Molecular Human Reproduction, Vol. 6, No. 1, 81-87,
January 2000
© 2000 European Society of Human Reproduction and Embryology
Uterus and pregnancy |
Tie-2 and angiopoietin-2 expression at the fetal–maternal interface: a receptor ligand model for vascular remodelling
1 Department of Obstetrics and Gynecology, and 2 Department of Pathology, Hadassah-University Hospital, Mt Scopus, POB 24035, Jerusalem 91240, Israel
Abstract
The blood vessels at the fetal–maternal interface widen dramatically during pregnancy in order to increase blood flow to nourish the developing fetus. This vessel remodelling destroys normal vessel integrity and encompasses the dissolution of vessel muscle and elastic tissue. It also includes the displacement of endothelial cells by fetal trophoblasts that invade the maternal arteries of the uterus. Interaction between the endothelial cell receptor, Tie-2, and its recently discovered antagonist ligand, angiopoietin-2 (Ang-2), has been implicated in the loosening of vessel structure. Using Northern blot hybridization and RNA in-situ hybridization analysis the expression pattern of Tie-2, and Ang-2 in the placenta throughout pregnancy, was investigated. We found Ang-2 expressed in the syncytiotrophoblast during the first trimester. In addition to the expected expression of the Tie-2 receptor in both fetal and maternal endothelial cells, we observed Tie-2 expression in endovascular invasive trophoblasts. These cells of epithelial origin invade the uterine spiral arteries and acquire endothelial cell properties. The temporal- and lineage-specific pattern of expression of Tie-2 and Ang-2 suggests that this receptor–ligand pair functions during the critical phase of development of the fetal vasculature and reworking of the maternal vessels during normal placentation.
angiopoietin-2/placenta/spiral artery/Tie-2/trophoblast
Introduction
Remodelling of blood vessels at the fetal–maternal interface is an essential determinant in establishing and maintaining a healthy pregnancy. The vessels of the uterus dramatically widen to allow for efficient blood flow to the placenta. This well-characterized alteration is termed conversion of the maternal spiral arteries. It includes loss of the muscle and elastic tissue surrounding the vessels (disruption of the tunica muscularis and elastica) and replacement of the endothelial cell lining with fetal trophoblasts that have invaded the maternal vessels (Brosens et al., 1967
; Boyd and Hamilton, 1970; Pijnenborg, 1990). This process leads to the development of vessels with low resistance and high capacity, thereby enhancing blood flow to the intervillous space to meet the increasing fetal demands for nutrient and gas exchange.
The trophoblasts which displace the maternal endothelial cell lining and colonize the spiral arteries of the uterus are fetal cells of epithelial cell origin. These trophoblasts are derived from a stem cell population of cytotrophoblasts that have embarked on an invasive pathway of differentiation (reviewed in Damsky et al., 1993; Cross et al., 1994). The invasive trophoblasts form cell columns which anchor the placenta to the uterus and invade the uterine tissue (interstitial invasion) and the maternal spiral arteries to the first third of the myometrium (endovascular invasion). The cytotrophoblast stem cells also differentiate to form syncytiotrophoblast of the chorionic villi which is in direct contact with maternal blood in the intervillous space. The syncytiotrophoblast is important for nutrient and gas exchange and production of many of the hormones and growth factors necessary to maintain the pregnancy.
The clinical significance of appropriate interstitial and endovascular trophoblast invasion is demonstrated when this process goes awry. For example, both interstitial and endovascular invasion is defective in pre-eclampsia, a toxaemia of pregnancy. Trophoblast invasion is shallow and does not proceed beyond the decidual portions of the spiral arteries. The mean external diameter of the myometrial vessels is less than half that of similar vessels from normal pregnancies and the number of vessels that show evidence of trophoblast invasion is decreased (Brosens et al., 1972; Gerretsen, 1981; Khong et al., 1986; Moodley and Ramsaroop, 1989). Lack of endovascular invasion by the trophoblasts leads to failure of conversion of the spiral arteries. This could lead to a placenta that is relatively hypoxic, with possible deleterious effects on the fetus, e.g. intrauterine growth retardation.
At the molecular level, the invasive pathway of trophoblast differentiation is associated with a change in adhesion receptor phenotype away from that of epithelial cells (integrins ß4 and ß5), to adhesion receptors typical of invasive cells and endothelial cells [vascular endothelial (VE)-cadherin, ß1 and ß3 integrins, vascular cell adhesion molecule 1 (VCAM-1), platelet-endothelial adhesion molecule 1 (PECAM-1) and intracellular adhesion molecule-1 (ICAM-1)] (Damsky et al., 1992; Burrows et al., 1994; Zhou et al., 1997a). This transformation is demonstrated in both in-vivo and in-vitro invasion assays. Furthermore, when trophoblast invasion does not proceed far enough, as in the case of pre-eclampsia, the switching in adhesion molecule phenotypes is impaired (Genbacev et al., 1996; Lim et al., 1997; Zhou et al., 1997b).
The changes in blood vessel structure at the fetal–maternal interface include conversion of the uterine spiral arteries and also the remodelling of the fetal placental vessels. This remodelling of villous fetal vessels includes loss of elastic membranes, thinner muscular coats and dispersion of muscle cells (Benirschke and Kaufman, 1995). Similarly, the conversion of the maternal vessels at the fetal–maternal interface leads to a vessel with loss of the musco–elastic coat of the vessel wall (disruption of the tunica media). The molecular mechanisms which govern conversion of the maternal spiral arteries and widening of the fetal placental vessels are mostly unknown.
Blood vessel formation can be divided into two main processes, vasculogenesis and angiogenesis (reviewed in Hanahan, 1997). In vasculogenesis the primitive vascular network is established. Angiogenesis is the remodelling of an existing vascular network. Conversion of the spiral arteries is a remodelling process more related to angiogenesis than vasculogenesis. Angiogenesis is mediated in part by the endothelial cell receptor tyrosine kinases (RTKs) Tie-1 and Tie-2 (Dumont et al., 1992; Partanen et al., 1992; Iwama et al., 1993; Maisonpierre et al., 1993; Sato et al., 1993; Schnurch and Risau, 1993; Ziegler et al., 1993) and the recently discovered Tie-2 agonist ligand angiopoietin-1 (Ang-1) and antagonist ligand angiopoietin-2 (Ang-2), (Davis et al., 1996; Maisonpierre et al., 1997). Inactivation of Tie-2 in mice leads to disrupted vessel structure and embryonic lethality (Dumont et al., 1994; Sato et al., 1995). This occurs at a later stage and with a phenotype distinct from those associated with inactivation of the vascular endothelial growth factor receptor tyrosine kinases (VEGF-R1 and VEGF-R2) which are involved in vasculogenesis (Fong et al., 1995; Shalaby et al., 1995). Of note is a novel VEGF-R1 soluble variant in the plasma of pregnant women (Banks et al., 1998) and the detection of specific patterns of VEGF isoforms in endometrial stromal cells (Huang et al., 1998). In mice with null mutations for Ang-1, vessel structure is defective primarily in the lack of smooth muscle cells and pericytes which support the vessel wall (Suri et al., 1996). Over-expression of Ang-1 in the skin of transgenic mice leads to increased vascularization (Suri et al., 1998). Over-expression of Ang-2, the antagonist ligand, in transgenic mice leads to a loosening and disruption of vessel structure and peri-endothelial support cells (Maisonpierre et al., 1997). A normal role for Ang-2 in vascular remodelling is displayed during rat ovarian follicle development. Full maturation necessitates vascular sprouting and growth that hypervascularizes the corpus luteum. The expression pattern of Ang-2 RNA is increased at the leading edge of vessels invading the corpus luteum, perhaps blocking the function of Ang-1 and allowing for vessel plasticity (Maisonpierre et al., 1997). The alterations in vessel structure coincident with normal Ang-2 expression, Ang-2 transgene over-expression and lack of Ang-1 parallel the remodelling of vessels at the fetal–maternal interface. This led us to investigate Ang-2 and Tie-2 expression during placental development.
In this study we describe the RNA expression patterns of the receptor Tie-2 and the ligand Ang-2 in the development of the human placenta. The expression of Tie-2 in fetal and maternal vasculature, and the temporal and cell lineage regulated expression of the ligand Ang-2 in the syncytiotrophoblast of the placenta, implicate a ligand receptor interaction at the fetal–maternal interface which may be essential for vascular remodelling in the placenta. In addition, Tie-2 was not expressed in trophoblasts other than those that have begun the endovascular invasion of the spiral arteries. This finding may reflect the new role that these trophoblasts must undertake in displacing and replacing the endothelial cells lining these arteries and remodelling vessels wide enough to nourish the fetus.
Materials and methods
First and second trimester placentas were obtained from elective pregnancy terminations, normal third trimester placentas were taken from normal deliveries or Caesarean sections all in accordance with the protocol of the Human Subjects Committee approved by our institution.
Tissue culture
Ishikawa, a human endometrial adenocarcinoma cell line, was grown as previously described (Hochner-Celnikier et al., 1997). JEG-3, a human choriocarcinoma cell line, was grown in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal calf serum and L-glutamine and gentamicin (all tissue culture reagents were supplied from Biological Industries, Beit Haemek, Israel) at 37°C in a humidified 5% CO2 incubator.
Northern blot hybridization
Total RNA was isolated with Tri Reagent (MRC Inc, Cincinnati, OH, USA) 20 µg total RNA was separated by formaldehyde/agarose gel electrophoresis (Ausubel et al., 1991) and transferred to a genescreen plus membrane (New England Nuclear, Boston, MA, USA) hybridized with a radioactive DNA probe, washed (according to the instructions provided with the membrane) and exposed to RX medical X-Ray film (Fuji Photo Film Co. Ltd, Japan).
Probes for Northern blot hybridization
The Tie-2 probe was a 1 kb cDNA fragment of the human gene, encoding amino acids 70–380. The Ang-2 probe was a 640 bp cDNA fragment encompassing 360 bp of 5' untranslated region and the coding region for the first 99 amino acids. Radioactive probes were prepared by random priming with a kit purchased from Boehringer Mannheim Biochemicals (Mannheim, Germany) and [32P]-dCTP (New England Nuclear).
In-situ hybridization
Tissues were formalin fixed and paraffin embedded. Sections (5 µm) were floated onto Superfrost/Plus slides (Menzel-Glaser, Germany). Radioactive RNA in-situ hybridization was carried out according to a previously described method (Millen and Hui, 1996), with some modifications. We investigated chromogen methods and found that given the high levels of alkaline phosphatase in the placenta the radioactive method that we employed gave the more reliable results. Prior to in-situ hybridization, the slides were baked for 40 min at 55°C, deparaffinized in xylene, and rinsed in graded alcohols followed by 0.5x sodium chloride/sodium citrate (SSC). Slides were treated with proteinase K, 20 µg/ml. (Sigma Chemical Corp, St Louis, MO, USA) for 7.5 min at 37°C. Acetylation of the slides was for 10 min at room temperature in 0.1 mol/l triethanolamine hydrochloride (TEA) (Sigma)/acetic anhydride. Slides were rinsed in phosphate-buffered saline (PBS) and 0.5x SSC, dehydrated through graded alcohol, and air-dried at room temperature. Hybridization with [35S]-UTP labelled antisense riboprobe was performed in 50% formamide at 55°C in a humidified chamber. After an overnight incubation the slides were rinsed in 2x SSC/0.1% 2-mercaptoethanol and treated with 20 µg/l RNAse A (Sigma Chemical Corporation) for 30 min at 37°C. The slides were washed for 2 h in 0.1x SSC/0.1% 2-mercaptoethanol at 55°C, rinsed in 0.5x SSC and air-dried. Slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak Co, Rochester, NY, USA) and exposed for 1–2 weeks at 4°C, and developed according to the manufacturer's instructions at 15°C. The slides were counterstained with haematoxylin and eosin and photographed under bright field and dark field microscopy.
In-situ ribobrobes
In-situ antisense riboprobes were generated from the 1 kb Tie-2 and 640 bp Ang-2 cDNAs (described above) in the plasmid bluescript (Stratagene, CA, USA). Stability of the Tie-2 probe was increased when the 5' most 350 bp of the 1 kb Tie-2 cDNA was subcloned into bluescript. Antisense radioactive riboprobes were synthesized from linearized plasmids with T3 or T7 RNA polymerase (Boehringer Mannheim) according to the orientation of the gene and digested with RNAse-free DNAse (Boehringer Mannheim). [35S]-UTP (New England Nuclear) was incorporated into the riboprobe labelling reactions.
Immunohistochemistry
Anti-cytokeratin immunohistochemistry with a broad spectrum keratin monoclonal mouse primary antibody was performed on serial sections according to the Histostain Plus kit (Zymed Lab-SA System, San Francisco, CA, USA), and developed with their AEC chromogen mixture. Control experiments were performed with non-immune serum to determine non-specific background staining.
Results
Northern analysis of Ang-2 and Tie-2 in the human placenta
We initially studied Ang-2 and Tie-2 expression in the human placenta by Northern blot hybridization of total RNA isolated from first, second and third trimester placentas. RNA from a choriocarcinoma cell line (JEG-3, which preserves many trophoblast properties) was also studied. In the case of Tie-2, we also used RNA from the human endometrial cell line, Ishikawa. We observed expression of the 2.1 kb Ang-2 RNA in only first trimester placenta (Figure 1). There was a faint lower molecular weight band of about 1.8 kb visible in all of the lanes. RNA integrity was verified by hybridizing the blot to a human ß-actin probe (Figure 1). Using the Tie-2 probe (Figure 2) we detected the 4 kb Tie-2 transcript in first, second and third trimester placental RNA but not in the JEG-3 or Ishikawa cells. The quality of the RNA was verified when the blot was hybridized with a ß-actin probe (Figure 2).
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Expression of Ang-2 in trophoblasts
To determine which cell types in the placenta expressed the Ang-2 we used RNA in-situ hybridization analysis on tissue sections of human placenta. In first trimester and early second trimester placenta, Ang-2 RNA was detected in the syncytiotrophoblast (Figure 3A,B
). Ang-2 was not detected in the cytotrophoblastic cells (Figure 3C,D), trophoblast cell columns or the interstitial extravillous trophoblasts. Expression of Ang-2 often appeared uneven within the syncytiotrophoblast of an individual section and this patchy expression was observed occasionally within an individual villus. We did not detect Ang-2 in the syncytiotrophoblast nor in interstitial trophoblasts of third trimester placenta. Examination of a placenta accreta revealed Ang-2 expression in the myometrium in small capillaries and veins (data not shown). No hybridization above background levels was detected when a sense Ang-2 probe was used for in-situ analysis (data not shown).
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Expression of Tie-2 in fetal and maternal vessels
We proceeded to examine Tie-2 expression in the placenta by RNA in-situ hybridization. In first trimester placenta (8 weeks of gestation) we detected Tie-2 RNA in areas of fetal blood vessels (Figure 4A,B
). Later in the first trimester the lumens of fetal vessels have widened and Tie-2 expression in endothelial cells is clearly observed (Figure 4C,D). Positive Tie-2 expression was found in the endothelial cells lining vessels of the decidua in third trimester and in the vessels of the myometrium in sections of uterus with placenta accreta (data not shown). When we used a sense Tie-2 probe we did not detect hybridization above background levels (data not shown).
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Expression of Tie-2 in trophoblasts invading maternal vessels
In early second trimester placenta we could detect vessels at the placental bed undergoing remodelling, where trophoblasts had entered the vessel and were in the process of displacing the endothelial cells. Tie-2 expression (Figure 5A–D
) was detected in these endovascular trophoblasts and in endothelial cells. We confirmed that these Tie-2-positive cells were indeed trophoblasts by positive immunohistochemical staining of the cells with an anti-cytokeratin antibody (Figure 5E).
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Discussion
We describe the temporal and cell lineage restricted expression of both the ligand Ang-2 and its receptor Tie-2 in the human placenta. Using both Northern blotting and RNA in-situ hybridization analysis we found that Ang-2 expression was restricted to the first trimester of human pregnancy. This expression was limited to the syncytiotrophoblast of the chorionic villi. We would like to know how the protein expression of Ang-2 is correlated with RNA expression. However, in order to get maximum resolution of tissue morphology we studied paraffin-embedded sections, but we did not have an antibody that would bind to Ang-2 in paraffin sections. Tie-2 expression was observed in fetal and maternal endothelial cells. Intriguingly, Tie-2 was also expressed in fetal trophoblast cells that invade the maternal blood vessels and replace the endothelial cells which line the vessels. Appropriately timed vascular remodelling at the fetal–maternal interface is essential for the continuation of the pregnancy, and nears completion by early second trimester. Ang-2 produced by the fetal syncytiotrophoblast may initiate the changes in both fetal and maternal vessels where the receptor is found. This finding suggests cross-talk across the fetal–maternal interface for the ligand–receptor pair Ang-2 and Tie-2. Ang-2 expressed in the syncytiotrophoblast can theoretically reach both fetal and maternal endothelial cells. Interaction with the maternal side of the placenta would occur through the syncytiotrophoblast, which is in direct contact with maternal blood of the intervillous space. This blood reaches the maternal vessels where Tie-2 is found. On the fetal side, we assume that Ang-2, like the growth factors and hormones produced by the syncytiotrophoblast, can reach the fetal vessels of the chorionic villi where Tie-2 is expressed. We propose that Tie-2 and Ang-2 may be involved in the remodelling of the maternal vessels at the fetal–maternal interface and may also play a role in the formation of the placental fetal vascular tree (Figure 6). Thus, the fetus mediates the remodelling of the maternal vessels necessary for sustaining the pregnancy. Failure of this process would lead to defective placentation and may be one of the primary causes for the relatively high rate of pregnancy loss at the end of the first trimester.
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When we studied expression of the receptor Tie-2 we found that it is expressed in endothelial cells of both fetal and maternal vessels. We further observed that trophoblasts colonizing the maternal spiral arteries expressed Tie-2, whereas trophoblasts invading decidual tissue did not express detectable levels of Tie-2 (Figures 6 and 7
). Here we have evidence of trophoblasts expressing an endothelial cell specific marker while undergoing endovascular, but not interstitial, invasion. This supports earlier studies which demonstrate that the invasive trophoblasts were associated with a change in the expression of the repertoire of adhesion molecules and acquisition of endothelial cell markers (Damsky et al., 1992; Burrows et al., 1994; Zhou et al., 1997a). We report that Tie-2 is expressed only in endovascular invasive trophoblasts. This is the first marker to be found that can distinguish between trophoblasts that invade tissue and those that invade blood vessels. This is in agreement with the acquisition by trophoblasts (specialized epithelial cells) of properties of an endothelial cell (Zhou et al., 1997a). The reason for trophoblast invasion of the spiral arteries and displacement and replacement of the maternal endothelial cells is unknown. However, it is a necessary precondition for subsequent widening of the maternal vessels. For example, when trophoblasts do not appropriately invade the spiral arteries they are narrower than normal, leading to a relatively hypoxic placenta as in the case of pre-eclampsia. We can only speculate why the endovascular trophoblast expresses Tie-2. In Tie-2 knock-out mice, angiogenesis is defective; most notably the vessels lack proper association with the peri-endothelial support cells. Perhaps in the first trimester of pregnancy, when the antagonist Ang-2 is expressed, binding of Ang-2 to the receptor Tie-2 on the endovascular invasive trophoblasts elicits the loosening of the pericyte as a prerequisite to widening of the vessels (Figure 7), coincident with loss of the muscle and elastic tissue supporting the vessel.
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In summary, the expression of Tie-2 by fetal and maternal endothelial cells and endovascular invasive trophoblasts, and the expression of the ligand Ang-2 by syncytiotrophoblast, suggests that the fetus supplies the ligand for widening of both fetal and maternal vessels of the placenta. The Ang-2 and Tie-2 receptor ligand system may play a pivotal role in remodelling of the vessels at the fetal–maternal interface.
Acknowledgments
We thank our colleague Mrs Ruth Har-Nir for advice and assistance and Mrs Mally Sappir for technical assistance. We thank Dr Samuel Davis of Regeneron Pharmaceuticals, Tarrytown, New York, USA, for providing us with the Tie-2 and Ang-2 plasmids.
Notes
3 To whom correspondence should be addressed 
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Submitted on May 18, 1999; accepted on October 8, 1999.
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 A. A. Ashkar, J. P. Di Santo, and B. A. Croy Interferon {gamma} Contributes to Initiation of Uterine Vascular Modification, Decidual Integrity, and Uterine Natural Killer Cell Maturation during Normal Murine Pregnancy J. Exp. Med., July 17, 2000; 192(2): 259 - 270. [Abstract] [Full Text] [PDF]
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