Research, University of Texas Health Science Center San Antonio, San. Antonio, TX ... influence of invasive trophoblast and call for further studies using fresh.
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Abstracts / Placenta 36 (2015) 469e521
explaining these risks are not well understood. Pgf / mice have less decidual and placental vascular branching and connectivity than controls and most experience stroke after unilateral common carotid artery occlusion. Thus, we hypothesized that PGF deficiency, which manifests in PE, diminishes brain vascular development and leads to impaired cognition and elevated stroke risk. Pgf / mice brain vasculature were found to be deficient compared to Pgf+/+ controls during gestation and as adults. The circle of Willis in Pgf / mice brains was often incomplete, explaining the stroke phenomenon after carotid occlusion. Cognitive behavioral testing performed in Pgf / mice revealed impairments in numerous cognitive domains, which also displayed sexually dimorphic differences. We have subsequently recruited children born to PE or uncomplicated pregnancies to determine if similar brain vascular and cognitive impairments are present in humans. Preliminary analyses have shown brain vascular anomalies to be common in PE children. Analyses of cognitive function in these children are currently ongoing. Our data produced to date are compelling in linking placental paracrine signaling to alterations in the development and proper functioning of the offspring. Although more data are needed to draw firm conclusions in this regard, our preliminary work has uncovered a previously unknown link between PGF expression and brain development. Currently ongoing studies will aim to further understand this process and potentially augment it in utero.Supported by NSERC, CIHR, the CRC program and Kingston General Hospital Foundation. MINI COURSE A. MCA-1. VASCULAR REGRESSION IN THE FETOPLACENTAL VASCULAR BED, AND ITS POSSIBLE IMPLICATIONS FOR FETAL GROWTH RESTRICTION John Aplin, Stefanie Swietlik, Jayne Charnock, Mahmoud Khalid, Melissa Westwood, Edward Johnstone. Maternal and Fetal Health Centre, University of Manchester, UK A consistent feature of placenta in fetal growth restriction (FGR) is reduced vascularisation (Chen et al Am J Obstet Gynecol. 187:764, 2002). This is not uniform in the villous tree; in late onset FGR, peripheral areas are less well vascularized than central areas (Junaid et al Placenta 35:808, 2014). Early in gestation, primitive extraembryonic mesenchymal cells differentiate into endothelium and form the first vascular cords. This process occurs throughout the villous placenta as well as in the chorionic plate (CP). However, as demonstrated by thin sectioning (including early implantation site specimens fixed in situ) and whole mount immunofluorescence, vascular regression occurs at 9-12 weeks in villi overlying the superficial aspect of the placenta as well as in the associated CP. These areas give rise to the CL. We used first trimester villous explants and primary mesenchymal cell cultures to examine potential mechanisms. Over 72 h, explants showed declining vessel integrity and altered endothelial morphology, while surrounding stroma and trophoblast remained intact. Vascular endothelial growth factor (VEGF) delayed this decline. CD31-positive endothelial cells were isolated from villous first trimester digests and cultured on collagen with or without VEGF. Within 24h, they acquired aSMA-positive stress fibres indicative of an endothelial-mesenchymal transition (EndMT). Thus VEGF extends the lifespan of vascular cells in tissue, but not in culture. In first trimester villi, double-positive cells were present in vessel walls, and at 9-12 weeks vascular regression was observed in areas far from the cord insertion. Thus endothelial cells in placental tissue abutting the capsular decidua undergo EndMT, express aSMA and produce the avascular CL. We suggest that exaggerated regression may occur in fetal growth restriction.
MCA-2. WHOLE MOUNT IMMUNOFLUORESCENCE OF THE HUMAN PLACENTA Shawn P. Murphy 1, 2, Meghan E. Bushway 2, Paula Zozzaro-Smith 1, Ian D. Scott A. Gerber 2, Richard K. Miller 1, Edith M. Perry 2, 2 1 Lord . Department of Obstetrics and Gynecology, University of Rochester School of Medicine, Rochester, NY, USA; 2 Department of Microbiology and Immunology, University of Rochester School of Medicine, Rochester, NY, USA
Objective: The human placenta plays multiple critical roles in successful pregnancy, and it is well established that defects in placental development and/or function are associated with severe complications of pregnancy. However, the molecular mechanisms underlying development of the human placenta are incompletely understood. Furthermore, the methods currently utilized to image the human placenta lack the capacity to simultaneously examine cellular phenotype, morphology and spatial arrangement of cells within intact placental structures. We therefore developed a novel whole mount immunofluorescence (WMIF) method to examine the placental microenvironment over the course of human gestation. Methods: Intact explants were dissected from 1st trimester (7e12 weeks), 2nd trimester (16e22 weeks) and term (39 weeks) placentas, fixed, and stained overnight with fluorescently-conjugated antibodies against specific cell surface or intracellular proteins. Samples were subsequently prepared as whole mounts and visualized by either conventional or confocal microscopy. Results: All of the major cell populations within the human placenta can be distinguished by WMIF, including immune cells, blood vessel endothelial cells, fibroblasts and several subpopulations of trophoblast cells. Significant differences in the morphology of placental blood vessels were observed over the course of pregnancy. Furthermore, both qualitative and quantitative differences were readily detected in placental expression of a number of markers over the course of gestation by WMIF. Our laboratory is currently successfully utilizing WMIF to compare marker expression in normotensive versus preeclamptic placentas, signal transduction in response to proinflammatory cytokines through the JAK-STAT pathway in intact placental explants, and infection of placental explants by pathogenic bacteria. Conclusions: Our collective studies demonstrate that WMIF can be successfully used to study the placental architecture over the course of gestation, and in normal versus pathological pregnancies. Moreover, this method provides a powerful approach for examining the effects of various factors on placental signal transduction and morphometry. MCA-3. THE IMMUNOMODULATORY PROPERTIES OF HUMAN PLACENTA: IMPLICATIONS FOR ITS USE IN REGENERATIVE MEDICINE O. Parolini, M. Magatti, S. Pianta, A. Silini, A. Cargnoni. Centro di Ricerca E. Menni, Fondazione Poliambulanza, Brescia, Italy Objectives: We have shown that mesenchymal (hAMSC) and epithelial (hAEC) cells can be isolated from the amniotic membrane of human term placenta and we have contributed to their characterization [1]. Our research is aimed at studying the immunomodulatory capabilities of these cells, and specifically on their derivatives, such as conditioned medium. Methods: Cell isolation from human term placenta, cell characterization, and generation of conditioned media were performed as previously described [2]. In vitro immunomodulation tests and in vivo models [3,4] were performed as described elsewhere. Results: We have previously demonstrated that hAMSC reduce T cell proliferation and recent findings indicate that hAMSC alter T helper cell subsets by specifically inducing polarization towards T regulatory cells [5]. Additionally, we have demonstrated that hAMSC can inhibit monocyte maturation towards dendritic cells [6], and promote macrophage polarization towards an M2 phenotype [6]. Importantly, we have recently shown that the conditioned medium derived from hAMSC and hAEC culture is also able to modulate immune cells, thus supporting the hypothesis that hAMSC and hAEC produce and secrete paracrine-acting factors which act on/ communicate with immune cells [7]. Furthermore, we have shown that the application of cells or their conditioned medium in animal models of disease can significantly attenuate progression of several inflammatoryassociated diseases, such as in animals with ischemic hearts [8] and lung and liver fibrosis [3,9]. Very recently, we have shown that hAMSC can ameliorate clinical progression of autoimmune diseases in experimental mouse models, such as multiple sclerosis and rheumatoid arthritis [4]. Importantly, we have also shown that hAMSC can suppress the inflammatory responses in cells isolated from patients with rheumatoid arthritis [4].
Abstracts / Placenta 36 (2015) 469e521
Conclusions: Cells and derivatives from human term placenta could represent a valuable therapeutic tool in diseases based on inflammatory processes and also in those with altered immune reactions, such as autoimmune disorders. References [1]. Parolini O, et al. Stem Cells Dev 2010 [2]. Rossi D, et al. PLoS One 2012 [3]. Cargnoni A, et al. Cytotherapy 2014 [4]. Parolini O, et al. Arthritis Rheumatol 2014 [5]. Pianta S, et al. Stem Cells Rev and Reports 2014 [6]. Magatti M, et al. Cell Transplantation, 2009 [7]. Magatti M, et al. Cell Transplantation, 2014 [8]. Cargnoni A, et al. Cell Transplantation 2009 [9]. Sant'Ansssna LB, et al. Cell Transplantation 2011
MINI COURSE B. THE ANATOMY AND PHYSIOLOGY OF A MANUSCRIPT Yoel Sadovsky 1, L. Myatt 2, Graham Burton 3. 1 Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA; 2 Center for Pregnancy and Newborn Research, University of Texas Health Science Center San Antonio, San Antonio, TX 78229, USA; 3 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK
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Placentation in Neotropical (platyrrhine) primates will be discussed in the context of evolution of invasive placentation in primates, which culminates in the deep penetration of the endometrium and its arteries in hominoids. In Old World monkeys and apes, columns of trophoblast cells advance to the base of the implantation site and spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts enter the spiral arteries and are responsible for remodeling them to form wide, low resistance conduits. Additionally, in human and great apes, there is invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. In platyrrhines, on the other hand, there is no cytotrophoblastic shell. Instead the fetal-maternal interface is formed by a thick pad of syncytiotrophoblast resting directly on the undecidualized endometrium. The conventional wisdom is that trophoblast does not invade the placental bed and its vessels. If this is the case, how is an adequate blood supply maintained? A distinctive feature of the platyrrhine placenta is the presence within it of large maternal capillaries with multiple openings into the intertrabecular space. It is known that they are derived from subepithelial uterine capillaries, but scant attention has been paid to the arteries supplying them. Our preliminary observations suggest there is transformation of the arterial wall consistent with widening of the lumen to reduce resistance to blood flow. We believe this occurs under the influence of invasive trophoblast and call for further studies using fresh material to test this hypothesis. The results would have intrinsic value in relation to the primate radiation in the Neotropics as well as broader implications for understanding the evolution of trophoblast invasion in higher primates, including the human species.
KEYNOTE LECTURES. K1. FORMATION OF HUMAN EXTRAVILLOUS TROPHOBLAST: MECHANISMS CONTROLLING PROLIFERATION, DIFFERENTIATION AND MIGRATION € fler. Department of Obstetrics and Fetal-Maternal Medicine, Martin Kno Reproductive Biology Unit, Medical University of Vienna, Austria Signaling pathways regulating human extravillous trophoblast (EVT) migration have been intensively investigated. However, key regulatory mechanisms controlling proliferation of trophoblast progenitors of placental anchoring villi and their differentiation towards the EVT lineage remain largely elusive. Since failures in the intrinsic EVT differentiation program could contribute to gestational diseases with abnormal placentation, such as preeclampsia or intrauterine growth restriction, a better understanding of the underlying mechanisms could be beneficial for future diagnosis and treatment. Recent studies in the laboratory suggested that the developmental pathways Notch, Wnt, Hippo and EGF may interact in an integrated manner to regulate cell column proliferation and differentiation. Development of EVTs is associated with nuclear recruitment of Wnt-dependent b-catenin and the co-activators of Hippo signaling YAP and TAZ. Accordingly, canonical Wnt signaling and the Hippo-Off state were shown to promote trophoblast migration and EVT marker expression. In contrast, canonical Notch activity could be required for balanced rates of column proliferation and EVT formation involving the key regulatory transcription factor RBPJk. However, Notch receptors, such as Notch1 and Notch2, have distinct expression patterns in individual trophoblast subtypes and exhibit differential roles. Notch1 controls trophoblast survival, whereas Notch2 regulates EVT motility. Similar to Notch receptors, trophoblasts switch the expression of EGF receptor family members during EVT differentiation. Functional analyses showed that distinct combinations of EGF receptors promote cell column proliferation and suppress EVT apoptosis, respectively, in a ligand-dependent manner. Moreover, our recent analyses indicated that, like in breast cancer cells, formation of EVT is associated with DNA amplification of EGF receptor 2 (HER-2) suggesting a physiological role of the gene in EVT function or differentiation.
K2. WHAT CAN NEOTROPICAL PRIMATES TEACH EVOLUTION OF INVASIVE PLACENTATION?
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A.M. Carter. Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark
K3. ANTIAPOPTOTIC EFFECTS OF LEPTIN ON TROPHOBLASTS n Toro 1, Malena Schanton 1, Julieta Maymo 1, Antonio Pe rezAyele 2 3 Bernardo Maskin , Víctor Sanchez-Margalet 2, Cecilia Perez , gica, FCEN, UBA, IQUIBICEN Varone 1. 1 Departamento de Química Biolo edica y CONICET, Buenos Aires, Argentina; 2 Departamento de Bioquímica M Biología Molecular, Universidad de Sevilla, Sevilla, Spain; 3 Hospital Nacional Alejandro Posadas, Buenos Aires, Argentina Leptin produced by the placenta has many roles as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblast cells. In the present work, we aimed to study the mechanisms that mediate the antiapoptotic effect of leptin in the placenta. Methods: BeWo and Swan cells, cultured under standard conditions, as well as human placental explants were used. Western blot, qRT-PCR and transfection assays with reporter constructs and expression vectors were carried out. All the procedures were approved by the ethical review committee at the Posadas National Hospital (Buenos Aires, Argentina). Results: Recombinant human leptin added to the BeWo cell line and human placental explants showed a decrease in Caspase-3 activation in a dose- dependent manner. Moreover, inhibition of endogenous leptin expression with 2 mM of an antisense oligonucleotide reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. Placental explants cultured in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. We found that under serum deprivation conditions, leptin increased the anti-apoptotic Bcl-2 protein expression and downregulated the pro-apoptotic Bax and Bid protein expression in Swan71 cells and placental explants. In both models, leptin augmented the Bcl2/Bax ratio. Moreover, we demonstrated that p53, one of the key cell cyclesignaling proteins, is downregulated in the presence of leptin under serum deprivation. Leptin also reduced the phosphorylation of Ser-46 p53, which plays a pivotal role in the apoptotic signaling by p53 and augments the levels of Mdm2 protein, a regulator of p53 half-life. Furthermore, leptin antiapoptotic effect and p53 regulation by leptin involved MAPK and PI3K signaling pathways. Conclusions: These results improve the understanding of leptin function during pregnancy and further support the importance of leptin in pregnancy.
