abortion were monitored until delivery. Results showed, that the hCGtreated group had significantly less premature deliveries compared to the magnesium treated one. An in vitro study investigated whether hCG could influence the proliferation of vascular endothelial cells and thus be a regulator of angiogenesis. HCG significantly stimulated the proliferation of human umbilical vein endothelial cells. The signalling mechanism seemed to be related to the PKA pathway. The eicosanoid synthesis in endothelial cells was also affected by hCG. These data clearly show the functional relevance of endothelial hCG/LH receptors and suggest a role of hCG in the implantation process with long-term benefits during the course of pregnancy. ALTERED MATERNAL SERUM CONCENTRATIONS OF PREGNANCYASSOCIATED PROTEINS IN EARLY PREGNANCY: FROM CHROMOSOMAL ABNORMALITIES AND GROWTH RESTRICTION TO ASSISTED REPRODUCTION N. Bersinger reported studies and potential clinical uses of different pregnancy-associated proteins in early pregnancy.
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The discovery of low maternal serum levels of PAPP-A in the first trimester of pregnancies affected by fetal trisomy 21 [11] led to the now widely established routine application of PAPP-A in biochemical screening for this pathology. It has been shown that the concentration of this protein was also reduced in other trisomies and in abnormal pregnancies without fetal chromosomal abnormalites, such as IUGR or subsequent pre-eclampsia. A longitudinal study showed that reduced PAPP-A levels could be observed in early pregnancy in asymptomatic patients that will develop pre-eclampsia or IUGR [12]. It has been suggested that the serum levels of PAPP-A were lower in pregnancies achieved by IVF treatment than in gestational age-matched spontaneous pregnant controls [13]. In this workshop presentation, the variability of PAPP-A levels in chromosomally abnormal pregnancies, in threatened abortion, pre-eclampsia, IUGR and after various protocols for assisted reproduction were shown and compared to other placental (SP1, hPL, pGF, inhibin A, activin A) and nonplacental (IGFBP-1, soluble E-selectin, VEGF) proteins. It was concluded that while some markers (e.g. PAPP-A) were sensitive in early pregnancy but not specific to a defined pathology, others were more restricted to a specific disease but useful also in later gestation.
PAPP-A: 30 YEARS LATER J.G. Grudzinskas gave an historical overview on pregnancyassociated plasma protein A (PAPP-A) and tried to answer the question: ‘Why did it take so long for PAPP-A to become a routine test?’ PAPP-A was discovered in 1974 [14] and since then a total of 487 studies (PubMed) have been published. In the late seventies, most PAPP-A studies were devoted to placental functions, whereas in the early nineties trisomy 21 was the main interest of these types of studies. PAPP-A is now investigated in men as a marker of myocardial infarction. This speaker proposed that it took a long time for PAPP-A to become a routine test because of (1) methodological problems, (2) divergent interests of investigators, and (3) business interests. Due to the molecular complexity of PAPP-A, all antibodies available in the late seventies were cross-reacting with other molecules, mainly the proform of the major basic protein that was later shown to be covalently linked to PAPP-A and to other serum proteins. Investigators in the PAPP-A field had divergent interests: scientists were searching for a biological function for PAPP-A, whereas obstetricians wanted a marker of placental function and gynecologists a marker of endometrial function. Business interests were also a cause for the late use of PAPP-A tests. Some investigators had patented the use of serum markers in the second trimester of pregnancy and, in order to avoid competition, PAPP-A was not accepted as a potentially new marker.
Than et al.: From Basic Research to Clinical Application of Placental Proteins
GENOMIC, PROTEOMIC AND FUNCTIONAL STUDIES OF HUMAN PLACENTAL PROTEIN 13 (PP13)/GALECTIN-13 N.G. Than presented his research group’s data on placental protein 13 (PP13). This member of the so-called ‘pregnancyrelated protein’ family was purified and characterized physicochemically in 1983. It was found to be composed of two identical 16 kDa subunits. On average, human term placenta contained 3.7 mg of PP13 [15]. Neither RIA [2] nor Western blot analyses could detect PP13 in maternal or fetal serum, or in amniotic fluid. PP13 was mainly found in placenta, but also in fetal liver and spleen. By cloning and sequencing from a human placental cDNA library, PP13 turned out to be 139 residues long [16]. As sequence analyses, alignments and computational 3D modeling showed, PP13 conserved structural and functional homology to members of the galectin family, and it was therefore denoted galectin-13 [17]. Similar to its homologue Charcot Leyden Crystal protein, weak lysophospholipase activity of PP13 was observed. Sugar binding assays revealed that similar to other galectins, N-acetyl-lactoseamine had the strongest binding affinity to PP13, which also effectively agglutinated erythrocytes. Using affinity chromatography, PAGE and MALDI-TOF mass spectrometry, beta/gamma actin and annexin II were identified as proteins specifically bound to PP13 in placental tissue and fetal hepatic cells. Perinuclear PP13 staining of syncytiotrophoblasts proved its expression in these cells, while strong labelling of the brush border membrane confirmed its galectin-like externalization to cell surface. Based upon its similar localization and specific binding to annexin II, PP13 was proposed to be secreted by ectocytosis into microvesicles containing actin and annexin II. Its unique dimerization via disulphide bonds might affect its activity upon the oxygenization changes in placenta. As other galectins are involved in immunobiological functions, PP13 and its placental homologues also have immune functions at feto-maternal interfaces
CONCLUSION AND FUTURE DIRECTIONS Placental proteins are not only important molecules for the development of the placenta and the embryo, but exert also placental and general regulatory functions. For more than three decades, extensive investigations by leading laboratories in the field have added not only important physico-chemical, sequential, structural and functional data to our knowledge, but have also helped clinicians in their daily routine work. In this context, the extensive use of placental proteins for diagnostic and therapeutic purposes in different pregnancy dis-
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orders is coming back into focus. The exciting new results and broad reviews presented at the workshop underlined the importance of this research field. The ‘historic work of the pioneer’, as well as the present molecular biological investigations and clinical studies reflect the enormous efforts that have already made (and will hopefully continue to give) a large impact in science. The workshop provided a good platform for helpful discussions and starting points for new collaborations between the participating research groups. REFERENCES [1] Bohn H. Detection and characterization of pregnancy proteins in the human placenta and their quantitative immunochemical determination in sera from pregnant women. Arch Gynakol 1971;210:440–57. [2] Than GN, Bohn H, Szabo´ DG. Advances in pregnancy-related protein research. Functional and clinical applications. Boston/Boca Raton (USA): CRC Press; 1993, p. 1–333. [3] Grudzinskas JG, Teisner B, Seppa¨la¨ M. Pregnancy proteins. Biology, chemistry clinical application. Sydney (Australia): Academic Press; 1982, p. 1–463. [4] Bischof P. Placental proteinsBasel (Switzerland): Karger; 1984, p. 1–96. [5] Bischof P, Klopper A. Proteins of the placenta. Biochemistry, biology and clinical application. Basel (Switzerland): Karger; 1985, p. 1–208. [6] Castellucci M, de Matteis R, Meisser A, Cancello R, Monsuro V, Islami D et al. Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion. Mol Hum Reprod 2000;6:951–8. [7] Islami D, Bischof P, Chardonnens D. Modulation of placental vascular endothelial growth factor (VEGF) by leptin and human chorionic gonadotropin. Mol Hum Reprod 2003;9:395–8. [8] Rao CV, Lei ZM. Gonadotropin receptors in nongonadal reproductive tissues. ARTA 1990;1:241–53. [9] Toth P, Li X, Rao CV, Lincoln SR, Sanfilippo JS, Spinnato JA et al. Expression of functional human chorionic gonadotropin/human luteinizing hormone receptor gene in human uterine arteries. J Clin Endocrinol Metab 1994;79:307–15. [10] Toth P. Clinical data supporting the importance of vascular LH/hCG receptors of uterine blood vessels. Semin Reprod Med 2001;19:55–61. [11] Bersinger NA, Brizot ML, Johnson A, Snijders RJ, Abbott J, Schneider H et al. First trimester maternal serum pregnancy-associated plasma protein A and pregnancy-specific beta 1-glycoprotein in fetal trisomies. Br J Obstet Gynaecol 1994;101:970–4. [12] Bersinger NA, Ødega˚rd RA. Second- and third-trimester serum levels of placental proteins in pre-eclampsia and small-for-gestational age pregnancies. Acta Obstet Gynecol Scand 2004;83:37–45. [13] Bersinger NA, Wunder D, Crazzolara S, Chanson A, Pescia G, Hanggi W. Serum medians for placental proteins (HCG, PAPP-A) in pregnancies achieved by IVF are different from those of spontaneous pregnancies: implications for fetal trisomy screening. Hum Reprod 2000; 15(suppl.1):171. [14] Lin TM, Halbert SP, Kiefer D, Spellacy WN, Gall S. Characterization of four human pregnancy-associated plasma proteins. Am J Obstet Gynecol 1974;118:223–36. [15] Bohn H, Kraus W, Winckler W. Purification and characterization of two new soluble placental tissue proteins (PP13 and PP17). Oncodev Biol Med 1983;4:343–50. [16] Than NG, Sumegi B, Than GN, Berente Z, Bohn H. Isolation and sequence analysis of a cDNA encoding human placental tissue protein 13 (PP13), a new lysophospholipase, homologue of the human eosinophil Charcot-Leyden crystal protein. Placenta 1999;20:703–10. [17] Visegrady B, Than NG, Kilar F, Sumegi B, Than GN, Bohn H. Homology modelling and molecular dynamics studies of human placental tissue protein 13 (galectin-13). Protein Eng 2001;14:875–80.