Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

Summary In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the intial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the initial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Endoglin (CD105) expression is regulated by the liver X receptor alpha (NR1H3) in human trophoblast cell line JAR

Human implantation involves invasion of the uterine wall and remodeling of uterine arteries by extravillous cytotrophoblasts. Defects in these early steps of placental development lead to poor placentation and are often associated with preeclampsia, a frequent complication of human pregnancy. One of the complex mechanisms controlling trophoblast invasion involves the activation of the liver X receptor beta (or NR1H2, more commonly known as LXRbeta) by oxysterols known as potent LXR activators. This activation of LXRbeta leads to a decrease of trophoblast invasion. The identification of new target genes of LXR in the placenta could aid in the understanding of their physiological roles in trophoblast invasion. In the present study, we show that the endoglin (ENG) gene is a direct target of the liver X receptor alpha (NR1H3, also known as LXRalpha). ENG, whose gene is highly expressed in syncytiotrophoblasts, is part of the transforming growth factor (TGF) receptor complex that binds several members of the TGFbeta superfamily. In the human placenta, ENG has been shown to be involved in the inhibition of trophoblast invasion. Treatment of human choriocarcinoma JAR cells with T0901317, a synthetic LXR-selective agonist, leads to a significant increase in ENG mRNA and protein levels. Using transfection and electrophoretic mobility shift assays, we demonstrate that LXR (as a heterodimer with the retinoid X receptor) is able to bind the ENG promoter on an LXR response element and mediates the activation of ENG gene expression by LXRalpha in JAR cells. This study suggests a novel mechanism by which LXR may regulate trophoblast invasion in pathological pregnancy such as preeclampsia.     

Intrauterine trophoblast migration: A comparative view of humans and rodents

ABSTRACT

Trophoblast migration and invasion through the decidua and maternal uterine spiral arteries are crucial events in placentation. During this process, invasive trophoblast replace vascular endothelial cells as the uterine arteries are remodeled to form more permissive vessels that facilitate adequate blood flow to the growing fetus. Placentation failures resulting from either extensive or shallow trophoblastic invasion can cause pregnancy complications such as preeclampsia, intrauterine growth restriction, placenta creta, gestational trophoblastic disease and even maternal or fetal death. Consequently, the use of experimental animal models such as rats and mice has led to great progress in recent years with regards to the identification of mechanisms and factors that control trophoblast migration kinetics. This review aims to perform a comparative analysis of placentation and the mechanisms and factors that coordinate intrauterine trophoblast migration in humans, rats and mice under physiological and pathological conditions.     

The rat as an animal model for fetoplacental development: a reappraisal of the post-implantation period

Following implantation in rodents, the uterine stromal fibroblasts differentiate into densely packed decidual cells. This process, called decidualization, is well-orchestrated and progresses both antimesometrially and mesometrially, creating two regions with distinctive cellular morphologies. In addition, subsequent placental development is dependent on the invasion of the trophoblast, the process intimately linked to the endometrial tissue remodelling and depending largely on the environment created by the decidua; this phenomenon is crucial for the establishment and maintenance of pregnancy. The key mechanisms underlying the maternal tissue remodelling and trophoblast invasion remain poorly understood. The rat, just like human beings, exhibits a highly invasive type of placental development, the haemochorial placentation. For obvious ethical reasons, the studies of endometrial tissue remodelling throughout pregnancy in humans are greatly limited. Although the rat differs somewhat from humans with regards to the implantation process, it is an appropriate model for studying the mechanisms of decidualization as well as subsequent remodelling of the uterine tissues and fetoplacental development. As decidual remodelling is very closely linked to placentation and the maternal-fetal interactions in the rat show several important similarities to human placentation, the morphological alterations occurring during the post-implantation period in the rat have been addressed in the present review.     

Function and control of human invasive trophoblast subtypes: Intrinsic vs. maternal control

ABSTRACT

The establishment of a functional placenta is pivotal for normal fetal development and the maintenance of pregnancy. In the course of early placentation, trophoblast precursors differentiate into highly invasive trophoblast subtypes. These cells, referred to as extravillous trophoblasts (EVTs), penetrate the maternal uterus reaching as far as the inner third of the myometrium. One of the most fundamental functions of EVTs is the transformation of spiral arteries to establish the uteroplacental blood circulation assuring an adequate nutrient and gas supply to the developing fetus. To achieve this, specialized EVT subpopulations interact with maternal immune cells, provoke elastolysis in the arterial wall and replace the endothelial cells lining the spiral arteries to induce intraluminal vascular remodeling. These and other trophoblast-mediated processes are tightly controlled by paracrine signals from the maternal decidua and furthermore underlie an intrinsic cell-type specific program. Various severe pregnancy complications such as preeclampsia or intrauterine growth retardation are associated with abnormal EVT function, shallow invasion, and decreased blood flow to the placenta. Hence a better understanding of human trophoblast invasion seems mandatory to improve therapeutic intervention. This approach, however, requires a profound knowledge of the human placenta, its various trophoblast subtypes and in particular a better understanding of the regulatory network that controls the invasive phenotype of EVTs.     

Regulation of human extravillous trophoblast function by membrane-bound peptidases

During human placentation, the invasion of extravillous trophoblasts (EVTs) into maternal decidual tissues, especially toward maternal spiral arteries, is considered an essential process for subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVTs from further invasion have not been well understood. Recently, we found that two cell surface peptidases, dipeptidyl peptidase IV (DPPIV) and carboxypeptidase-M (CP-M,) are differentially expressed on EVTs. DPPIV expression was mainly observed on EVTs that had already ceased invasion. CP-M was detected on migrating EVTs including endovascular trophoblasts in the maternal arteries. The enzymatic inhibition of these peptidases affected the invasive property of choriocarcinoma-derived cell lines, BeWo and JEG3 cells. In addition, a chemokine, RANTES, that is one of the substrates for DPPIV, enhanced invasion of EVTs isolated from primary villous explant culture and its receptor, CCR1, was specifically expressed on migrating EVTs toward maternal arteries. Furthermore, a novel membrane-bound cell surface peptidase, named laeverin, was found to be specifically expressed on EVTs that had almost ceased invasion. These findings suggest that membrane-bound peptidases are important factors regulating EVT invasion during early placentation in humans.     

Expression of SWAP-70 in the uterus and feto-maternal interface during embryonic implantation and pregnancy in the rhesus monkey (Macaca mulatta)

SWAP-70 is a unique signaling protein involved in multiple processes including lymphatic cell activation, migration, adhesion, and cytoskeleton organization. Its role in reproductive system remains to be unclear. In the present study, the spatial and temporal expression of SWAP-70 in the uterus during normal menstrual cycle as well as on the feto-maternal interface during pregnancy was investigated in the rhesus monkey by in situ hybridization and immunohistochemistry. It was shown that SWAP-70 was mainly expressed in glandular epithelial cells of uterine endometrium, and the level peaked at the mid-secretory stage. At the beginning of embryonic implantation, SWAP-70 was intensely expressed at the implantation site, mainly localized in glandular and luminal epithelial cells, as well as in primary trophoblasts and epithelial plaque. High level of SWAP-70 was observed in villous cytotrophoblast (VCT), syncytiotrophoblast (ST), column cytotrophoblast, trophoblast shell, interstitial trophoblast, and endovascular trophoblast during gestational days 15–25. From gestational day 50 to term, expression of SWAP-70 decreased evidently and was restricted in VCT cells. What’s more, SWAP-70 co-localized with F-actin on the feto-maternal interface, especially in highly motive extravillous trophoblasts. The data indicate that SWAP-70 may be involved in regulating motility of trophoblast cells during embryonic implantation and placentation.     

Placental villous mesenchymal cells trigger trophoblast invasion

The successful transformation of uterine spiral arteries by invasion trophoblasts is critical for the formation of the human hemochorial placenta. Placental trophoblast migration and invasion are well regulated by various autocrine/paracrine factors at maternal–fetal interface. Human placental multipotent mesenchymal stromal cells (hPMSCs) are a subpopulation of villous mesenchymal cells and have been shown to produce a wide array of soluble cytokines and growth factors including HGF (hepatocyte growth factor). The function of hPMSCs in placental villous microenvironment has not been explored. The interaction between hPMSCs and trophoblasts was proposed in vitro in a recent article. HGF produced by hPMSCs was able to engage c-Met receptor on trophoblast and induced the trophoblast cAMP expression. The cAMP activated PKA, which in turn, signaled to Rap1 and led to integrin β1 activation. The total integrin β1 protein expression by trophoblasts was not affected by HGF stimulation. Hypoxia downregulated HGF expression by hPMSCs. HGF and PKA activator 6-Bnz-cAMP increased trophoblast adhesion and migration that were inhibited by PKA inhibitor H89 or Rap1 siRNA. Thus, hPMSCs-derived paracrine HGF can regulate trophoblast migration during placentation. These findings provided insight revealing at least one mechanism by which hPMSCs implicated in the development of preeclampsia.     

Expression and localization of SWAP-70 in human fetomaternal interface and placenta during tubal pregnancy and normal placentation.

SWAP-70 has been demonstrated as a multiple functional signaling protein involved in formation of membrane ruffling induced by signal cascade of tyrosine kinase growth factor receptors. In the present study, the spatial and temporal expression pattern of SWAP-70 on human fetomaternal interface was investigated using specimens collected from tubal and normal pregnancies by in situ hybridization, immunohistochemistry, and Western blotting. Data showed an intense expression of SWAP-70 in trophoblasts at weeks 3-6 of fallopian implantation and at weeks 6-7 of normal pregnancy. The most intense expression was exhibited by those highly motile and invasive extravillous trophoblasts. From gestational week 8 on, the level of SWAP-70 in trophoblasts decreased significantly, and the signal was restricted in villous cytotrophoblast cells. In the in vitro cultured human trophoblast cell line, B6Tert-1, colocalization of SWAP-70 with F-actin was verified. Data in human placenta were similar to what we recently reported on rhesus monkey fetomaternal interface. Our results suggest that SWAP-70 may be involved in regulating migration and invasion of trophoblast cells during the processes of embryonic implantation and placentation in primates.     

Differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta

The molecular mechanisms and pathologic significance of placental viral infections are poorly understood. We investigated factors that regulate placental infection by adenovirus, which is the most common viral pathogen identified in fetal samples from abnormal pregnancies (i.e., fetal growth restriction, oligohydramnios, and nonimmune fetal hydrops). We also determined the pathologic significance of placental adenovirus infection. Northern hybridization, flow cytometry, and immunostaining revealed that placental expression of the coxsackievirus and adenovirus receptor (CAR) varied with gestational age and trophoblast phenotype. The CAR was continuously expressed in invasive or extravillous trophoblast cells but not in villous trophoblast cells. We postulate that the villous syncytiotrophoblast, which does not express CAR and is resistant to adenovirus infection, limits the transplacental transmission of viral pathogens, including adenovirus. Conversely, extravillous trophoblast cells underwent apoptosis when infected by adenovirus in the presence of decidual lymphocytes (which simulated the maternal immune response to viral infection). Thus, adenovirus infection and/or the maternal immune response to adenovirus infection induced the death of placental cell types that expressed CAR. Consequently, we speculate that adenovirus infection of extra-villous trophoblast cells may negatively impact the process of placental invasion and predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.     

The role of invasive trophoblast in implantation and placentation of primates

We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human.     

Osteopontin expression in uterine stroma indicates a decidualization-like differentiation during ovine pregnancy

Osteopontin (OPN) is a component of the extracellular matrix that interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function. In pregnant mice and primates, OPN has been detected in decidualized stroma and is considered to be a gene marker for decidualization. Decidualization involves transformation of spindle-like fibroblasts into polygonal epithelial-like cells that are hypothesized to limit conceptus trophoblast invasion through the uterine wall during invasive implantation. Decidualization is not considered characteristic of species with noninvasive implantation, such as domestic animals. However, the extent of trophoblast invasion between sheep and pigs differs, with sheep exhibiting erosion of the uterine luminal epithelium (LE) and fusion of trophectoderm with LE to form syncytia, and pigs maintaining an intact LE throughout pregnancy. Therefore, the present study measured changes in the decidualization marker genes OPN, desmin, and alpha smooth muscle actin (alphaSMA) in ovine and porcine uterine stroma throughout pregnancy. The morphology of endometrial stromal cells in pregnant ewes changes following conceptus attachment, with cells increasing in size and becoming polyhedral in shape by Day 35 of pregnancy. Expression of OPN mRNA and protein, as well as desmin and alphaSMA proteins, was observed in this same uterine stromal compartment. In contrast, no morphological changes in uterine stroma nor induction of OPN mRNA and protein, or desmin protein, were detected during porcine pregnancy. Interestingly, alphaSMA protein was absent on Day 20, but prominent in uterine stroma of pregnant pigs on Day 45. Collectively, these results indicate that the uterine stroma of sheep undergoes a program of differentiation similar to decidualization in invasive implanting species, whereas porcine stroma exhibits differentiation that is more limited than that in sheep, rodents, or primates. Results suggest that uterine stromal decidualization is common to species with different types of placentation, but the extent is variable and correlates with the depth of trophoblast invasion during implantation.     

The role of corticotropin-releasing hormone in blastocyst implantation and early fetal immunotolerance.

During blastocyst implantation, the maternal endometrial response to the invading semi-allograft has characteristics of an acute, aseptic inflammatory response. However, once implanted, the embryo suppresses this response and prevents rejection. Simultaneously, the mother's immune system prevents a graft VS. host reaction deriving from the fetal immune system. We have shown that embryonic trophoblast and maternal decidua cells, i.e., cells located in the interface between the fetal placenta and the maternal endometrium, produce corticotropin-releasing hormone (CRH) and express Fas ligand. CRH may play a crucial role in the implantation and the anti-rejection process that protects the fetus from the maternal immune system, primarily by killing activated T cells through the Fas-FasL interaction. In experimental animals, type 1 CRH receptor (CRH-R1) blockade by antalarmin, a specific type 1 CRH receptor antagonist, decreased implantation sites by approximately 70%. CRH is also involved in controlled trophoblast invasion, by downregulating the synthesis of the carcinoembryonic antigen-related cell adhesion molecule 1 by extravillous trophoblast cells. IN VITRO findings showed that CRH-R1 blockade by antalarmin increased trophoblast invasion by approximately 60%. Defective uterine CRH/CRH-R1 system during early pregnancy may be implicated in the pathophysiology of recurrent miscarriage, placenta accreta, and preeclampsia.     

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.     

Expression of P450 aromatase and 17beta-hydroxysteroid dehydrogenase type 1 at fetal-maternal interface during tubal pregnancy

Steroidogenesis in the placenta has been studied widely, but little is known about steroid metabolism in ectopic pregnancy. Previous studies have indicated that trophoblast invasion and placentation in the uterus and the fallopian tube may be controlled by similar mechanisms. As far as 17β-estradiol (E₂) production is concerned, it has been well demonstrated that its biosynthesis in the placenta involves the action of P450 aromatase (P450arom) and 17β-hydroxysteroid dehydrogenase type 1 (17HSD1). The purpose of this study was to characterize the expression pattern of P450arom and 17HSD1 at the fetal–maternal interface, particularly in various trophoblast cells, in tubal pregnancy. Using in situ hybridization, P450arom mRNA was localized in syncytiotrophoblast (ST) cells, which are mainly responsible for hormone production during pregnancy, whereas no signal was detected in villous cytotrophoblast (VCT), column CT and extravillous CT (EVCT) cells. Immunohistochemical assays revealed that 17HSD1 is present in ST cells, a large portion of EVCT cells and 20% of column CT cells. On the other hand, no expression of 17HSD1 was detected in VCT cells. In addition, 17HSD1 was found in epithelial cells of the fallopian tube. Interestingly, the expression level of 17HSD1 in fallopian tube epithelium during tubal pregnancy was significantly higher than that during normal cycle. Our data provide the first evidence that normal and tubal pregnancies possess identical expression of P450arom and 17HSD1 in ST cells and therefore, similar E₂ production in the placenta. Further, the association of 17HSD1 with EVCT cells indicates that 17HSD1 perhaps play a role in trophoblast invasion. Finally, increased expression of 17HSD1 in epithelial cells of fallopian tube may lead to a local E₂ supply sufficient for the maintenance of tubal pregnancy.