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Characterizing ADAM28 function in trophoblast differentiation De Luca, Lauren Celeste 2016

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Characterizing ADAM28 Function in Trophoblast Differentiation   by   Lauren Celeste De Luca   B.Sc., The University of British Columbia, 2013      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIRMENTS FOR THE DEGREE OF    MASTER OF SCIENCE  in  The Faculty of Graduate and Postdoctoral Studies  (Experimental Medicine)     THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     July 2016     © Lauren De Luca, 2016    	   ii	  ABSTRACT  Human placental development is a complex process dependent on continuous cross talk between fetal and maternal uterine compartments. Trophoblasts, placental cells of fetal epithelial lineage, invade the maternal uterine lining, forming the maternal-fetal interface. This interface is vital for controlling nutrient, gas, and waste exchange between maternal and fetal circulatory systems. The differentiation of progenitor trophoblasts into highly-specific cell subsets important for placental function is a highly-regulated developmental process. Trophoblast differentiation into invasive subtypes is essential for enhancing optimal uterine artery physiology, maternal-fetal nutrient exchange, and immunotolerance in pregnancy. Trophoblast invasion is controlled by cell-cell and cell-matrix interactions, as well as through production of various growth factors, cytokines, and hormones. Insufficient trophoblast invasion results in insufficient arterial remodeling that underlies cellular events responsible for the development of pregnancy disorders such as preeclampsia and fetal growth restriction.  A Disintegrin and Metalloproteinase 28 (ADAM28) is a multi-functional protein belonging to the metzincin superfamily of metalloproteinases. ADAM28 exists as two alternative splice variants: a full-length transmembrane form (ADAM28-L) and a truncated secreted variant (ADAM28-S). Recently, ADAM28 has been shown to be differentially expressed in two distinct trophoblast subtypes ex vivo; however despite this knowledge, little is known about the role of ADAM28 in placental development or trophoblast biology. My research has demonstrated that both ADAM28 isoforms are highly expressed in invasive trophoblasts of distal regions in placental columns. Utilizing a loss-of-function strategy, I demonstrate that ADAM28 promotes trophoblast column outgrowth, and directs trophoblast cell migration and survival. Collectively, these findings describe a novel role for ADAM28 in regulating the differentiation of progenitor trophoblasts into extravillous trophoblast subsets and highlight ADAM28 as a key protease in human placental development.  	   iii	  PREFACE  Dr. Alexander G. Beristain and myself generated the hypothesis for this work. Dr. Alexander G. Beristain and I designed the experiments in Chapters 3 and 4. I performed the experiments in Chapter 3, except for the FACS purification of placental cells, which was performed by both Sofie Perdu and myself. I conducted the experiments in Chapter 4, with help from Dr. Alexander G. Beristain and Dr. Hoa Le. Dr. Barbara Castellana and myself collected and processed tissue specimens for this study. Dr. Alexander G. Beristain, Dr. Hoa Le and I collected and analyzed data. Future publication will arise from work presented in Chapters 3 and 4 of this thesis. This thesis was read and approved by Drs. Alexander G. Beristain, Julian Christians, and Calvin Roskelley.  This research was conducted with ethical approval from the University of British Columbia/Children’s and Women’s Health Centre of British Columbia Research Ethics Board (Certificate H1300640).                      	   iv	  TABLE OF CONTENTS   ABSTRACT………………………………………………………………………………..………………ii PREFACE…………………………………………………………………………………………………iii TABLE OF CONTENTS…………………………………………………………………………………iv LIST OF TABLES………………………………………………………………………………….……vii LIST OF FIGURES……………………………………………………………………..………………viii LIST OF ABBREVIATIONS………………………………………………………………………… …ix ACKNOWLEDGEMENTS………………………………………………………………………………xii DEDICATION……………………………………………………………………………………………xiii CHAPTER 1. INTRODUCTION…………………………………………………………………………1 1.1 Overview…………………………………………………..………………………………………1 1.2 Implantation and placental development………………………………………………….…..3 1.2.1 Uterine decidualization……………………………………………………………….…4 1.2.2 Blastocyst implantation……………………………………………………………….…5 1.2.3 Early placental development…………………………………………………………...6 1.3 Models to study human placentation…………………………………………………...…….10 1.3.1 Animal models………………………………………………………………………….10 Rodent models…………………………………………………………...…..11 Non-human primate models………………………………………..………12 1.3.2 Ex vivo and in vitro models……………………………………………………………13 Primary trophoblast cells and villous explant cultures…………………...13 Immortalized trophoblast cell lines…………………………………………15 Choriocarcinoma cell lines…………………………………………..…...…16 1.4 Metzincin metalloproteinases regulating trophoblast invasion…………………….………17 1.5 The role of ADAMs in Development and disease……………………………………..……19 	   v	  1.5.1 ADAM deficient mice………………………………………………………………..…20 1.5.2 ADAMs in placental development……………………………………………………21 1.6 ADAM28……………………………………………………………………………..………..…23 1.6.1  Catalytic activity of ADAM28……………………………………………………...….24 1.6.2  ADAM28 in Cancer……………………………………………………………..…..…25 1.7 Hypothesis and rationale……………………………………………………………..…..……26 CHAPTER 2 MATERIALS & METHODS…………………………………………………….………28       2.1 Placental tissue collection…………………………………………………………….….……28       2.2 FACS purification of placental cells………………………………………………………..…28       2.3 RNA purification and qPCR analysis…………………………………………………………29       2.4 Immunofluorescence microscopy………………………………………………….…………30       2.5 Cell culture…………………………………………………………………………….……..…31       2.6 Cell lysis and immunoblot analysis…………………………………………………..……….32       2.7 ADAM28 siRNA transfection………………………………………………………….………33       2.8 Placental villous explant culture………………………………………………..…………..…33       2.9 Cell viability……………………………………………………………………………….….…35       2.10 Migration assay…………………………………………………………………………….…35       2.11 Statistical analyses………………………………...…………………………………….…...36 CHAPTER 3 EXAMINING ADAM28 EXPRESSION IN FIRST TRIMESTER TROPHOBLAST SUBTYPES………………………………………………………………………………………………38       3.1 Rationale……………………………………………………………………………….…..……38       3.2 Results……………………………………………………………………………….…….……39 3.2.1   ADAM28 localizes to HLA-G+ trophoblasts within first trimester anchoring  placental columns…………………………………………………………………...…39   3.2.2   ADAM28 expression in trophoblast cell lines………………………………………46       3.3 Summary……………………………………………………………………………………..…49 	   vi	   CHAPTER 4 EXAMINING THE ROLE OF ADAM28 IN TROPHOBLAST BIOLOGY……….…50       4.1 Rational………………………………………………………………………………………….50       4.2 Results…………………………………………………………………………….…………….51 4.2.1   Loss of ADAM 28 reduces trophoblast column outgrowth……………………….51 4.2.2   ADAM28 promotes JEG3 cell migration……………………………………………53 4.2.3   ADAM28 promotes JEG3 cell proliferation and survival………………….………56 4.2.4   ADAM28 promotes cell survival in ex vivo placental explants………………...…58       4.3 Summary………………………………………………………………………….…………….60 CHAPTER 5 DISCUSSION……………………………………………………………………………61       5.1 ADAM28 expression in first trimester trophoblast subtypes………………………….……61       5.2 The role of ADAM28 in trophoblast biology……………………………………………....…65       5.3 Study limitations and shortcomings………………………………………………………..…68       5.4 Experimental Strategy……………………………………………………………………....…70       5.5 Importance of Findings (Biological inferences of ADAM28 in healthy pregnancy) …..…71       5.6 Summary………………………………………………………………………………….….…72 REFERENCES…………………………………………………………………………………………..74          	   vii	  LIST OF TABLES Table 2.1 Accession numbers and target sequences for ADAM28-directed siRNAs…………...33 Table 2.2 Antibodies used in this thesis…………………………………...…………………………37                        	   viii	  LIST OF FIGURES  Figure 1.1 Illustration of trophoblast differentiation………………………………………………..…7 Figure 1.2 Structure of ADAM28-L and ADAM28-S isoforms……………………………………..24 Figure 2.1 Inverted images of first trimester chorionic villous explant imbedded on Matrigel.....34 Figure 3.1 Isolating flow cytometry purified trophoblast populations….…………………………..41 Figure 3.2 ADAM28 localizes to invasive column extravillous trophoblasts (colEVTs) in first trimester human placenta ………………………………………………………………………….…..43 Figure 3.3 ADAM28 localizes to HLA-G+ distal column trophoblasts in vivo………………..…..45 Figure 3.4 ADAM28 is preferentially expressed in choriocarcinoma trophoblastic cell lines modeling column EVTs……………………………………………………………………………...….48 Figure 4.1 Loss of ADAM28 inhibits trophoblast column outgrowth………………………………52 Figure 4.2 ADAM28-siRNA mediated knockdown in JEG3 cells………………………………….54 Figure 4.3 ADAM28 promotes JEG3 cell migration…………………………………………………55 Figure 4.4 ADAM28 promotes JEG3 cell proliferation and cell survival………………………….57 Figure 4.5 ADAM28 promotes cell survival in ex vivo placental explants……………………..…59 Figure 5.1 Schematic diagram demonstrating ADAM28 localization in human placental villi….62          	   ix	  LIST OF ABBREVIATIONS  ADAM    A Disintigrin and Metalloproteinase  ADAMTS   A Disintigrin and Metalloproteinase with ThromboSpondin motif  cAMP    Cyclic adenosine 3’-5’- monophosphate  Cdx2   Caudal type homeobox 2  CNS   Central nervous system  CTGF   Connective tissue growth factor  DAPI   4’, 6-Diamidino-2-phenylindole   ECL   Enhanced chemiluminescence  ECM   Extracellular matrix  EGF   Epidermal growth factor  ErbB   Erb-b2 receptor tyrosine kinase  EVT   Extravillous trophoblast  FACS   Fluorescence activated cell sorting  FBS   Fetal bovine serum  GCM1   Glial cell missing homolog 1  HB-EGF  Heparin-binding EGF-like growth factor  HBS    HEPES buffered saline   HBSS   Hank’s balanced salt solution  hCG   Human chorionic gonadotropin  HELLP   Hemolysis elevated liver enzymes  HER   Human endogenous retroviris  HLA-G   Major histocompatibility complex, class I, G  hPL   Human placental lactogen  HRP    Horseradish peroxidase  	   x	   hTERT   Human telomerase reverse transcriptase   ICM    Inner cell mass  IGF   Insulin-like growth factor  IGFBP   Insulin-like growth factor binding protein  IL   Interleukin  IUGR   Intrauterine growth restriction  KRT7   Cytokeratin-7  LIF   Leukemia inhibitory factor  MBP   Myelin basic protein  MDC   Metalloproteinase disintegrin cysteine-rich  MEK   Mitogen-activated protein kinase kinase  MMP   Matrix metalloproteinase  mTOR   Mammalian target of rapamycin  NS   Non-silencing   OCT   Optimal cutting temperature compound  PAPP-A  Pregnancy-associated plasma protein-A  PBS   Phosphate-buffered saline  PCDG   Protocadherin  PE   Preeclampsia  PFA    Paraformaldehyde  PI3K   Phosphatidylinositol 3-kinase  PSGL-1  P-selectin glycoprotein ligand -1  RT    Room temperature   siRNA   Small interfering RNAs  SV40   Simian Virus 40  	   xi	  SVMP   Snake venom metalloproteinases  synCT   Syncytiotrophoblast  TGC   Trophoblast giant cells  TIMP   Tissue inhibitor of metalloproteinase  TNFα   Tumor necrosis factor - α  uNK   Uterine natural killer   uPA    Urokinase plasminogen activator  vCT   Villous cytotrophoblast  vEGF   Vascular endothelial growth factor                   	   xii	  ACKNOWLEDGEMENTS  I would like to express my sincere gratitude to my supervisor, Dr. Alexander G. Beristain, for his continuous support, encouragement, and guidance throughout my MSc studies. To my supervisory committee members, Drs. Julian Christians and Calvin Roskelley, I am extremely grateful for all of your unwavering guidance, suggestions, and encouragement over the past two years. To Dr. Vincent Duronio, my sincerest thanks for your caring support.  To all the current and former members of Beristain Lab: Mahroo Aghababaei, Sofie Perdu, Grace Lai, Kathy Chan, Taylor Bahen, and Drs. Hoa Le and Barbara Castellana, thank you so much for all of your help and kindness along the way. I am so grateful to have been surrounded by such caring, hardworking women. You have made my time at CFRI unforgettable. A special thank you to Dr. Hoa Le, for being my mentor. You’ve taught me more than you know.  I would like to extend my utmost gratitude to my family and loved ones for their unconditional love and endless encouragement. To my Mom and Dad, thank you for believing in me and motivating me to persevere. I strive every day to emulate your hard work. Every obstacle I overcome and every goal I achieve is because of your love and support. To my beloved brothers Francco, Michael and Robert, thank you for making me smile and for shaping me into the person I am today.  Finally, I wish to express a sincere thank you to all the women who participated in our study. This project could not have been completed without you.       	   xiii	        Dedicated to my beloved parents, Pasquale and Celeste                             	   1	  Chapter 1. Introduction   1.1 Overview  Normal human reproduction is an inefficient process, where clinical pregnancy rates are a mere 25% per monthly cycle, and pregnancy loss rates are 32% for all conceptions1–3. Implantation failure undoubtedly contributes to the inefficient nature of human reproduction; however, placentation during early gestation is one of the most critical determinants of pregnancy outcome 4.  Abnormal placental development and subsequent inadequate fetal-maternal interactions can lead to many adverse pregnancy-related complications including miscarriage, preterm birth, preeclampsia, intrauterine growth restriction (IUGR), and intrauterine death4–6. Thus, the formative steps of placental development are pivotal for a successful pregnancy. The placenta serves a multitude of vital roles for the survival and health of both embryo and mother. It anchors the conceptus to the uterus, creating the maternal-fetal interface and securing adequate oxygen and nutrients to the fetus4,7. The placenta serves as an endocrine organ, synthesizing a broad range of pregnancy-associated hormones and growth factors, altering maternal physiology to support the growth and development of the fetus and parturition7,8. Importantly, the placenta acts as an immunological barrier, preventing its rejection by the maternal immune system and ensuring the protection of the fetus from maternal immune attack8–10. The human placenta is a highly specialized organ that bridges maternal and fetal circulatory systems by complex cellular interactions4.  It is a haemochorial villous organ whereby maternal blood directly interacts with fetal-derived epithelial cells of the placenta called trophoblasts11. Compared to all eutherian mammals, the human placenta is the most invasive placenta type8,12. Highly invasive trophoblasts extensively infiltrate uterine tissue and blood vessels, and by doing so, establish the placenta as the site of transfer of nutrients, gases and wastes between the mother and developing fetus4,9. The differentiation of trophoblast 	   2	  progenitors into highly specialized terminally differentiated cell subsets is essential for optimal placental function. In particular, the acquisition of pro-invasive cellular characteristics facilitates placental attachment to uterine tissues and regulates many of the physiological changes that occur within the fetal-maternal interface by extensive uterine infiltration and maternal arterial remodeling. Notably, the underlying cellular and molecular processes important in controlling trophoblast differentiation into invasive cell subsets are under strict control; however an in-depth knowledge of critical pathways central to trophoblast differentiation does not exist.  Proteases play key roles in cell migration and invasion and additionally promote biological processes such as angiogenesis, leukocyte mobility and embryonic development 13–15. Additionally, protease systems play fundamental roles in tumorigenesis, controlling cell proliferation, growth, survival and cancer cell metastasis to distant organ sites 16,17. Placentation involves many, if not all, of the above described cellular processes and not surprisingly requires regulated expression of proteases by diverse subtypes of trophoblasts. Protease-derived trophoblasts play roles in coordinating cell differentiation, migration, invasion, and tissue remodeling12.  Among these proteases controlling trophoblast function are the metzincins, a superfamily of zinc-dependent metalloproteinases, including matrix metalloproteinases (MMP) and the a Disintigrin and Metalloproteinase (ADAM) families18. MMPs are widely accepted for their critical role in trophoblast invasion because of their ability to degrade extracellular matrix (ECM) thereby promoting cell movement through uterine stroma18,19. Alternatively, ADAMs primarily act as surface “sheddases” that function analogous to molecular scissors; ADAMs cleave various growth factors, hormones, and cytokines, and thus have numerous physiological and pathological implications in diverse cell systems such as central nervous system (CNS) development, neovascularization, the inflammatory response, and tumorigenesis20. Notably, multiple ADAM proteases (ADAM9, -10, -12, -15, -17, -19, and -28) play critical roles in tumor formation and progression20. However, the function of ADAM proteases in trophoblast biology remains poorly understood.  	   3	  A recent study comparing global gene expression in distinct trophoblast subpopulations identified ADAM8, -12, -19, and -28 to be highly expressed in invasive trophoblasts21,22; however, few studies have elucidated the importance of these genes in trophoblast biology. Over recent years, our lab has investigated the importance of members of the ADAM family in directing key cellular events critical in early placental development. Specifically, our lab has shown that ADAM12 is highly expressed by invasive trophoblast subsets and plays critical roles in directing cell invasion and cell-cell fusion23,24. Additionally, preliminary data show mRNA levels of another ADAM family member, ADAM28, are abundantly expressed by highly invasive as well as proliferative trophoblast populations within first trimester placenta. Building upon these findings, my master’s thesis research aims to interrogate the role of the ADAM family member ADAM28 in trophoblast differentiation and function.  The following section will provide an overview of early human gestational development, highlighting blastocyst implantation and placentation, as well as the research models currently used to study placental development and trophoblast function. Additionally, I will describe the importance of metzincin metalloproteinases and their role in regulating trophoblast differentiation into invasive trophoblast subsets. The ADAM family of metalloproteinases will be introduced and their diverse roles in physiological and pathological processes will be described. Lastly, the importance of ADAMs expressed in placental development, notably ADAM28, will be discussed.  1.2 Implantation and placental development  Successful implantation is contingent on the proper development of both the embryo and an endometrium that is receptive to the embryo25.  After fertilization, the one-cell zygote undergoes a series of mitotic cell divisions, eventually forming a differentiated tissue called the blastocyst26. The blastocyst is comprised of two distinct cellular populations: the pluripotent inner cell mass (ICM) and an outer layer of polarized epithelial trophectoderm surrounding the 	   4	  ICM26,27. The ICM gives rise to fetal and embryonic tissues, whereas the placenta and embryonic membranes are derived from the trophectodermal cells, thereby establishing the primary interaction with the uterine epithelium26.   1.2.1 Uterine decidualization The process of implantation is initiated by synchronized cross-talk between the blastocyst and luminal epithelia of the uterus. Uterine receptivity, the time during which the uterine environment is conducive to blastocyst acceptance and implantation, exists only briefly, and is termed the “window of implantation”, extending from 6-10 days post ovulation28–30. In preparation for implantation, the endometrial stroma (supportive cellular architecture consisting of fibroblastic cells and tissue-resident immune cells) undergoes extensive differentiation through a process referred to as decidualization. A number of cellular and vascular changes occur as uterine mesenchymal lineage-derived stromal cells differentiate into enlarged/secretory “decidual cells.” Decidualization is also characterized by the swelling of connective tissue and uterine vasculature, and extensive restructuring of the ECM11,31,32. Concurrently, the decidual reaction also leads to drastic changes in uterine leukocyte content characterized by massive infiltration of primarily innate immune cell subsets such as macrophages and uterine natural killer (uNK) cells; together macrophages and uNK cells facilitate uterine angiogenesis and maintain maternal-fetal tolerance31,33. These processes are regulated by maternal steroid hormones estrogen and progesterone, as well as growth factors and cytokines, establishing a unique uterine environment to support blastocyst attachment, growth, and succeeding events of implantation25,26,33. The molecular interactions facilitating embryo/uterine intercommunication are further described in the following section.     	   5	  1.2.2 Blastocyst implantation The process of embryo implantation involves three key phases: apposition, adhesion, and invasion. During apposition, the initially free-floating blastocyst positions itself adjacent to the endometrium, leading to weak/unstable adhesion to the apical surface of the uterine lumen25,32. Thereafter, adhesion of the blastocyst to the uterine wall is strengthened through engagement of crucial cell-cell and cell-matrix interactions; these interactions will be described in more detail below. It is thought that the blastocyst’s outer trophectodermal layer adheres to the receptive luminal epithelium by establishing contact via micro protrusions on the apical surface of the endometrial epithelium called pinopodes25,27. Although the exact role of pinopodes in human fertility remains unknown, it is suggested that the human blastocyst preferentially adheres to pinopode-presenting cells in vitro28. Following the establishment of blastocyst-endometrial interaction, trophectodermal penetration and invasion into the uterine stroma initiates the establishment of the early fetal-maternal interface34. Primitive multinucleated trophoblasts, referred to as the primitive syncytium, invade the uterine wall and superficial uterine stroma thus enabling embryo embedment beneath the uterine surface30,35.  The complex cell-cell interactions occurring during blastocyst implantation are regulated by a variety of molecules, including cell-adhesion molecules, growth factors, cytokines, chemokines, and hormones; these molecules play a critical role in preparing the blastocyst and endometrium receptivity25. Selectins and integrin families of adhesion receptors and their ligands are considered to mediate the initial apposition and stable adhesion of the blastocyst33,36,37. Specifically, adhesion molecule L-selectin plays a functional role in binding receptors expressed on trophoblasts to oligosaccharide ligands expressed on uterine luminal epithelium during apposition27,33. Following the initial connection mediated by L-selectin, integrin adhesion molecules α1β1, α4β1, and ανβ3 are believed to play a critical role in initiating a stable blastocyst attachment to the endometrial lining29,33,35. Cytokines and growth factors present in the embryo-maternal interface, including leukemia inhibitory factor (LIF), Interleukin-11 (IL-11), 	   6	  and the heparin-binding epidermal growth factor/Erb-b2 receptor tyrosine kinase 4 (HB-EGF/ErbB4) complex, have been shown to enhance blastocyst development, promote adhesion of the blastocyst and uterine epithelial surface, and drive decidualization processes 30,32,33. These key roles during implantation are thought to be regulated by α2 integrin expression at the human implantation site 33. Studies have shown that aberrant or altered expression of these factors is linked to recurrent miscarriage and infertility29,32,38.  1.2.3 Early placental development After successful implantation, cellular processes contributing to placenta establishment are initiated. During placentation, progenitor cytotrophoblasts residing within the trophectodermal layer differentiate along one of two main cellular pathways: (i) the villous pathway or (ii) the invasive pathway8,39 (Figure 1.1A and B). These distinct pathways give rise to all distinct trophoblast subtypes found within the fetal-maternal interface and are thus critical for placental function. It is widely accepted that early in gestation, cytotrophoblasts are bipotential progenitors capable of differentiating into either of these pathways, however the factors that drive selective differentiation remain poorly described40–42. Notably, in contrast to their term placenta equivalents, first trimester cytotrophoblasts favor the invasive pathway over the villous pathway, suggesting that trophoblast cell differentiation is dynamic and alters throughout gestation8. During villous pathway differentiation, villous cytotrophoblasts (vCTs) establish an epithelial monolayer characterized by mitotically active vCTs 8,40,42. It is thought that proliferating vCTs undergo a unique process of cell-cell fusion with neighboring vCTs to form an overlying multinucleated cell layer called the syncytiotrophoblast (synCT)8 (Figure 1.1B). The synCT is mitotically inactive but regenerates, grows, and is maintained by continuous fusion of underlying vCTs with the overlaying syncytium; this growth/renewal is in part balanced by shedding of apoptosed synCT fragments called syncytial knots into the blood-filled intervillous space8,43. 	   7	                       Figure 1.1 Illustration of trophoblast differentiation. (A-C) Mononuclear villous cytotrophoblasts differentiate into multinucleated syncytiotrophoblasts (B) or invasive EVTs (C). Along the invasive pathway, trophoblasts differentiate and migrate into and remodel decidual stroma and arterioles. Trophoblast-mediated remodeling of both maternal stroma and vasculature is essential for establishing adequate blood flow to the placenta and growing fetus. EC, endothelial cell; EG, endometrial gland; cEVT, columnar extravillous cytotrophoblast; eEVT, endovascular extravillous cytotrophoblast; iEVT, interstitial extravillous cytotrophoblast; Mϕ, macrophages; mSA, maternal spiral artery; synCT, syncytiotrophoblast, TGC, trophoblast giant cell; uNK, uterine Natural Killer cell; vCT, villous cytotrophoblast. 	   8	  The syncytial layer is essential for facilitating the exchange of gases, nutrients and waste across the maternal-fetal interface8, and is the primary source of growth factors, cytokines and hormones, such as human chorionic gonadotropin (hCG), human placental lactogen (hPL) and progesterone, which together are required for maintenance and immunological adaptation of pregnancy44–46. Activation of the transcription factor glial cell missing homolog 1 (GCM1) and two of its downstream targets, the human endogenous retrovirus (HERV) envelope proteins Syncytin-1 and -2, are thought to be the primary molecular drivers initiating trophoblast syncytialization47–50. Reduced expression levels of these factors has been linked to severe  pregnancy-associated anomalies including preeclampsia (PE) and hemolysis, elevated liver enzymes, low platelet count (HELLP) syndrome, highlighting the importance of these molecules in placentation and suggesting that insufficient placental function due to inadequate syncytialization is linked to severe complications of pregnancy51,52. Trophoblast differentiation along the invasive pathway initiates at locations of placental-uterine attachment characterized by cellular projections (columns) located at the tips of first trimester chorionic villi; collectively these villi are referred to as anchoring villi4,14. At these attachment points, vCT progenitors adopt a highly proliferative phenotype forming a multi-layer cellular column of cells, herein referred to as the trophoblast column 42. Columnar trophoblast at the distal portion of the anchoring column breach the syncytial layer and differentiate into invasive extravillous trophoblast (EVT) subtypes responsible for uterine stromal and vascular remodeling34,40,8 (Figure1.1A and B). Invasive trophoblasts acquire expression of distinct EVT markers, such as HLA-G (major histocompatibility complex protein), α1 and β1 integrins, and ECM-degrading proteases MMP2 and MMP953,54. Additionally, invasive EVT subtypes exhibit expression of specific integrins, including loss of α6β4 integrin expression within the distal trophoblast column and the induction of integrin α5β1 within distal column trophoblasts 42,55,56. EVT-specific α1 and β1 integrins are thought to associate with ECM-associated proteins, 	   9	  promoting cell invasion and migration, as well as the stabilization of the growing and differentiating placental cell column.  Column trophoblasts give rise to two distinct populations of EVTs: interstitial EVTs (iEVTs), and endovascular EVTs (eEVTs). iEVTs invade into and remodel maternal uterine stroma, while eEVTs migrate into uterine spiral arteries and physically replace blood vessel endothelium, thus promoting the morphogenesis of narrow/vasoconstrictive arteries to dilated wide-bore conduits essential for increasing the volume of blood delivered to the intervillous space8,43 (Figure 1.1B and C). Maternal factors produced by leukocytes, including macrophages and uterine natural killer (uNK) cells, as well as from secretions by endometrial glands, namely epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), promote trophoblast differentiation along the invasive pathway6,22,33. iEVT and eEVT invasion into maternal tissue continues as far as the inner third of the myometrium, where terminal differentiation along the invasive pathway is characterized by the formation of multinucleated  trophoblasts called trophoblast giant cells (TGC)31,57. This cellular event is thought to limit excessive trophoblast invasion, and is regarded as the last step along extravillous pathway11. Although the exact function of TGCs remains unknown, evidence suggests that they contribute to the production of local pregnancy-associated hormones, such as hCP and hPL, and therefore support the maintenance of healthy pregnancy57,58.   It is noteworthy that trophoblast differentiation along the villous and invasive pathways initially occurs in a low oxygen environment equalling 1-3% oxygen 6,59,60. This hypoxic environment, lasting up to 10 weeks of gestation, is thought to be important in limiting trophoblast invasion/vascular remodeling, while concurrently promoting trophoblast proliferation and limiting fetal tissue damage to reactive oxygen products11. At approximately 11 weeks of gestation, trophoblast-directed breaching of maternal blood vessels leads to a spike in oxygen levels (∼8%) at the fetal-maternal interface that is thought to accelerate invasive characteristics of trophoblasts 59,60. Deficient or inadequate EVT invasion and arterial remodeling is associated 	   10	  with pregnancy disorders linked to placental insufficiency and hypoxia, including maternal hypertension, preeclampsia (PE) and intrauterine growth restriction (IUGR)5,6,35.    1.3 Models to study human placentation The early stages of human implantation and placental development are a crucial period for establishing a healthy pregnancy, however these formative stages are largely unknown since access to samples are extremely limited for obvious ethical reasons. However, numerous in vivo animal models and in vitro tissue and cell culture systems have been developed to provide important insight into both cellular and molecular events that occur during gestation and throughout early pregnancy.  1.3.1 Animal models  Animal models of placentation have been successfully used to study specific processes of placental development such as cell invasion, vascular remodeling, cell fusion, trophoblast-immune cell interaction, as well as gas, nutrient, and drug delivery. In order to effectively evaluate these processes across different species, it is important to consider the components of the interhemal barrier functioning throughout gestation 61. There are three main structural types of placenta in mammals according to the cell layers that comprise the interhemal area: i) epitheliochorial, ii) endotheliochorial, and iii) hemochorial type 62. In the epitheliochorial type of placenta, seen in horses, pigs, and ruminants (including sheep and cows), uterine tissue apposed to the trophoblast layer remains intact and is directly exposed to trophoblast cells throughout the duration of pregnancy61. This is the most superficial type of placenta, where maternal blood does not come into direct contact with the chorionic villus, and the uterine lining lacks substantial invasion or destruction, and no layers are removed62. These species are therefore considered useful models for initial blastocyst apposition and attachment phases in 	   11	  early gestation63. An endotheliochorial placenta refers to loss of uterine epithelium resulting in direct contact of trophoblasts with the maternal endometrium61,62. Carnivores possess an endotheliochorial placenta, where trophoblasts are able to partially erode the maternal epithelial layer and underlying connective tissue64. Humans, most non-human primates, and rodents (mice and rats) have hemochorial placentae, which are the most invasive62. In hemochorial placentae, maternal blood directly bathes trophoblast cells, leading to extensive erosion of all maternal tissue layers61,62 Although animal models may not perfectly represent human implantation and trophoblast invasion, they prove to by very useful tools in elucidating the molecular and physiological events involved in maternal-fetal interactions described in the following sections. Rodent models Rodents have been used extensively as experimental models of human reproduction due to their size, high fecundity rates, brief gestation period, low cost of maintenance, as well as diverse sets of tools allowing for sophisticated genetic manipulation61. They are frequently used to examine blastocyst and trophoblast lineage development, and genetic and transgenic studies utilizing knockout and over-expression models have provided researchers powerful platforms to identify essential molecular processes in placentation63. For example, the fibroblast growth factor (FGF) signaling pathway has been shown to be essential in trophoblast stem cell maintenance, where mutations in FGF4/Fgfr2 proteins or in downstream targets – specifically the transcription factors caudal type homeobox 2 (Cdx2) and eomesodermin (Eomes) – cause defects in trophoblast stem cell formation and trophectoderm differentiation65–69. While FGF signaling is important for maintaining trophoblast stemness, suppression of FGF signaling leading to the expression of GCM1, Hand1, and Stra13 transcription factors initiates trophoblast lineage maturation70. Whether pathways identified as being essential in placental development using mouse genetics can also be applied to human placental development requires further 	   12	  study. However, gene expression studies have identified gene homologues in both human and mouse that share key roles in placentation. For example, the retroviral envelope proteins syncytin-1 and -2 (human) and syncytin-A and –B (mouse) play critical roles in trophoblast fusion and synCT morphogenesis during placental development48,51,71. The structural and functional homologies and dissimilarities between rodent and human placenta are well understood, and provide insight into the appropriateness of rodents as experimental models of human implantation and placentation. Although rodents and humans share similar hemochorial placentae apposed to a well-decidualized environment, there are several key differences. During implantation, mouse and rat uterine epithelium is displaced prior to trophoblast penetration of the basal lamina (displacement implantation) whereas human trophoblasts penetrate between epithelial cells (intrusive implantation)63,72. Moreover, within the rodent labyrinth (the region of maternal-fetal exchange), three layers of trophoblasts in the interhemal membrane separate materal and fetal circulations, compared to only one synCT layer and one vCT layer forming the human placental barrier, highlighting the disparities in trophoblast subset organization between rodent labyrinth structures and human villous tree73. As in humans, both mouse and rat trophoblasts invade via two routes: A vascular and an interstitial route31,72. It is important to note that in rats, spiral arteries delivering maternal blood to the placenta are less abundant than in humans where hundreds of spiral arteries contribute to maternal-fetal exchange 31. Taking into account these disparities, it may be difficult to draw analogies from findings obtained from rodent models and determine their usefulness in physiological mechanisms in healthy human implantation and placentation. Non-human primate models    Non-human primates are considered the most closely related animal model to study early human pregnancy and placental development63,74. As with humans, non-human primates possess invasive hemochorial placentae; however invasion remains superficial and is 	   13	  significantly shallower than in human placentae, leading to less extensive decidual erosion and shaping of the basal plate 31. An animal that is thought to particularly model humans is the great ape, where placental villous structure shows high concordance with human placentation as characterized by substantial trophoblast invasion5,73. Additionally, the baboon has proven to be an important model for studying molecular mechanisms in decidualization, as their pseudo-pregnancy can be artificially induced by infusion of hCG into the uterine cavity, initiating stromal fibroblast differentiation into a decidualized phenotype 63,75. Studies involving these non-human primates are extremely costly and undoubtedly are a great ethical concern due to their genetic likeness with humans73.  Furthermore, due to the close phylogenetic association between humans and nonhuman primates, human reagents, particularly antibodies, cross-react with non-human primate tissues, leading to unsatisfactory results from some antibodies reacting to irrelevant antigens within these tissues76.  1.3.2 Ex vivo and in vitro models In order to apply the findings from animal model studies to the events of human placenta development, a number of ex vivo and in vitro models have been developed. Human placental tissues and trophoblastic cell lines have been commonly used in examining the cellular and molecular mechanisms contributing to trophoblast differentiation, further described in the following sections. Primary trophoblast cells and villous explant cultures Primary trophoblast cultures are important tools for analyzing cellular processes in placental development. Primary trophoblasts isolated from first trimester placental specimens can be instructed to differentiate along an EVT-like pathway through exposure to specific distinct ECM substances like Matrigel, fibronectin and collagen. ECM-integrin engagement is thought to accelerate differentiation of trophoblasts along the invasive pathway, where primary 	   14	  trophoblasts propagated in vitro express invasive EVT-specific markers like HLA-G (major histocompatibility complex protein), α1 and β1 integrins, and ECM-degrading proteases MMP2 and MMP953,54. Primary trophoblast cultures allow for standard molecular manipulations, such as gene transfection or siRNA-mediated gene silencing, and thus establish simple single-cell based systems to gain mechanistic insight into the roles of individual genes and gene pathways in trophoblast biology.  Trophoblasts isolated from human term placenta specimens are useful for studies examining trophoblast terminal differentiation along the villous pathway, where mononuclear term trophoblasts spontaneously fuse in culture to form multinucleated structures resembling the syncytiotropholast77,78. Thus primary trophoblast cultures derived from term placentae facilitate investigation of key molecular mechanisms and pathways involved in trophoblast differentiation and syncytialization8,77–79. Although primary trophoblast models are thought to closely resemble and maintain the molecular pathways important in trophoblast function in vivo, limitations in these models still exist. For example, most primary cultures are established and maintained as single cell types depleted of other cell types such as immune cell populations, thus greatly simplifying the heterogeneous cell make-up existing in vivo. Further, primary trophoblast cultures are normally short-lived and have limited passage capacity, and undergo senescence following terminal differentiation, making such cultures challenging to work with8,54. Placental tissue culture systems, also referred to as placental explants, additionally serve as a primary culture platform used to study placentation 80,81. Placental explants derived from first trimester placental tissue imbedded in Matrigel ECM allow for the precise examination of trophoblast differentiation along the invasive pathway 55,56. For example, Matrigel-imbedded placental explants recapitulate trophoblast column formation as well as iEVT invasion within a three dimensional context and greater cell complexity than in two dimensional cell culture systems80–82. Molecular characteristics conserved between explants and in vivo placentation include: the loss of α6β4 integrin expression within the distal trophoblast column; the induction 	   15	  of integrin α5β1 within distal column trophoblasts and iEVTs; and the de novo expression of HLA-G by invasive trophoblast42,55,56. Although explants provide excellent systems to phenocopy aspects of in vivo placental development, they are difficult to genetically manipulate and must be studied immediately after establishment, thus limiting their experimental accessibility. Immortalized trophoblast cell lines Many human trophoblast cell lines have been developed to study trophoblast function. Of these, several cell lines have been established from first trimester placentae where trophoblast cells are immortalized by the expression of exogenous genes, like the Simian Virus 40 (SV40) or human telomerase reverse transcriptase (hTERT)55,83. Among these are HTR8/SVneo, SGHPL-4, SGHPL-5, and Swan 71 cell lines 83–85.  The EVT-derived HTR8/SVneo cells display an unlimited life span in culture, and express some molecular signatures common to the invasive trophoblast cell type, including EVT specific integrins such as α1 and β1 integrins85–87. However, HTR8/SVneo cells produce mesenchymal cell marker vimentin, where negative vimentin expression is indicative of primary trophoblasts 88–90. HTR/SVneo cells also lack trophoblast marker cytokeratin-7, although it is thought to be re-expressed when stimulated by growth on different matrices 91,92. SGHPL-4 and -5 are well-characterized, primary trophoblast-derived cell lines that preserve some EVT features including integrins associated with the invasive phenotype like α3, α5, αvβ3, and β184,93,94. Similar to HTR8/SVneo, these cell lines express the mesenchymal cell marker vimentin, however they lack expression of trophoblast marker cytokeratin-7 or EVT-specific marker HLA-G 92,95. SGHPL-4 and SGHPL-5 cell lines have a limited passage lifespan as they eventually undergo senescence and are thus commonly used until passage twenty-five93,94. Another putative model of primary trophoblasts are SWAN 71 cells, which express both cytokeratin-7 and HLA-G83. Unlike SGHPL cells, SWAN 71 cells can be maintained for more than 100 passages, while still retaining attributes of primary first trimester trophoblast cells83.  	   16	   Choosing an appropriate cell model to study human trophoblast biology and differentiation by loss-of-function and gain-of-function experiments is critical to understanding different physiological and pathological processes contributing to healthy placental function. However, it is important to note differences to their primary counterparts. Prolonging the cellular lifespan of these cell lines in culture has lead to aneuploidy and changes in gene expression that are not observed in EVTs in vivo or in primary trophoblast cultures. Studies comparing the gene expression signatures of different cellular models show that there is a wide diversity of gene expression profiles amongst trophoblastic cell lines, and between primary EVTs82. Researchers therefore suggest that experiments carried out on a cell line of choice should be used with caution as a secondary model to primary cells 82. Choriocarcinoma cell lines  In addition to immortalized cells, there are a number of cell lines derived from choriocarcinomas, tumors of placenta trophoblast cells, including BeWo, JEG3 and JAR cell lines, which are valuable models in studying trophoblast biology. The first developed choriocarcinoma cell line, the BeWo cell line, maintains several cytotrophoblast-like features in culture such as expressing cytokeratin-7 and integrin α687,96. In addition to mimicking early trophoblast cell morphology, BeWo cells sustain functional hormone synthesis in vitro, producing the placental hormones hCG, hPL, progesterone and estrogens96,97. BeWo cells can be induced to syncytialize in culture by the addition of cAMP or forskolin, and are hence widely used as a fusogenic model of trophoblast syncytialization46,96–98  The JEG3 choriocarcinoma cell line is highly-proliferative and expresses multiple trophoblast-specific factors like hCG, hPL, progesterone, and HLA-G99–101. Based on this extravillous-like phenotype, JEG3 is commonly used as a model of primary columnar trophoblasts and EVTs99,100. Additionally, JEG3 cells have been used to examine placental hormone function on trophoblast invasion. 	   17	   JAR cells share many characteristics with early vCTs such as expression of cytokeratin-7 and integrin α6, while lacking the EVT-specific marker HLA-G82,87. In fact, JAR cells completely lack expression of any HLA class molecules, and therefore are considered better models of vCT cells than EVTs. JAR cells are able to differentiate into syCT-like cells in vitro, synthesizing hCG, hPL as well as a variety of steroid and peptide hormones102–104. Additionally, JAR cells constitutively synthesize various ECM-degrading enzymes103,104.  Trophoblastic cell lines are useful surrogates for primary trophoblasts for in vitro studies of trophoblast biology, providing insight into mechanisms controlling different paths of trophoblast differentiation in healthy and pathologic pregnancies. However, choriocarcinoma cells in culture, as with immortalized trophoblasts, do not truly represent primary trophoblast populations, and phenotypic or functional experiments performed on these cells should be properly interpreted.    1.4 Metzincin metalloproteinases regulating trophoblast invasion Similar to processes that involve cellular movement through tissues such as cancer cell invasion, the invasion of trophoblasts into decidual stroma necessitates the upregulation of diverse subtypes of proteases that help regulate ECM binding and degradation 12,105. Among these proteases are members of the metzincin metalloproteinase gene family which play pivotal roles in proteolytic degradation of decidual ECM and promote cell invasion18,22,35. Metzincins are characterized by a zinc-binding motif, HEXXHXXGXXH, present in the catalytic domain; the three histidine residues act as zinc ligands that bind to the catalytic zinc atom where glutamic acid acts as the catalytic base during hydrolysis106–108. Additionally, members of the metzincin family have a conserved methionine residue present in a downstream 1,4-β-Met-turn, which is critical for catalytic function and maintaining the structural architecture of the protease13,108,109.  	   18	  Members of the metzincin superfamily are subdivided at the structural level into seven families: adamalysins, astacins, leishmanolysins, matrixins, serralysins, snapalysins, and pappalysins107. These multidomain proteins are characterized by an N-terminal prodomain, a catalytic protease domain, and farther downstream domains engaged in protein-protein and cell-cell interactions and other regulatory functions107. Matrixins, comprising the endopeptidase matrix metalloproteinases (MMPs), are the main contributors of matrix proteolysis and degradation, and are known to play key roles in trophoblast invasion104. Both MMPs and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), are expressed and secreted by trophoblasts and together regulate cell invasion depth and extent of ECM remodeling.110. Perhaps the best described matrixins in regulating trophoblast invasion are the gelatinases, MMP2 and MMP9, and together with TIMP1, -2, -3, and -4, establish a central protease-inhibitor system fundamental to healthy placental development 110–112. For example, the importance of MMP9 in placentation was recently demonstrated using a mouse genetic knock-out strategy showing MMP9 deficient mice to have impaired trophoblast invasion and under-remodeled uterine arteries reminiscent of placental pathologies associated with pre-eclampsia 113. In addition to MMPs, other protease subtypes are highly expressed in the human placenta, one of which is pregnancy-associated plasma protein-A (PAPP-A)114. A member of the pappalysin subfamily, PAPP-A is a secreted protease that confines to vCT, synCT, and EVT subtypes, and is detectable in maternal serum throughout the duration of pregnancy114. Poor pregnancy outcomes such as preeclampsia, IUGR, and extreme premature delivery have been correlated to low levels of PAPP-A in maternal serum114,115. PAPP-A controls trophoblast biology in part by regulating the bioavailability of IGFs via cleavage of insulin-like binding growth factor proteins (IGFBP)-4 and -5; IGFBP-IGF complexes sequester IGFs preventing them from binding to their cognate receptors114,116. IGF signaling in trophoblasts promotes trophoblast invasion, cell 	   19	  growth and survival, and thus proteolytic processes regulating IGF gradients are considered to be essential to healthy placenta development114,116,117.  Most studies have focused on the significance of MMPs in trophoblast biology, however the adamalysin family has recently been recognized as an important protease family in controlling trophoblast differentiation. For example, A Disintegrin and Metalloproteinase with ThromboSpondin repeats (ADAMTS)-12, promotes trophoblast invasion through integrin-ECM interactions118. More recently, members of the ADAM family of proteases, namely ADAM10, -12, and -19, have been examined for their abilities to control trophoblast cell adhesion, migration, and signaling with respect to placental development in healthy and compromised pregnancies20.  1.5 The role of ADAMs in development and disease The ADAM family has emerged as a key proteases that promote ectodomain shedding, cleaving numerous growth factors, hormones, and chemokines from the surface of cells, and thus is implicated in controlling many biological and pathological processes20. ADAMs, along with ADAMTS and snake venom metalloproteinases (SVMPs), form the adamalysin subfamily20. Forty ADAM family members have been identified in the mammalian genome, 21 of which have been described in the human genome20. The typical active zinc-binding consensus site is encoded by only 12 human ADAM genes – ADAM8, -9, -10, -12, -15, -17, -19, -20, -21, -28, -30 and -33 – which are predicted to be functional metalloproteinases20.  ADAMs are approximately 750 amino acids in length and are characterized structurally by conserved protein domains that are predicted to control intrinsic protease activity, cell-cell and cell-matrix adhesion, and intracellular signaling cascades20. ADAM consensus domains are organized as follows: An N-terminal signal sequence, followed by a prodomain, metalloproteinase domain, disintegrin domain, cysteine-rich region, EGF domain, transmembrane domain, and cytoplasmic tail20. ADAM domain structure contributes to the function significance of these proteases in directing various cellular processes including surface shedding (metalloproteinase domain), cell-cell/cell-	   20	  matrix adhesion (disintegrin, cysteine-rich and EGF domains) and cell signaling (cytoplasmic tail) 20. The majority of ADAM proteases are transmembrane proteins, however a few subtypes express secreted gene splice-variants in addition to membrane-anchored isoforms20. Select ADAMs have been shown to be highly expressed during different stages of human pregnancy and development.  Their importance, as well as studies of their adhesive and proteolytic functions in knockout mice, will be described in the following sections.  1.5.1 ADAM deficient mice   Studies analyzing the phenotype of ADAM deficient mice have revealed important information regarding potential functions of these proteases in normal development. ADAM1a, ADAM2, and ADAM3 deficient mice are viable, however male mice show several defects in fertility, primarily due to impaired sperm biology. Specifically, sperm of ADAM1a, -2 and -3 knock-out mice show altered membrane protein modifications, have reduced sperm motility, and are characterized by impaired fusion with female gametes119,120. ADAM8 and ADAM12 are among the ADAM proteases that are prominently expressed by trophoblasts and maternal decidua during placentation 121,122. While ADAM8-/- mice show no major defects and no apparent pathological phenotypes, one third of ADAM12 deficient mice die in early embryonic stages121,123. Of the viable homozygotes from ADAM12-/- mice, 30% show defects in adipocyte function translating to reduced weight gain and fewer number of adipocytes; these changes are linked to enhanced insulin sensitivity compared to wild-type mice123.  Targeted ADAM deficiencies in mice have also shed light on their roles in the development of the central nervous system (CNS) and cardiovascular system. Deletion of ADAM10 results in early embryonic lethality at embryonic day (E)9 characterized by numerous defects in CNS, somites, and heart development, mimicking biological defects associated with impaired Notch signalling124. Similarly, ADAM15 and ADAM19 depletion leads to congenital heart defects and alterations in neovascularization125,126. ADAM17-/- mice are also non-viable, 	   21	  die perinatally, and are characterized by significant abnormalities in lung and myocardial development127,128. ADAM11 and ADAM22 are highly expressed in brain and nervous system, and are known to be essential for regulating neuronal death/survival and proper structure and function of the CNS129,130. ADAM11-/- mice show significant cognitive insufficiencies in spatial learning and motor control, while ADAM22 null mice suffer from severe ataxia and hypomyelination of peripheral nerves131,132.  In addition to studies of ADAM deficient mice, numerous studies of malignancy suggest that ADAM9, -10, -12, -15, and -17 play a role in tumor formation and progression, where focus has been on identifying key cellular and molecular processes promoting tumor cell migration, proliferation, and survival133. While the detailed mechanistic processes controlled by ADAM proteases in tumorigenesis are beyond the scope of this thesis, it is sufficient to say that many (if not most) of the cellular mechanisms fundamental to tumorigenesis including cell proliferation, invasion, growth, and survival are also highly conserved during placentation 54. Thus it is highly likely that cellular processes regulated in part by ADAM proteases in physiological and pathological conditions are conserved in human trophoblast biology and placentation. However, to date, very little is known with respect to the role of ADAM proteases in trophoblast biology.   1.5.2 ADAMs in placental development  The biological significance and function of ADAM proteases in placental development remains poorly understood, however there is increasing evidence that ADAM gene subtypes control crucial cellular processes contributing to successful gestation. This includes early studies of ADAMs in the formative stages of pregnancy in mice, including spermatogenesis and fertilization119,120,134. The immuno-localization of ADAM8, -9, -10, -12, -15, and -17 in the implantation site of uterine tissues in mice demonstrates that their expression levels change throughout gestation, suggesting a potential role in implantation or uterine remodeling during the 	   22	  peri-implantation period122. Additionally, there have been a few studies highlighting the significance of ADAM deficiency in pregnancy disorders, suggesting ADAM gene dysregulation may drive abnormal placental development. ADAM12, expressed as two isoforms, a membrane-bound (ADAM12L) and a secreted variant (ADAM12S), is highly expressed in the human placenta, where the secreted isoform is also detectable in maternal blood135. Associations between ADAM12S levels in maternal blood and fetal health exist, where low levels predict trisomy 18 and 21chromosomal abnormalities135,136. Low levels of maternal blood ADAM12S positively correlate with severe pregnancy disorders (i.e. preeclampsia, IUGR, and preterm birth) that are often associated with inadequate placental development, presumably due to the effect of ADAM12-reduction on trophoblast invasion and subsequent insufficient uterine remodeling; however the usefulness of ADAM12 as a reliable biomarker for these disorders remains controversial, as it has poor predictive ability137–139.  Both ADAM10 and ADAM17 expression are elevated in placentae from patients with preeclampsia, where ADAM10- and -17 directed cleavage of adhesion factors and cytokines, such as protocadherin (PCDH)12 and tumor necrosis factor-α (TNFα), is thought to contribute inadequate placental function characterized in preeclampsia development18,140,141,142. Apart from these associative studies, few studies have experimentally examined the role of ADAM proteases in trophoblast biology or placental development.  A microarray expression study comparing global gene signatures between EVTs and vCTs propagated from human first trimester placental tissues identified the preferential expression of ADAM8, -12, -19 and -28 in highly-invasive EVTs12,21. More recently, ADAM10 expression was described in proliferative vCTs from third-trimester tissue 18. Together, these findings establish insight into putative roles that these ADAM subtypes play during trophoblast differentiation along both invasive and villous pathways.   Along with our lab’s recent work in characterizing the importance of ADAM12 in controlling trophoblast invasion and fusion, these formative bodies of work have paved the way for interrogating the importance of related ADAM 	   23	  proteases previously described as being highly expressed in invasive EVTs. To this end, my thesis has focused on the biological importance of the least characterized ADAM protease expressed in the placenta, ADAM28.  1.6 ADAM28  ADAM28, also referred to as metalloproteinase disintegrin cysteine-rich (MDC)-L due to its specific expression in human lymphocytes143,  is expressed as two distinct splice variants that encode either a full-length transmembrane subtype (ADAM28L) or a truncated secreted isoform (ADAM28S). ADAM28S lacks the canonical transmembrane domain and cytoplasmic tail regions143,144 (Figure 1.2).  The functional differences between these two isoforms as well as their specific developmental roles in placental biology remain elusive. Interestingly, upon comparison of protein sequence to metzincin proteases, ADAM28 was found to be among the few ADAM family members that share close homology to snake venom metalloproteases (SVMPs) than to other ADAMs; putative substrates are thus thought to overlap with SVMPs and include integrin cell adhesion molecules and components of the ECM145. ADAM28 is predicted to have intrinsic catalytic activity due to the presence of the HEXXXHXXGXXH zinc-binding motif143,144.        	   24	    Figure 1.2: Structure of ADAM28-L and ADAM28-S isoforms. Depicted are generic ADAM28 membrane-bound (ADAM28-L) and secreted (ADAM28-S) domain structures. ADAM28S lacks the canonical transmembrane domain and cytoplasmic tail regions.    1.6.1 Catalytic activity of ADAM28  Similar to other ADAM species, ADAM28 is first synthesized as an inactive zymogen (proADAM28), which requires proteolytic removal of the pro-domain to confer activity146. In contrast to most other ADAM proteases that necessitate pro-domain removal via furin-like proprotein convertase, pro-domain removal and maturation of ADAM28 is constitutively self-activated through an autocatalytic mechanism144,145. However, proADAM28 can also be activated by MMP-7146. Numerous substrates have been identified for ADAM28, including insulin-like growth factor binding protein -3 (IGFBP-3), connective tissue growth factor (CTGF), tumor necrosis factor-α (TNF-α), myelin basic protein (MBP), and clusters of designation (CD) 23 glycoprotein146–151. ADAM28 also regulates IL-1β-induced MMP-13 expression by cleaving proMMP-13, which affects cellular proliferation, differentiation, and survival 152. With respect to endogenous inhibition, similar to related MMPs and related adamlysins, ADAM28 is inhibited by TIMP-3 and TIMP-4 146,149.   	   25	   In addition, ADAM28 plays a key role in inflammation. ADAM28 is known to be a sheddase of TNF-α, one of the major pro-inflammatory cytokines involved in the metabolic syndrome151. Moreover, the disintegrin domain of ADAM28 serves as a ligand for the leukocyte integrin α4β1 expressed on macrophages, which are a key contributing source of pro-inflammatory cytokine TNF-α151,153. Further emphasizing ADAM28’s role in inflammation is its ability to bind to P-selectin glycoprotein ligand-1 (PSGL-1) present on leukocytes, promoting the PSGL-1/P-selectin mediated adhesion following migration of leukocytes into tissues 151.  There are few studies that have interrogated the mechanism(s) fundamental to ADAM28 activation or expression. However, recent studies have shown that Src is an inducer of ADAM28 mRNA expression acting via mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK), and also through phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways154. Dysregulated activation of Src and these downstream signaling pathways are critical in driving overexpression of ADAM28 in human carcinoma cells, detailed in the following section154.  1.6.2 ADAM28 in cancer  ADAM28 promotes key cellular processes in tumorigenesis that are common to placental development, including proliferation, invasion, and survival. ADAM28 is highly expressed in numerous cancers, most notably in human non-small cell lung, breast, ovary, kidney, and colon carcinomas, whose RNA expression levels show direct association with carcinoma cellular proliferation and metastasis154,155. Active forms of ADAM28 are overexpressed in lung carcinomas, where ADAM28 serum levels in non-small cell lung cancer greatly increase throughout tumor grade severity, lymph node metastasis, and cancer relapse154,156. ADAM28 also promotes tumor metastasis and cell survival by cleaving and thereby inactivating the pro-apoptotic von Willebrand factor (vWF) in carcinoma cells 155. vWF induces apoptosis by binding to ανβ3 integrin on tumor cells, stimulating downstream signaling 	   26	  pathways involving phosphorylation of TP53 and activation of CASP3155,157,158. Both transmembrane ADAM28-L and secreted ADAM28-S active isoforms are overexpressed in human breast carcinoma, promoting cell proliferation and tumor development by promoting IGF signaling by cleaving IGFBP-3154,159. ADAM28 plays a prominent role in regulating critical cellular processes such as cell migration, invasion, proliferation and survival in a number of human cancer systems, those of which are also highly preserved during placental development. This may ultimately provide insight into the involvement of ADAM28 in healthy placentation and trophoblast biology.   1.7 Hypothesis and rationale  In humans, establishment of the placenta is achieved in part through extensive uterine infiltration by invasive trophoblasts. Differentiation of trophoblast progenitors into invasive cell subtypes requires coordinated regulation of pathways that control processes fundamental to cell invasion, cell-cell adhesion, and cell-matrix interaction. The ADAM family of metalloproteinases includes good candidates in regulating these cellular processes, predominantly acting as molecular sheddases that cleave membrane associated proteins like growth factors, cytokines, and cell adhesion molecules16. ADAMs have been described as key regulators in cancers and other tissue systems, promoting cell proliferation, migration, invasion, and survival154,156,159.  Recent studies have shown that ADAM8, -10, -12, -19, and -28 subtypes are expressed in the human placenta21,22. Among these ADAMs, the roles of ADAM8, -10, -12, and -19 in placental development have been examined in ADAM-deficient mouse models122. As mentioned previously, our lab’s work has highlighted the importance of ADAM12 in regulating trophoblast invasion and fusion in human trophoblast biology23,24. However, little work has been done in examining the role of ADAM28 in placental development. Notably, ADAM28 is highly expressed by ex vivo-derived column trophoblasts21, however the importance of ADAM28 in placentation and trophoblast biology is unknown. Given that preliminary data shows ADAM28-S and 	   27	  ADAM28-L mRNA levels are abundantly expressed by highly invasive as well as proliferative trophoblast populations within first trimester placenta, I set out to test the hypothesis that ADAM28 plays a central role in placental development by controlling trophoblast differentiation into proliferative and invasive EVT subsets.  The specific aims of my research are as follows:  Aim 1: To characterize ADAM28 expression and localization within distinct subsets of trophoblasts.  Aim2: To define the role of ADAM28 through its effects on invasive trophoblast cell migration, proliferation, and survival.   These aims interrogate the role of ADAM28 in regulating trophoblast differentiation and invasion, an essential process in early establishment of healthy placenta where dysregulation leads to severe pregnancy complications and pathologies.            	   28	  Chapter 2. Materials & Methods   2.1 Placental tissue collection   First trimester placental tissue samples between 5-12 weeks of gestation were obtained from women aged 19-35 undergoing elective surgical terminations of pregnancy at BC Women’s Hospital’s Comprehensive Abortion and Reproductive Education (CARE) Program. All women who participated in our study offered written informed consent. The University of British Columbia Research Ethics Board provided ethical approval for this study.  2.2 FACS purification of placental cells  Single cell suspensions from placental villi were generated by enzymatic digestion of newly obtained first trimester placental specimens (N=5) and analyzed by flow cytometry, following protocols modified from Aghababaei et al. Briefly, placental villi were digested in Hanks Balanced Salt Solution (HBSS), 750 U/mL collagenase and 250 U/mL hyaluronidase, for 1.5h at 37°C. To obtain single cells, specimens were vortexed and subjected to red blood cell lysis in 0.8% ammonium chloride, with additional dissociation in 0.25% trypsin for 2min and 5mg/mL dispase with 0.1mg/mL DNase I for 2 min, then filtered through a 40 µm mesh. EasySep CD45-conjugated magnetic beads (all reagents from StemCell Techologies Inc, Vancouver, Canada) were used to remove contaminating immune cells from the cell admixture. Following magnetic bead exclusion, 2.5 x 106 cells were blocked with Fc receptor antibody (eBiosciences, San Diego, CA, USA), and incubated with the following antibodies on ice for 30min: anti-CD45 (clone 2D1, eBioscience), anti-CD49F-PE-Cy7 (clones GoH3, eBioscience) and anti-HLA-G-PE (clone 87G, eBioscience). Additionally, by staining with 7AAD (eBioscience), dead cells were excluded from analysis. The cell surface markers CD49f (analogous to α6 integrin) and HLA-G were used to identify placental trophoblast cell populations, while CD45 was used to identify and exclude contaminating immune cells that were 	   29	  not completely removed by EasySep magnet purification. FACS analysis was performed using FACSDiva (BD, San Diego, CA, USA) and FlowJo software (Tree Star, Inc. Ashland, USA); cell sorting was performed on a FACsAria (BD) flow cytometer and samples were directly sorted into 1.5mL Eppendorf tubes containing 1mL TRIzol LS reagent (Invitrogen).  2.3 RNA purification and qPCR analysis   Total RNA was extracted from trophoblastic cell lines HTR8/Svneo, JEG3, BeWo, and Jar using TRIzol reagent (Life Technologies). Total RNA was prepared from FACS purified primary placental cell populations and micro-dissected placental explant columns using TRIzol LS reagent (Invitrogen) and RNeasy MinElute Cleanup Kit (Qiagen). RNA purity was confirmed using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). One microgram of RNA was reverse-transcribed using a qScriptTM cDNA synthesis kit (QuantaBiosciences); synthesized cDNA was subjected to quantitative PCR (ΔΔCT) analysis, using Perfecta SYBR Green FastMix (QuantaBiosciences) on an ABI 7500 Sequence Detection System (Applied Biosystems).   Primers used for qPCR analysis include: ADAM8 (forward 5’-ATGTGTGACCTCG AGGAGTTCT-3’, reverse 5’-GGA TGTCATAGGAGAGACAGGA-3’), ADAM10 (forward 5’-GGATTGTGGCTCATT GGTGGGCA-3’, reverse 5’-ACTCTCTCGGGGC CGCTGAC-3’), ADAM12L (forward 5’–GACAATGGGAGACTGGGC-3’, reverse 5’-GTGGAT CTGGGCACT TGG-3’), ADAM19 (forward 5’-ACCTCGCAGGATGAAAAGGG-3’, reverse 5’-CGTCCTGG TCTCGTCGATTC-3’), ADAM28 (forward 5’-CCCTACCTGCCACC AAACTA-3’, reverse 5’-AGAGAACACTCCCCCTCCAT-3’), ADAM28S (forward 5’-AATGGCTTCCCTTGCCA TCA-3’, reverse 5’-TTGTCCTCCTACCTGGTCCC-3’), ADAM28L (forward 5’-TCGCAGAGTGG ATGA CCACAC-3’, reverse 5’-GGA GCTCATGGTCACACACA-3’), VIM (vimentin) (forward 5’-AAAGTGTGGCTGCC AAGAACCT-3’, reverse 5’-ATTTCACGCATC TGGCGTTCCA-3’), HLA-G (forward 5’-TTGCTGGCCTGGTTGTCCTT-3’, reverse 5’-TTGCCACTCAGTCCC ACACAG-3’), KRT7 (cytokeratin-7) (forward 5’-GGACATCG AGATCGCCACCT-3’, reverse 5’-ACCGCCA 	   30	  CTGCTACTGCCA-3’), and GAPDH (forward 5’-AGG GCTGCTTT TAACTCTGGT-3’, reverse 5’-CCCCACTTGATTTT GGAGGGA-3’). Cycle threshold (CT) values were normalized to endogenous GAPDH transcripts.  2.4 Immunofluorescence microscopy  Villous explants were fixed in 2% paraformaldehyde overnight at 4°C, paraffin embedded, serially sectioned at 5µm thickness and mounted on glass microscope slides. Tissues underwent antigen retrieval by heating slides in a microwave for 5 X 2 minute intervals in a citrate buffer (pH 6.0). Following antigen retrieval, sections were incubated with sodium borohydride for 5min at room temperature (RT) and permeablized with 0.2% Triton X-100 for 5min at RT. Sections were then blocked in 5% normal goat serum/0.1% saponin for 1h at RT and incubated overnight at 4°C with antibodies directed against rabbit monoclonal Ki67 (1:100, clone SP6; Thermo Scientific, Waltham, MA) and rabbit polyclonal cleaved caspase-3 (1:100, clone 5A1E; Cell Signaling). After overnight incubation with primary antibodies, sections were washed with PBS and incubated with Alexa Fluor goat anti rabbit-488/-586- and goat anti mouse-488/-568 conjugated secondary antibodies (Life Technologies) for 1h at RT. Sections were washed with PBS and coverslips were mounted onto the glass slides with mounting media containing 4’,6-Diamidino-2-Phenylindole (DAPI; Life Technologies).   JEG3 cells were washed with cold PBS and fixed in 4% paraformaldehyde for 20mins at 4°C. After additional washing with PBS, PFA-fixed cells were subjected to IF immunolocalization processes described above. JEG3 cells were incubated with mouse monoclonal antibody directed against caspase-cleaved Kertain-18 (1:20, clone M30; Roche, Penzberg, Germany) overnight at 4°C.  Placental villi were fixed in 2% paraformaldehyde for 30min at 4°C, then washed with cold PBS and incubated in 30% sucrose at 4°C until the placental tissue was completely 	   31	  saturated and sunk to the bottom of the 15mL Falcon tube (3-4 days). Since our antibodies directed against ADAM28 were only usable on frozen cryosections, placental specimens were embedded in optimal cutting temperature compound (OCT), frozen using dry ice and ethanol, serially sectioned at 16µm thickness and collected onto SuperFrost+ slides. Sections were washed with HEPES buffered saline (HBS) and incubated with ‘HBST” (HBS, 0.1% Tween 20, 5% goat serum, 0.05% BSA) for 1hr at RT. Frozen sections were incubated with combinations of the indicated antibodies overnight at 4°C: antibodies directed against ADAM28 included a rabbit polyclonal antibody (RP1, Cat# IU-011024; Triple Point Biologics) and a mouse monoclonal antibody (clone 297-2F3, kindly gifted from Dr. Yasunori Okada, Keio University, Tokyo, Japan156); rabbit monoclonal cytokeratin-7 (1:75, clone SP52; Ventana); mouse monoclonal cytokeratin-7 (1:100, clone C46; Santa Cruz Biotechnology, Dallas, TX); rabbit monoclonal Ki67 (1:100, clone SP6; Thermo Scientific, Waltham, MA); mouse monoclonal HLA-G (1:100, clone 4h84; ExBio). Following overnight incubation, sections were washed with HBST and incubated with Alexa Fluor goat anti rabbit-488/-586- and goat anti mouse-488/-568 conjugated secondary antibodes (Life Technologies) for 1.5h at RT. Similar to paraffin embedded samples, sections were washed in HBS, and mounted with coverslips using DAPI. All IF images were imaged using an AxioObserver inverted microscope (Carl Zeiss) using 20x objective (0.8NA) and the Apotome.2 structured illumination device; all images were obtained using an Axiocam 506 monochrome camera.  2.5 Cell culture  The immortalized trophoblast HTR8/SVneo cell line was kindly gifted from Dr. Charles H. Graham from Queen’s University, Canada. The HTR8/SVneo cells were cultivated in RPMI media with 25mM glucose, L-glutamine, 10% FBS, and 0.1% antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin). The human choriocarcinoma JEG3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in DMEM 	   32	  media containing 25mM glucose, L-glutamine, 10% FBS, and 0.1% antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin). The fusogenic BeWo choriocarcinoma cells (B30 clone) were kindly provided by Jerome Strauss III. BeWo cells were maintained in Ham’s F12 media (Invitrogen) including 25mM glucose, L-glutamine, 10% FBS, and 0.1% antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin). JAR cells were generously provided by Dr. Caroline Dunk, Lunenfeld Tanenbaum Research Institute, Toronto. JAR cells were cultured in RPMI media containing 0.5% D-glucose, 1% pen strep, 1% Sodium Pyruvate, 1% 1M Hepes, and 10%FBS.  2.6 Cell lysis and immunoblot analysis  Cells washed with ice-cold PBS were lysed with RIPA (radioimmunoprecipitation assay)  buffer (50mM Tris-HCL (pH7.4), 50mM NaCl, 5mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.05% SDS, 1mM phenylmethylsulphonyl fluoride, 5mM NaF, 1mM Na3VO4) and Complete Mini, EDTA-free protease inhibition cocktail (7-fold dilution; Roche). Protein concentrations were determined using a DCTM Protein Assay kit (Bio-Rad); 20-30µg of cell protein lysate was resolved by SDS-PAGE and transferred to nitrocellulose membrane (Pall Life Sciences). The membranes were probed with a mouse monoclonal antibody directed against ADAM28 (clone 297-2F3, kindly gifted from Dr. Yasunori Okada, Keio University, Tokyo, Japan156). Primary antibodies were incubated overnight at 4°C. Following incubation, the membranes were probed with an anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology). Signals were distinguished using enhanced chemiluminescence substrate (ECL; PerkinElmer). To normalize the amounts of protein loaded, the blots were stripped and re-probed with an HRP-conjugated monoclonal antibody directed against β–actin (Santa Cruz Biotechnology).   	   33	  2.7 ADAM28 siRNA transfection  siRNA’s targeting ADAM28 (Ambion; s21322 and s21323, Cat# 4392420) as well as non-silencing (NS) siRNA (Dharmacon, ON-TARGET plus Non-targeting siRNA#1, Cat# D001810-01-20) were used in this study. Table 2.1 shows the target sequences and accession numbers for both siRNAs. The choriocarcinoma cells line, JEG3 was grown to 40-50% confluency and reverse transfected with 20nM of siRNA oligos using Lipofectimen RNAiMAX reagent (Life Technologies). Cells transfected with NS siRNA served as negative controls. In villous explant cultures, siRNA-mediated ADAM28 silencing was performed after explants were allowed to adhere to the Matrigel. After establishing the explants for 24h in culture, siRNA transfection was carried out using 200nM of siRNA oligos.  Table 2.1 Accession numbers and target sequences for ADAM28-directed siRNAs ADAM28-siRNA Accession number Target Sequence A28si-2 S21322 5’-GGCCTTAGTTGGTATGGAA-3’ A28si-3 S21323 5’-CGACTATTCTTGCAAGTGT-3’   2.8 Placental villous explant culture  Ex vivo placental villous cultures were established by obtaining placental villi (5-7 weeks gestation) from placentae of women undergoing elective terminations of pregnancy. Placental villi were dissected, washed in cold phosphate-buffered saline (PBS) and placed onto Millicell cell culture inserts (12mm, 0.4µm pores, EMD Millipore, Darmstadt, Germany) containing 200µL of growth factor-reduced Phenol-red free Matrigel (BD Biosciences, Mississauga, Canada).  All explants were allowed to establish overnight in a humidified 37°C trigas incubator at 3% oxygen, containing 400µL DMEM/F12 (1:1) medium in the outer chambers, containing 25mM glucose, L-glutamine, 15mM HEPEs, and supplemented with 0.1% anitbiotics (100 U/mL 	   34	  penicillin, 0.1 mg/mL streptomycin, and Antibiotic-Antimycotic). After ~16hours of culture, explants were transfected with siRNA for 24h. Growing explant cultures were imaged at three time points using Nikon SMZ 7454T triocular dissecting microscope equipped with a digital camera: 0h, 24h and 48h. EVT column outgrowth area and length were quantified used ImageJ software. Outgrowth area was measured as a ratio of column area at 48h to 0h; area was determined from the base of each column, encompassing all invading EVTs to the invasive front (Figure 2.1). Quantification of EVT outgrowth length was measured as a difference in column lengths at 48h and 0h measured from the base of each column to the invasive front of the invading EVTs at 5 distinct locations within each column (Figure 2.1).             Figure 2.1 Inverted images of first trimester chorionic villous explant imbedded on Matrigel. EVT column outgrowth length is calculated as the difference in column length at 48 and 0 h (µm) measured from the base of each column to the invasive front at 5 distinct locations (red lines). Column outgrowth area is measured as a ratio of column area at 48 to 0h, encompassing all invading EVTs from the base of each column to the invasive front (yellow dotted area). Black arrows indicate the direction of trophoblast invasion.    	   35	  2.9 Cell viability  Cellularity was measured in JEG3 cells with the thiazol blue tetrazolium bromide (MTT) assay (Sigma-Aldrich). Briefly, cells were seeded in seven replicate wells at a density of 1 x 104 cells/well into opaque 96-well microplates containing 100µL of DMEM media supplemented with 10% FBS. The cells were then incubated at 37°C for 8, 24, 48, and 72h. The MTT assays were performed according to the manufacturer’s instructions, and luminescence was measured using a Perkin Elmer EnSpire 2300 multilabel reader.  Villous explants were dual-labeled with Ki67 and cleaved-caspase 3 antibodies, and the percentage of proliferating cells within an EVT column was calculated as a ratio of Ki67 positive cells to the total number of DAPI-positive cells. Likewise, the percentage of apoptotic cells was calculated as the proportion of cleaved-caspase 3 postive cells to number of DAPI positive cells.  The viability of JEG3 cells in culture was examined by immunofluorescent staining with M30 Cytodeath antibody (caspase-cleaved cytokeratin 18; Roche). The percent of apoptotic cells were determined by calculating the ratio of M30 positive cells to total number of cells present (DAPI positive) in each field of view. Percentages were determined as an average of ten random, non-overlapping fields of view per section using a 20x objective (0.8 NA).   2.10 Migration assay  Trophoblast migration assays were performed using Transwell inserts (6.5mm filters and 8µm pore size, BD Bioscience), which were uncoated, fibronectin-coated (gift from Dr. James Lim) or collagen-coated (Stemcell Technologies). Assays were performed according to the manufacturer’s protocol. Briefly, JEG3 cells were suspended in DMEM media containing 0.1% BSA and seeded in the upper chambers of Transwell inserts at 2.5 x 104 cells/chamber. DMEM media in the bottom chambers were supplemented with 10% FBS, serving as a chemoattractant. After 24-72h of incubation, cells were fixed first with 2% paraformaldehyde for 2mins, then with 100% methanol for 20mins and washed with PBS. Transwell inserts were 	   36	  washed with dH2O and cells residing on the upper surface of the membrane were removed using cotton swabs. The filters were then cut off from the inserts and mounted on glass slides with DAPI. Cell migration was determined by counting the number of DAPI positive cells in ten random fields using a 20x objective. Cells that had invaded into the underside of each Transwell membrane were quantified using ZenPro2 software (Carl Zeiss).  2.11 Statistical Analyses  Data are presented as mean ± S.E.M. All statistical analyses were performed using GraphPad Prism software. Comparisons were made using two-tailed Student’s t-test and ANOVA. Cellular migration indices were analyzed by multiple t-tests followed by the Mann Whitney test. P-values less than 0.05 were considered statistically significant.                	   37	  Table 2.2 Antibodies used in this thesis Antibody Clone/Source Applications	  (Concentrations)	   ADAM28 (gifted by Dr. Yasunori Okada)  297 2F3/mouse Immunoblotting (1:500) Immunofluorescence (1:200)  HLA-G (Exbio)   Monoclonal/Mouse  Immunofluorescence (1:100)  Ki67 (Thermo Scientific)   Monoclonal/Rabbit  Immunofluorescence (1:100)  Cytokeratin-7 (Santa Cruz)  Monoclonal/Mouse  Immunofluorescence (1:100)  Cytokeratin-7 (Ventana Medical Systems)  Monoclonal/Rabbit  Immunofluorescence (1:75)   β-actin (Santa Cruz)   Monoclonal/Mouse  Immunoblotting (1:10000)  Cleaved-caspase-3 (Cell Signaling)   Monoclonal/Rabbit  Immunofluorescence (1:100)  Caspase-cleaved cytokeratin-18 (M30 Cytodeath)    Monoclonal/Mouse   Immunofluorescence (1:20)          	   38	  Chapter 3. Examining ADAM28 expression in first trimester trophoblast subtypes  3.1 Rationale   In mammalian development, the placenta forms the mechanical and physiological link between maternal and fetal circulations. Correct placentation, required for transfer of oxygen and nutrients to the developing fetus, underlies fecundity and fitness of ~ four thousand species of placental mammals 4,7. In rodents and higher order primates (including humans) this transfer is achieved through extensive uterine infiltration by highly invasive fetal-derived epithelial cells called trophoblasts 31. Trophoblast differentiation into specialized cell populations is essential for correct placentation, and the underlying processes are therefore strictly regulated 54. These critical cellular and molecular processes affect cell invasion, cell-cell interaction, and cell-matrix adhesion; however, knowledge of their components and contributions to placental development is severely lacking.  ADAMs are multi-functional proteins that belong to the metzincin superfamily of metalloproteinases and are structurally characterized by an N-terminal signal sequence followed by a pro-domain, a metalloproteinase domain, a disintegrin domain, a cysteine rich region, an epidermal growth factor (EGF) domain, a transmembrane domain and a cytoplasmic tail16 (Fig 1.2). Unlike ECM-degrading MMPs, ADAMs primarily function as surface “sheddases” responsible for cleaving (shedding) proteins from plasma membranes 16,20. By directing surface shedding (metalloproteinase domain), cell-cell/cell-matrix adhesion (disintegrin, cysteine-rich and EGF domains) and signaling (cytoplasmic tail), ADAMs affect diverse cellular processes, many of which are critical in placental development 20. However, the roles that ADAMs play in trophoblast biology are unknown. Recent gene microarray studies identified the ADAM subtype, ADAM28, as a gene highly expressed by invasive trophoblasts cultured on Matrigel matrix 12,21. In other cell systems, including cancer, ADAM28 plays central roles in directing cell survival, proliferation and invasion 	   39	  151,154,155. Functioning as an active sheddase, ADAM28 controls cellular processes through ectodomain cleavage of growth factors, cytokines, and peptide hormones146,148,149,151. ADAM28 is expressed as two splice variants: a full-length transmembrane protein (ADAM28L) and a truncated secreted variant (ADAM28S) that lacks both transmembrane and cytoplasmic domains.  Both isoforms are proteolytically active and have the potential to regulate cell-cell and cell-matrix interactions through their disintegrin and cysteine-rich ancillary domains 146,159. Despite evidence that ADAM28 may play roles in directing cellular processes fundamental in placental establishment, the importance of ADAM28 in regulating trophoblast biology has currently not been investigated.  In this chapter I aim to elucidate the significance of ADAM28 in trophoblast biology by directly addressing the following aim:  Aim 1: To characterize ADAM28 expression and localization within distinct subsets of trophoblasts.  3.2 Results  3.2.1 ADAM28 localizes to HLA-G+ trophoblasts within first trimester anchoring placental columns  As a first step to understanding the importance of ADAM28 in placental development, ADAM28’s localization within first trimester human placental villi was examined. To isolate distinct trophoblast cell populations from first trimester placental tissue, a fluorescence activated cell sorting (FACS) approach and quantitative PCR strategy were performed. Labeling placental single cell suspensions with antibodies directed against CD49f  (analogous to α6 integrin) and HLA-G, two surface markers used to differentiate between vCTs and column trophoblasts, three distinct populations of cells were identified; vCTs (CD49flo/HLA-G-), column trophoblasts 	   40	  (CD49fhi/HLA-G+), and placental mesenchymal cells (CD49f−/HLA-G−) (Figure 3.1A). Following sorting of these three populations, cell type composition was verified by qPCR gene expression analysis of VIM (mesenchymal marker), KRT7, and HLA-G. As expected, VIM was abundantly expressed in the mesenchymal cell fraction, while vCTs and column trophoblast fractions showed little/no expression (Figure 3.1B). The epithelial marker, KRT7, was strictly expressed in both vCT and column trophoblast populations, indicating that FACS isolation of these distinct trophoblast subtypes was successful (Figure 3.1C). Moreover, HLA-G, known to be specifically expressed by trophoblasts located at distal tips of anchoring villous columns and to invasive EVTs86, was exclusively expressed by column trophoblasts, again reaffirming the cell type of our sorted populations (Figure 3.1D).                  	   41	    Figure 3.1 Isolating flow cytometry-purified trophoblast populations. (A) Representative fluorescence activated cell-sorting (FACS) plot demonstrating the trophoblast isolation strategy used to purify mesencymal core (MC) cells, columnar EVTs (colEVT) and villous cytotrophoblasts (vCT). Cells were segregated by cell surface labeling of HLA-G and CD49f; cell subtype proportions are indicated in each gated population (percent of cells within FACS plot). (B-D) Trophoblast subtype purity was assessed by qPCR analysis targeting the trophoblast marker cytokeratin-7, the EVT-marker HLA-G, and the mesenchymal lineage marker vimentin (VIM). GAPDH mRNA was used for normalization. Results are presented as mean ± S.E.M in bar graphs (***P≤ 0.0005, ****P≤ 0.0001) from 5 distinct placental villi specimens (n=5).  	   42	   Following verification of trophoblast subtype isolation, I next set out to examine the expression of select ADAM proteases (ADAM8, -10, -12L -19, -28L, -28S) in vCTs and column trophoblasts; these ADAMs had previously been described as being highly expressed in ex vivo propagated EVTs 12,18,21. Notably, ADAM12L, previously shown in our lab to be highly expressed by invasive trophoblasts23 and serving as a positive control, was indeed specifically expressed by HLA-G+ cells. qPCR analysis using primer sets that detect ADAM28 and distinguish between ADAM28L and ADAM28S isoforms showed that ADAM28 is expressed at markedly higher levels in HLA-G+ column trophoblasts compared to vCTs (Figure 3.2A, B & C).  Although I observed considerable variation in expression of ADAM28 in column trophoblasts isolated from individual pregnancies, ADAM28 levels were consistently elevated in column trophoblasts compared with vCTs from the same patient, possibly highlighting effects of gestational age differences and/or genetic factors. The distribution in expression levels of the other ADAM subtypes examined was similar to ADAM12 and ADAM28, where ADAM8, -10 and -19 mRNA levels were higher in column cells (Figure 3.2D).  Taken together, these findings suggest that ADAM28 and its distinct isoforms are highly expressed in invasive placental cell types. While ADAM8 and ADAM10 expression are less defined to specific cell types, both ADAM12L and ADAM19, like ADAM28, are distinctly expressed by trophoblasts differentiating along the invasive pathway and thus may play important roles in EVT biology.        	   43	   Figure 3.2 ADAM28 localizes to invasive column extravillous trophoblasts (colEVTs) in first trimester human placenta. qPCR gene expression analysis of (A) ADAM28 and (B-C) it’s two isoforms, ADAM28S and ADAM28L, as well as (D) other placental ADAM subtypes (ADAM8, -10, -12L, -19) (B) in FACS-purified vCT and colEVTs. (A) ADAM28, ADAM28-S, and ADAM28-L expression is shown with normalization of vCT to the colEVT population from each individual patient (left) and normalization of vCT and colEVT expression to one single patient (right). GAPDH mRNA was used for normalization. Results are presented as mean ± S.E.M in bar graphs (***P≤ 0.0005, ****P≤ 0.0001) from 5 distinct placental villi specimens (n=5). 	   44	   Following the characterization of ADAM28 mRNA levels in FACS-purified trophoblasts from first trimester placental tissues, I next set out to confirm ADAM28 localization to column trophoblasts using an immunofluorescence microscopy approach. Serially sectioned placental villi were immunostained with antibodies directed against ADAM28, cytokeratin-7 (KRT7; trophoblast marker), HLA-G (EVT-specific marker) and Ki67 (proliferation marker) (Figure 3.3A and B). As expected, cytokeratin-7 labeled trophoblasts line the epithelial surface of chorionic villi, while cells within the villous mesenchymal core stained negative. HLA-G specifically localized to trophoblasts positioned at distal locations within placental columns; mesenchymal core cells, vCTs and the synCT outer layer were HLA-G-. Although mouse-derived antibodies directed against HLA-G and ADAM28 could not be co-stained within the same sample, it is evident that the ADAM28 signal tightly overlapped with HLA-G+ cells, affirming that ADAM28 expression is specific to column trophoblasts differentiating into invasive EVTs.             	   45	                 Figure 3.3 ADAM28 localizes to HLA-G+ distal column trophoblasts in vivo. (A-B) Representative serial sections of 6 weeks gestation human placental villi dual-labeled with HLA-G (green) and cytokeratin-7 (KRT7; red), ADAM28 (green) and cytokeratin-7 (KRT7; red), or ADAM28 (green) and Ki67 (pink). White arrows indicate ADAM28 expression. Nuclei are stained with 4’, 6-Diamidino-2-Phenylindole (DAPI; blue). KRT7, cytokeratin-7; colEVT, columnar extravillous cytotrophoblast; vCT, villous cytotrophoblast; MC, mesenchymal core. Scale bars = 100µm. (C) Percentage of Ki67+/- ADAM28 positive cells within cell columns of serially section placental villi. Results are presented as mean ± S.E.M in bar graphs (****P≤ 0.0001) from 28 trophoblast columns (n=28) from 6 distinct placental samples. 	   46	  Anchoring trophoblast columns are characterized by highly proliferative cells within the proximal (proximal to the base of the column) an central areas of the column. Proliferating column trophoblasts differentiate into senescent cells that gradually acquire the expression of invasive EVT markers, like HLA-G and subsets of integrin heterodimers. As ADAM28 localization was restricted to the apical/most distal regions of placental columns, I next set out to examine the relationship between ADAM28 positivity and cell proliferation. First trimester placenta villi were dual-labeled with ADAM28 and Ki67 antibodies; Ki67 specifically labels actively proliferating cells. Quantification within trophoblast columns revealed that ∼20% of ADAM28+ cells co-stained for Ki67, while 80% of ADAM28+ cells were non-proliferating trophoblasts (Figure 3.3C). These findings indicate that the biological role(s) of ADAM28 within placental columns may be independent of proliferation.  3.2.2 ADAM28 expression in trophoblast cell lines  Following examination of ADAM28 localization within first trimester placenta, I investigated the expression of ADAM28 and EVT-associated ADAMs (ADAM8, -10, -12L and -19) in HTR8/SVneo, JEG3 and JAR  trophoblast cell lines. Traditionally, these cell lines have been used to model invasive EVTs (HTR8/SVneo) and column trophoblasts (JEG3 and JAR). For these studies, RNA and protein were harvested from actively growing cells with a surface confluence of approximately 70%. qPCR gene expression analysis showed that ADAM28 and its isoforms (ADAM28-S, and ADAM28-L) were expressed at exceptionally high levels in JAR cells and at high levels in JEG3 cells (Figure 3.4A). In contrast to ADAM28’s high expression in these choriocarcinoma cell lines, levels were next to undetectable in HTR8/SVNeo cells (Figure 3.4A).    mRNA levels of the other ADAM subtypes examined showed distinct and similar expression patterns compared to ADAM28. For example, ADAM12L expression was specific to HTR8/SVNeo cells whereas levels were undetectable in JEG3 and JAR cells. ADAM8 and -10 	   47	  showed similar expression patterns to ADAM28, where mRNA levels were detectable in JEG3 and JAR cells and absent in HTR8SVNeo cells. ADAM19 levels on the other hand were detectable in all three cell lines examined (Figure 3.4B).   Protein levels of ADAM28 examined by immunoblotting were highly conserved with mRNA levels (Figure 3.4C). For these studies, an ADAM28 antibody directed against the catalytic domain thus targeting both full-length (L) and truncated (S) ADAM28 (clone 297-2F3) was used. Importantly, ADAM28 was detected in JAR and JEG3 protein lysates, while levels were undetectable in HTR8/SVNeo cells. 297-2F3 antibody recognized a single protein species, identifying a band equating to 42 kDa, which has been suggested to represent the active form of ADAM28-S156 (Figure 3.4C). Together, this work shows ADAM28 as a protease preferentially expressed by invasive subtypes of column trophoblasts in vivo, and identifies two cell lines, JAR and JEG3, as appropriate trophoblastic models to examine ADAM28 function.                                                    	   48	                      Figure 3.4 ADAM28 is preferentially expressed in choriocarcinoma trophoblastic cell lines modeling column EVTs. (A-B) qPCR analysis of ADAM28, ADAM28-S, ADAM28-L, and placental ADAMs (ADAM8, -10, -12L, -19) in trophoblastic cell lines HTR8/SVneo (n=3), JAR (n=3) and JEG3 (n=3), which model invasive EVT and proliferative colEVTs respectively. GAPDH mRNA was used for normalization. (C) Immunoblot analysis of ADAM28 in HTR8/SVneo, JAR, and JEG3 using a mouse monoclonal antibody (clone 297-2F3). Molecular weights are shown to the left. β-actin was used as the loading control.   	   49	  3.3 Summary  In this chapter, I examined the characterization of ADAM28 expression and localization within distinct subsets of trophoblasts isolated from first trimester placental villi. I show that ADAM28 isoforms, ADAM28-S and ADAM28-L, are both highly expressed in invasive column trophoblasts compared to non-invasive trophoblasts. I show that ADAM28 localizes to column trophoblasts differentiating into invasive HLA-G+ EVTs at distal regions of placental columns. Additionally, ADAM28 localization to specific column trophoblasts showed proportionately that more non-proliferating Ki67- cells expressed ADAM28 than did proliferating Ki67+ cells, suggesting that ADAM28 may not directly influence cell proliferation, but instead control other processes critical to trophoblast function within cell columns. In the following chapter, I interrogate the possible functional roles of ADAM28 in these processes, including its effects on cell migration, proliferation, and survival.               	   50	  Chapter 4. Examining the role of ADAM28 in trophoblast biology  4.1 Rationale First trimester anchoring villi form highly proliferative cell columns comprised of heterogeneous trophoblast populations that display a gradual differentiation from proximal to distal portions of the column. Within proximal regions of anchoring cell columns, rapidly proliferating vCT cells undergo a gradual process of differentiation into cells undergoing senescence and acquiring de novo expression of cell invasion-associated genes 8. Collectively, trophoblasts residing within the distal tips of anchoring villi are called colEVTs and are identified in part by their expression of HLA-G and α1/β1 integrins 42. In chapter 3, I characterized the cell-type expression of ADAM28 in human first trimester placenta by showing that ADAM28 localizes to HLA-G+ trophoblasts within distal portions of anchoring cell columns. I established novel insight into the possible roles ADAM28 may play in regulating the differentiation and/or function of invasive trophoblast cell subsets in placental development. My finding that ADAM28 preferentially localizes to Ki67- trophoblasts indicates that it may not play a direct role in regulating cell proliferation, but may instead control processes such as cell migration and/or survival, of which both are essential in trophoblast column formation. In this chapter I aim to dissect the molecular function(s) of ADAM28 in controlling anchoring column formation by addressing the following aim:  Aim 2: To define the role of ADAM28 through its effects on invasive trophoblast cell migration, proliferation, and survival.  ADAM28’s role in regulating cellular processes intrinsic to column formation and growth will be examined, namely cell proliferation, migration, and survival. ADAM28’s role in regulating 	   51	  these processes will be interrogated using ex vivo placental explant systems as well as JEG3 trophoblastic cell line.  4.2 Results  4.2.1 Loss of ADAM28 reduces trophoblast column outgrowth To examine the functional importance of ADAM28 in regulating trophoblast column establishment and growth, an siRNA knockdown strategy was performed in ex vivo Matrigel-imbedded placental explants using two sets of siRNA oligos both targeting ADAM28’s metalloproteinase domain. siRNA transfection efficacy in explants was examined by imaging RFP-siRNA positivity in the nuclei of column trophoblasts via placental explant whole-mount microscopy (Figure 4.1A). RFP-siRNA labeling of cell columns showed qualitatively that a majority of cells were transiently transfected, suggesting that transfection with ADAM28-siRNA oligos would be similarly effective.  The extent of ADAM28 knockdown in trophoblast column cells and Matrigel-invading EVTs was measured via qPCR analysis where transcript levels of ADAM28-L and ADAM28-S were measured. As shown in Figure 4.1B, both ADAM28 si-2 and si-3 oligos achieved > 80% knockdown efficiency of ADAM28-L/S mRNA transcripts over 48 hours in culture. Importantly, ADAM28 silencing impaired column size (µm2) and length (µm) compared to control siRNA-transfected explants (Figure 4.1C, D, and E).  These results demonstrate that ADAM28 is important in driving placental column formation.      	   52	                      Figure 4.1 Loss of ADAM28 inhibits trophoblast column outgrowth. (A) Representative image of Matrigel-imbedded chorionic villous explant cultures transfected with a siRNA-RFP control oligo. Nuclei are stained with DAPI (blue). (B) qPCR analysis of ADAM28, ADAM28-S, and ADAM28-L mRNA levels in chorionic villous explant cultures transfected with control (NS) or ADAM28-directed siRNAs. (C) Representative images of Matrigel-imbedded chorionic villous explant cultures transfected with control (NS) or ADAM28-directed siRNAs (si2 or -3). Images were taken 0 and 48 h post transfection. Images at 48 h were inverted to provide better visualization of invading EVT column. Column length is measured from the base of each column (white hashed lines) to the invasive front (black hashed lines) at five distinct locations (red hashed lines). Column area is measured as all invading column trophoblasts collectively between the base of each column (white hashed lines) and the invasive front (black hashed lines). Explants were established using four distinct placentae (n=30 columns). Scatter plot graphs represent the quantification of EVT column outgrowth area (D) and length (E). Villi, placental villi; column, villous trophoblast column. Scale bars = 100µm. Data are presented as mean ± S.E.M (**P≤ 0.001). 	   53	  4.2.2 ADAM28 promotes JEG3 cell migration  Following verification that ADAM28-mediated siRNA knockdown reduces column outgrowth area and length, I set out to test the effect of ADAM28 on migration using a trophoblastic cell model. JEG3 cell transfection efficiency was shown to be ~95% by immunofluorescent RFP-siRNA labeling within the cell nuclei, indicating that transient transfection using ADAM28-siRNA would likewise be achievable (Figure 4.2A). qPCR analysis 48h post transfection showed that both ADAM28 si-2 and si-3 generated knockdown (~70%) in the ADAM28-L variant, with only minimal knockdown in ADAM28-S (Figure 4.2B). ADAM28 siRNA knockdown was then examined over a ten day time period using ADAM28 si2 measuring both the mRNA and protein level by qPCR analysis and immunoblotting (Figure 4.2C and D). si2-mediated knockdown in ADAM28-L mRNA was seen as early as 24 h post transfection, while ADAM28-S mRNA knockdown was not seen until day 4 and 5 (Figure 4.2C). si2-mediated reduction of ADAM28 protein levels was detectable by one 42kDa protein band, representing the active form of ADAM28-S 156,159. ADAM28 reduction at the protein level was only evident during days 5 and 6 post transfection (Figure 4.2D). For this reason, all downstream ADAM28 siRNA experiments required JEG3 cells to be cultured for 4 days post ADAM28 siRNA transfection prior to experimental analysis to ensure adequate ADAM28 knockdown.  Once I established a siRNA-mediated knockout strategy in this cell system, I set out to test the effects of ADAM28 on trophoblast migration. JEG3 cells transfected with two ADAM28 siRNA (si2 and si3) and control (NS) siRNA were subjected to uncoated and matrix-coated Transwell migration assays (Figure 4.3A and B). ADAM28 knockdown led to a reduction in trophoblast migration on all membrane surfaces: uncoated, fibronectin-coated, and collagen-coated Transwell cell inserts. Together, these results suggest that endogenous ADAM28 expression is important for controlling trophoblast migration. The extent of migration on different matrices suggests that cell-matrix interactions, possibly via integrin engagement, counters ADAM28 inhibition.   	   54	            Figure 4.2 ADAM28-siRNA mediated knockdown in JEG3 cells. (A) The transfection ability of JEG3 cells tested with a siRNA-RFP oligo control. qPCR analysis of ADAM28 mRNA in JEG3 cells transfected with non-silencing control (NS), and ADAM28-directed siRNA (si2 and si3) over (B) 48h post transfection (n=3) and (C) over 10 days (n=3). (D) Immunoblot analysis of ADAM28 levels in JEG3 cells transfected with non-silencing control (NS), and ADAM28-directed siRNA (si2 and si3) over 10 days using mouse monoclonal antibody directed against ADAM28. Molecular weights (kDa) are shown to the right; β-actin serves as the loading control. Scale bars = 100µm. Data are presented as mean ± S.E.M (*P≤ 0.05). 	   55	     Figure 4.3 ADAM28 promotes JEG3 cell migration. (A) Representative images and (B) bar graph showing Transwell migration of JEG3 cells transfected with control siRNA for four days in culture (NS) and siRNA directed against ADAM28 (si2 and si3). Migration assays were performed for 72 h and repeated on three independent occasions using uncoated, fibronected-coated, and collagen-coated Millicell inserts (n=3). Bar graphs show the quantification of cell migration on uncoated, fibronectin-coated, and collagen-coated Transwell cell inserts. Left bar graph shows one control group (NS uncoated); right bar graph shows each NS group as a control. Scale bars = 100µm. Data are presented as mean ± S.E.M (*P≤ 0.05).        	   56	  4.2.3 ADAM28 promotes JEG3 cell proliferation and cell survival  To further elucidate the roles of ADAM28 isoforms in controlling column outgrowth and trophoblast function, I focused on how the loss of ADAM28 affects cell proliferation and survival in trophoblastic JEG3 cells and ex vivo placental explants. To measure the effects of ADAM28 on cell proliferation and cell survival, ADAM28-silenced JEG3 cells were subjected to an MTT assay (Figure 4.4A) and immunostained with an antibody directed against caspase-cleaved cytokeratin-18 (M30 Cytodeath) (Figure 4.4B and C), respectively. siRNA directed loss of ADAM28 inhibited JEG3 proliferation over 72 hours in culture, where inhibition of cell proliferation was achieved by both ADAM28-targeting oligos (Figure 4.4A). JEG3 cells that were subjected to ADAM28 siRNA transfection exhibited a 2-3-fold increase in caspase-cleaved cytokeratin-18 expression compared to control cells (Figure 4.4B and C).                	   57	                    Figure 4.4 ADAM28 promotes JEG3 cell proliferation and cell survival. (A) JEG3 cells transiently transfected with ADAM28-directed siRNAs (si2 and si3) and control (NS) siRNA for four days in culture were subjected to MTT assays over 72 h. MTT assays were performed in 7 replicate wells and repeated on three independent occasions. (B) Representative immunofluorescent images of JEG3 cells immunostained with an antibody directed against caspase-cleaved cytokeratin-18 (M30 Cytodeath). Nuclei are stained with DAPI (blue). (C) Bar graph indicating the percent of M30+ JEG3 cells transfected with control (NS) siRNA and ADAM28-directed siRNAs (si2 and si3) (n=3). Scale bars = 100μm. Bar graphs are presented as mean ± S.E.M (*P≤ 0.01).    	   58	  4.2.4 ADAM28 promotes cell survival in ex vivo placental explants  Next, I investigated if the loss of ADAM28 correlated with cell proliferation and survival within ex vivo Matrigel-imbedded placental explants.  Paraffin-embedded first trimester placental explant cultures transfected with ADAM28-directed siRNA oligos were subjected to immunofluorescence microscopy using the cell proliferation marker Ki67 (Figure 4.5A) and cell apoptosis marker cleaved caspase-3 (Figure 4.5C). The expression of these markers was analyzed in KTR-7+ trophoblast cells within placental cell columns. Although depletion of ADAM28 did not significantly affect the expression of Ki67 within trophoblast columns compared to controls (Figure 4.5B), loss of ADAM28 correlated with an increase in cleaved caspase-3 (Figure 4.5D). These results suggest that indeed ADAM28 may play a role in trophoblast column formation independent of proliferation, by mediating processes such as column trophoblast cell migration and survival.               	   59	     Figure 4.5 ADAM28 promotes cell survival in ex vivo placental explants. Representative images of EVT columns stained with (A) Ki67 (proliferation marker) or (C) cleaved caspase-3 (apoptosis marker), with cytokeratin-7 (trophoblast marker). Nuclei are stained with DAPI (blue). Placental explants were transiently transfected with ADAM28-directed siRNAs (si2 and si3) and control (NS) siRNA for 24 hours after explant establishment. Bar graphs represent the percentage of (B) proliferative and (D) apoptotic cells. The percentage of cellular proliferation and apoptosis within the trophoblast cell column was calculated as the number of Ki67 positive or cleaved-caspase-3 positive cells in the number of trophoblast column cells (cytokeratin-7 positive and DAPI stained cells), respectively (n=2). KRT7, cytokeratin-7; cleaved K3, cleaved caspase-3. Scale bars = 100µm. Bar graphs are presented as mean ± S.E.M.            	   60	  4.3 Summary  In this chapter, I set out to investigate functional roles of ADAM28 in mediating placental column formation and invasive column trophoblasts by examining its affects on key cellular processes including cell migration, proliferation, and survival. In ex vivo placental villous explant cultures, ADAM28 isoforms were shown to promote trophoblast column outgrowth. In vitro, ADAM28 promotes JEG3 cell migration when cultured on uncoated, fibronectin-coated, or collagen-coated Transwells, alluding to possible cell-matrix interactions involving ADAM28-regulated cell migration. MTT cellularity assays suggest that ADAM28 promotes cell proliferation, however Ki67 immunostaining within Matrigel-imbedded villous explants suggests that ADAM28’s function may not be central to regulating mitogenic events in trophoblasts. Importantly, preliminary work suggests that ADAM28 may actually promote column trophoblast survival. These findings provide valuable insight into the functional roles of ADAM28 in regulating trophoblast biology, as well as provide a foundation for future examinations of the molecular mechanisms regulating ADAM28 expression.               	   61	  Chapter 5. Discussion  5.1 ADAM28 expression in first trimester trophoblast subtypes  In Chapter 3 of this study, I aimed to characterize ADAM28 expression and localization within distinct subsets of trophoblasts. My findings show that ADAM28 is highly expressed in column trophoblast cells differentiating into invasive HLA-G+ EVTs at distal regions of placental columns (Figure 5.1). ADAM28 localization to specific column trophoblasts showed an inverse correlation with proliferating cells, suggesting that ADAM28 may be important in processes independent of proliferation in placental cell columns.  Previously, gene microarray studies identified ADAM28, along with ADAM8, -10, -12, and -19, to be highly enriched in ex vivo derived invasive trophoblasts21,22. However, my work represents the first study to examine the in vivo expression of ADAM28 isoforms in different cell populations within the placenta. vCTs and column EVT trophoblast subtypes were isolated from first trimester placental tissue from five patients spanning the gestational age range within early pregnancy: three were 7 weeks of gestation (7.1, 7.2, and 7.4 wks), one 10 weeks of gestation (10.3) and one 14 weeks of gestation (14.1). Although ADAM28 expression in column trophoblasts isolated from these individual pregnancies showed considerable variation, ADAM28 levels relative to each patient were consistently elevated in HLA-G+ column trophoblasts compared to vCTs. This may suggest that expression of ADAM28’s distinct isoforms in invasive cell types is prominent in both early and late first trimester of pregnancy, potentially playing a role in column formation and acquisition of trophoblast invasiveness throughout early gestation. Although ADAM28 levels did not show a trend according to gestational age in my experiments, I can speculate that the great variability in ADAM28 expression between patients may be due to a multitude of variables including ethnicity, maternal age, or external factors such as diet and other environmental factors.   	   62	         Figure 5.1 Schematic diagram demonstrating proposed ADAM28 localization in human placental villi. ADAM28 is primarily localized to invasive HLA-G+ EVTs at distal regions of placental columns. EC, endothelial cell; EG, endometrial gland; cEVT, columnar extravillous cytotrophoblast; eEVT, endovascular  extravillous cytotrophoblast; iEVT, interstitial extravillous cytotrophoblast; Mϕ, macrophages; mSA, maternal spiral artery; synCT, syncytiotrophoblast, TGC, trophoblast giant cell; uNK, uterine Natural Killer cell; vCT, villous cytotrophoblast.      	   63	  In trophoblastic cell lines, mRNA and protein levels of ADAM28 and its two isoforms showed highly consistent results, where ADAM28 levels are highly expressed in both JAR and JEG3 choriocarcinoma cells, and nearly undetectable in HTR8/SVNeo cells. Although derived from EVT cells, the immortalized HTR8/Svneo cells are poor models of primary column EVTs in vivo, as they lack expression of major EVT markers like HLA-G, cytokeratin-7; moreover, HTR8/SVNeo cells express mesenchymal markers like vimentin, possibly highlighting molecular changes attributable to viral-induced imortalization 89–92. JAR and JEG3 cells are more conventional models of column trophoblast cells, modeling progenitor vCTs and primary columnar EVTs, respectively 82,87,100. Both are highly proliferative and synthesize multiple trophoblast-specific factors and hormones, while only JEG3 cells express EVT-specific marker HLA-G 82,100. Taken together, these findings suggest the JEG3 cell line as an appropriate model to test the biological function of ADAM28 in controlling column formation and acquisition of invasive characteristics of column trophoblasts situated at the most distal regions of placental columns. ADAM28 is an active sheddase thought to control various cellular processes through ectodomain cleavage of growth factors, cytokines, and peptide hormones 146,148,149,151. ADAM28 and its splice variants have been implicated in numerous cancer systems, their expression positively correlating with cancer growth, progression, and metastasis 154–156,159. Since the cellular processes regulating tumorigenesis are well conserved in placentation, it is reasonable to postulate that the roles of ADAM28 in cancer cell progression are applicable to placental development and trophoblast differentiation. Studies have demonstrated ADAM28’s ability to cleave substrates important in promoting tumorigenic processes, including insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in cancer cell proliferation in non-small cell lung and breast carcinomas146–151. IGF-binding proteins IGFBP-1 and IGFBP-3 are highly expressed in the placenta, and are associated with regulation of fetal and placental growth and development160,161. These actions are controlled through IGF1 receptors, abundantly 	   64	  expressed on apical and basal surfaces of placental trophoblast cells, where critical processes including placental growth, maternal-fetal exchange, trophoblast invasion, and placental angiogenesis are highly regulated160,161. Of the IGFBPs expressed in the human placenta, IGFBP-3 is known to be highly expressed by invasive EVT subtypes 161. Additionally, maternal serum levels of IGFBP-3 are altered in pregnancies complicated by disorders like preeclampsia and diabetes161. Therefore, within the placenta, we can postulate that ADAM28 may modulate factors such as IGFBP-3, a well-known ADAM28 substrate critical to cancer cell proliferation, to help drive cell differentiation and proliferation within the growing trophoblast column or by facilitating trophoblast invasiveness at the column periphery. The function of other ADAMs within the placenta may also be hypothesized from the expression analyses I performed in different trophoblast subtypes. ADAMs subtypes within trophoblast populations show both similar and distinct expression patterns compared to ADAM28. Our lab has previously shown ADAM12L to be highly expressed by invasive trophoblast populations, as well as specific expression to HTR8/SVneo cells, and plays a critical role in directing cell invasion and cell-cell fusion 23,24. ADAM8 and -10 show similar expression patterns to ADAM28, with preferential expression in column EVTs, and choriocarcinoma cells, while ADAM19 expression was preferential to column EVTs but detectable in all three cell lines. We can speculate from our characterization of these ADAMs that their function roles in trophoblast biology may be similar to or work in concert with ADAM28 in regulating processes within the placental column.  Altogether, the work described in this chapter characterizes ADAM28 as a protease preferentially expressed by invasive subtypes of column trophoblasts in vivo, at distal regions of placental columns. My findings suggest that ADAM28 may play a role in cellular processes central to column formation, including cell differentiation, migration and survival, as well as acquisition of invasive features such as expression of the EVT specific marker HLA-G. I also 	   65	  identify two cell lines, JAR and JEG3, as suitable trophoblastic models to further elucidate ADAM28 function in the placenta.   5.2 The role of ADAM28 in trophoblast biology  In chapter 4, I investigated the functional roles of ADAM28 in regulating column formation and invasive column trophoblasts through its effects on cellular migration, proliferation, and survival. My findings show that ADAM28 isoforms promote trophoblast column outgrowth in ex vivo placental villous explant cultures. In vitro, I show that ADAM28 promotes JEG3 cell migration when cultured on uncoated, fibronectin-coated, or collagen-coated matrices. Subjecting JEG3 cells to MTT assays suggested that ADAM28 also promotes cell proliferation; however, further investigation using Matrigel-imbedded villous explant cultures suggests that ADAM28 promotes column outgrowth through cellular processes affecting cell survival with little effect on cell proliferation. The differences observed between these two experimental systems can in part be explained by the tumorigenic (and aneuploid) characteristics of the JEG choriocarcinoma cell line. As previously discussed, trophoblast cell differentiation and proliferation is dynamic and alters throughout early placental development 8. As the tips of anchoring villi form placental-uterine attachment points, vCT progenitors at the base of these villi adopt a highly proliferative phenotype forming the multi-layer cellular column of trophoblast cells 42. Columnar trophoblasts at the distal portions of anchoring columns differentiate into highly-invasive EVT subtypes responsible for extensive uterine infiltration and migration into uterine spiral arteries and stroma8,34,40 Trophoblast differentiation, migration and invasion are critical processes during early placental development that are strictly regulated by various cytokines and growth factors, such as EGF and VEGF, along the invasive pathway6,22,33. Since my findings show that ADAM28 expression is positively correlated with the promotion of trophoblast column outgrowth in placental explant cultures, as well as JEG3 cellular migration and cell survival, I speculate 	   66	  that ADAM28’s function as an active sheddase may be a central component in directing these critical processes. Moreover, my findings that ADAM28 expression is preferential to invasive EVT populations, in accordance with EVT-marker HLA-G, may suggest that ADAM28 could potentially also be a marker for EVT cells. Further experiments investigating ADAM28 as a potential EVT marker may include elucidating the specific subtypes of EVTs, iEVTs or eEVTs, which express ADAM28 by FACs isolation of these different cell types.  ADAM28 activity as a molecular sheddase is prominently studied in cancer systems, where ADAM28 promotes tumor metastasis and cell survival by cleaving and thereby inactivating the pro-apoptotic von Willebrand factor (vWF) plasma protein in carcinoma cells 155. Although little is known about the exact mechanism of function, vWF-induced cancer cell apoptosis is thought to involve an ανβ3 integrin-activated mitochondrial cell death pathway155,162. In a study done by Mochizuki et al., mouse models were used to show that in multiple human carcinoma cell lines, including human lung carcinoma PC-9 and breast carcinoma MDA-MB-231, cells expressing high levels of ADAM28 were resistant to vWF-induced apoptosis, however apoptosis occurred when ADAM28 expression or activity was blocked 155. These studies provide an understanding and insight into possible mechanisms of how ADAM28 expression may influence cell survival in trophoblast column systems, further detailed below.   Apoptosis is considered to be an essential process for normal placental development; constant morphological changes are characterized by the recycling of trophoblast cells via apoptosis163. Apoptosis is important for trophoblast cell turnover, where aging trophoblast cells are selectively removed and replaced by new populations of trophoblasts following proliferation and differentiation into specific cell subtypes 163. Importantly, higher rates of trophoblast apoptosis have been observed in pregnancy complications like preeclampsia and IUGR, inferring that accelerated/dysregulated trophoblast apoptosis may contribute to these pathological diseases 163,164. Pro-apoptotic vWF levels in maternal plasma are significantly increased during preeclampsia 165. However vWF is expressed in ADAM28− synCT cells 165, 	   67	  therefore suggesting cellular apoptosis occurs independent of ADAM28 control. Although, a possible alternative method of ADAM28-mediated trophoblast cell apoptosis may occur through ADAM28 substrate TNF-α, a known potent inducer of trophoblast apoptosis163. Death receptor TNF-R1 and ligand TNF-α are localized to both vCT and EVT subtypes, where they trigger the apoptotic cascade by activating effector caspases such as caspase-3, caspase-6, and caspase-7163. Since ADAM28 is preferentially localized to EVT subsets, these findings suggest possible mechanisms of ADAM28-regulated trophoblast cell survival, and provide future experiments to test these postulated mechanisms. ADAM28 expression has also been highly associated with promoting cell proliferation and tumor development in human breast and non-small cell lung carcinomas through ADAM28-mediated cleavage of IGFBP-3 154,159. As described previously, studies have shown that in these cancer systems, ADAM28 is overexpressed in its active form, promoting proliferation through enhanced bioavailability of IGF-I from the IGF-I/IGFBP-3 complex by selective cleavage of IGFBP-3146,159. ADAM28 overexpression in these systems is thought to be induced by Src via mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK), and also through phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways154. Understanding these key molecular mechanisms controlling ADAM28 expression and function in alternative cell systems are critical for elucidating such mechanisms within the placenta. I can speculate on the putative mechanisms mediating ADAM28 sheddase activity and its target substrates within the placenta, conceivably cleaving the aforementioned factors to drive cellular processes critical to trophoblast column growth and function.  Among these cellular processes, we analyzed the affect ADAM28 on cell migration on uncoated, fibronectin-coated, and collagen-coated matrices. It is thought that ECM-integrin engagement in developing placental columns involving laminin, fibronectin, or type IV collagen accelerates differentiation of trophoblasts along the invasive pathway26,54,166.  Notably, the expression of cell-surface α1/β1 integrins on trophoblasts in cell columns is important for 	   68	  regulation of trophoblast cell invasion, migration, and adhesion 166. Previous studies have shown that interactions of primary human trophoblasts involving the α1/β1 integrin receptor with type IV collagen promoted cell invasion, whereas interaction with fibronectin inhibited invasion but significantly promoted cell migration166,167. Moreover, binding of fibronectin to trophoblast α5β1 integrin is thought to play a role in the stabilization of the growing and differentiating placental cell column by interaction with the RGD (Arg-Gly-Asp) peptide sequence present in fibronectin proteins26,54. Our findings show that ADAM28 promotes cellular migration of JEG3 trophoblast cells on both fibronectin and collagen matrices. These preliminary results bring insight to the possible involvement of the aforementioned integrin-ECM associated processes involved in ADAM28 mediated cell migration, although more evidence is required. My work on identifying the localization and function of ADAM28 in placental cell biology shares similarities and distinctions to recent work from our lab on ADAM12. As with ADAM28, ADAM12 is highly expressed in invasive HLA-G+ trophoblasts localized to distal portions of placental cell columns, promoting column outgrowth and trophoblast invasiveness. However, unlike ADAM28 which is undetectable at alternative regions within placental villi, ADAM12 is also expressed in the syncytiotrophoblast layer of placental villi, promoting trophoblast fusion and syncytial formation through cleavage of E-cadherin23,24. Interestingly, ADAM12 is preferentially expressed in HLA-G− HTR8/Svneo trophoblastic cells with undetectable levels in JEG3 cells; these findings are the inverse to ADAM28’s differential expression within these two cell lines, where ADAM28 levels are only detectable in JEG3 cells. Both ADAM12 and ADAM28 have implications in trophoblast cell mobility and invasiveness, although further investigations are required in elucidating those exact roles in ADAM28.   5.3 Study limitations and shortcomings  One of the major limitations of this study was the inevitable reliance on appropriate human tissue specimens from the CARE program at BC Women’s Hospital. The number of 	   69	  specimens received on a weekly basis fluctuates drastically, and often times left us with no specimens that fit the guidelines of our study. Placental tissue samples that we collect vary in gestational age (between 5-12 weeks), size of specimen, and quality of tissues, which sometimes made finding an appropriate sample for isolating subtypes of trophoblasts quite challenging. Moreover, FACs isolated populations of trophoblast subsets yielded very low numbers of each target population of trophoblasts. For this reason, I chose to focus my efforts on isolating mRNA from these populations; however, future studies may include examining ADAM28 protein levels in these populations by immunoblot analysis.  The mouse monoclonal antibody directed against ADAM28 in this study was used in both immunoblot analysis and immunofluorescence microscopy. However, immunolabelling was only possible on placental specimens embedded in OCT and frozen into cryosections. Although this method was sufficient for first trimester placental specimens, Matrigel-embedded placental explant cultures were unable to be processed into frozen samples due to the inability of the Matrigel to solidify. For these reasons, we were unable to immunolabel ADAM28 in paraffin-embedded explant cultures, and resorted to testing other cellular markers within the invading trophoblast columns.  Another constraint to this study was developing an ADAM28 siRNA-mediated knockdown system in JEG3 trophoblastic cells. After much difficulty in achieving adequate knockdown for the ADAM28-S isoform, I resorted to analyzing ADAM28 mRNA and protein levels over a ten day time period using siRNA-mediated knockdown. ADAM28-S knockdown was seen at day 4 at the mRNA level, and day 5 and 6 at the protein level. For this reason, all downstream ADAM28 siRNA experiments required JEG3 cells to be cultured for 4 days post ADAM28 siRNA transfection prior to experimental analysis to ensure adequate ADAM28 knockdown. Although the reasons behind the delayed knockdown of this gene in trophoblastic cell lines are unknown, I speculate that ADAM28-S protein stability or turnover is prolonged. Studies have shown that extrinsic factors, such as microRNAs, play a role in the regulation of 	   70	  expression certain ADAM genes by increasing mRNA stability 168. It is also conceivable that factors specific to these choriocarcinoma cells lines, perhaps within their culture media, are affecting the prolonged stability of ADAM28-S. To negate this issue in future studies I would use a CRSPR-Cas9 ADAM28 inhibition system to perform functional experiments and continue to investigate the molecular mechanisms controlling ADAM28 expression within trophoblast biology.  5.4 Experimental Strategy  In this study, I utilized three major experimental strategies to test my project aims: i) FACS isolation of trophoblast subsets from first trimester placental villi, ii) Matrigel-embedded first trimester placental explant cultures, and iii) in vivo immunolabelling of serially sectioned placental villi and explant cultures. Although cell-sorting techniques are widely known, researchers have seldom used this approach in isolating trophoblast subpopulations from enzymatically digested placental specimens. Using this innovative approach allows for distinct isolation of target trophoblast populations based on expression of cell surface markers, with little contamination from other cell types. However, the procedure is cumbersome and subjects placental cell types to stress/strain, affecting cell viability. Additionally, in order to obtain sufficient amounts of cells within each sorted population, FACS procedures should directly follow enzymatic digestion of placental specimens. This is due to the freezing and thawing process of stored samples having significant affects on the viability of cells.  To study trophoblast function within placental cell columns, our lab uses a first trimester villous explant model, in which villous tissue is cultured on Matrigel. Unlike other models used for analyzing trophoblast cells within invasive columns, explant cultures provide a model in which trophoblast column outgrowth is two-dimensional and can therefore be readily quantified. Cellular outgrowth from explant models retains the morphology and cellular surface expression of in vivo trophoblast residing in placental cell columns, where this preservation is confirmed by 	   71	  immunostaining with antibodies such as cytokeratin-7, HLA-G, and Ki67 40. Compared to in vitro cell isolation strategies, explant cultures are advantageous in that column cells retain physical contact with the basal lamina, continuing to receive important growth factor signals from the underlying stromal cells, such as FGF, EGF, and VEGF 35,42. Although explant cultures are valuable models mimicking EVT differentiation and trophoblast column function, it is difficult to separately study the different ongoing cellular processes; ie proliferation, adhesion, migration and invasion 54. Separate experiments are required in alternative models to further dissect the role of these cellular processes within the placental column.  The study of human placental is pivotal not only for understanding healthy maternal-fetal interactions, but also in deciphering pregnancy-related disorders such as preeclampsia and IUGR. Many studies use animal models as their primary model to study the temporal development and function of the placental in normal and abnormal pregnancies. Although the fundamental processes of placentation are preserved from species to species, including trophoblast invasion, establishing maternal-fetal vasculature and nutrient transfer, and subsequent maternal vascular remodeling, the mechanisms and factors controlling these processes can be very distinct. Despite structural and morphological similarities between human and non-human primate placentae, these species display superficial implantation with less developed decidua, and minimal interstitial trophoblast invasion 72,169. Rodent models are widely used, accessible experimental models used to study many aspects of implantation and placentation. Although rodents and humans similarly have hemochorial placentae and well-decidualized environments, key structural differences in rodent placentae affect the translatability of findings to a human placental system. Disparities are present in the trophoblast subset organization between rodent labyrinth and trophospongium structures and human villous tree, highlighting differences in the regions of maternal-fetal exchange. Although no animal model resembles exact placental features or the deep trophoblast invasion and remodeling of maternal vessels found in humans, animal models are still warranted as appropriate models in 	   72	  studying the systemic effects of these processes throughout gestation and provide useful information that ex vivo studies cannot.  5.5 Importance of Findings (Biological inferences of ADAM28 in healthy pregnancy)  Sufficient placental column formation and trophoblast invasion are the epitome of successful placentation and a critical determinant of pregnancy outcome. Abnormalities within these processes lead to subsequent inadequate fetal-maternal interactions, resulting in many adverse pregnancy-related complications including miscarriage, preterm birth, preeclampsia, intrauterine growth restriction (IUGR), and intrauterine death4–6. These pregnancy-associated disorders greatly contribute to the low fertility rates of women in North America. Identifying key molecules and mechanisms controlling the proper development of placental subunits is critical to clinical research of placental biology. My work not only highlights ADAM28 as a protease promoting placental column outgrowth and trophoblast migration and survival, but also gives insight into other placental ADAMs that may have important roles in placental development. Furthermore, genes such as ADAM28 that are highly expressed in invasive trophoblast cells may be useful biomarkers in aberrant and high-risk pregnancy screenings. Taken together, the work presented in this thesis contributes to the field of placental research by furthering the role of ADAM family proteases in directing key cellular processes in placental development.  5.6 Summary  Utilizing first trimester placental tissues, trophoblastic cell lines and ex vivo explant cultures, ADAM28 expression was identified in HLA-G+ distal columnar EVTs and choriocarcinoma JAR and JEG3 trophoblastic cell lines. ADAM28 expression of both isoforms ADAM28-S and ADAM28-L was shown to promote column outgrowth in placental villous explant cultures. In elucidating the cellular processes contributing to ADAM28 correlated column growth and trophoblast invasiveness, I provided evidence that ADAM28 promotes cell migration in 	   73	  JEG3 cells when grown on matrix-free, fibronectin, and collagen matrices. Although MTT assays suggest that ADAM28 also promotes cell proliferation, immunolabelling in placental columns showed minimal correlation to proliferating Ki67+ cells. Additionally, immunolabelling in placental explant cultures suggests that ADAM28 plays a role in trophoblast column formation independent of proliferation, but by instead mediating processes such as cellular migration and survival. Further investigation of the functional roles of ADAM28 is required to examine the biological significance of ADAM28 during placental development and trophoblast differentiation. Moreover, studies elucidating the molecular mechanisms in inducing ADAM28 expression as well as identifying the molecular substrates that ADAM28 acts on within the placenta remain to be done. Since ADAM28 functions as a cell surface sheddase and modulates bioavailability of different growth factors and cell surface receptors, the findings presented in this thesis can be built upon further by identifying novel ADAM28 substrates and interacting proteins which may contribute to ADAM28-mediated trophoblast mobility and column growth. The findings presented in this thesis suggest a critical role of ADAM28 in regulating trophoblast function, and along with still unknown molecular mechanisms contributing to ADAM28 activity, highlight ADAM28 and related ADAM family members as attractive candidates in controlling placental development.          	   74	  REFERENCES  1. Evers, J. L. H. Female subfertility. Lancet (London, England) 360, 151–9 (2002). 2. Wang, X. et al. 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