Human Placentation Research Articles

Expression and Oxygen Regulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Human Placenta during Early Pregnancy

Angiogenesis is a key process of dynamic tissue remodeling occurring during placentation. Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be deregulated in preeclampsia. Recently a new angiogenic factor, endocrine gland-derived VEGF (EG-VEGF), also known as prokineticin 1 (PK1), has been identified, and its expression was shown to be restricted to endocrine glands, including the placenta. In this study we investigated the pattern of expression of EG-VEGF, its related factor Bv8/PK2, and their common receptors, PKR1 and PKR2, in human placenta during the first trimester of pregnancy. We also examined EG-VEGF and PKR1 regulation by oxygen tension in isolated trophoblast cells (TCs). Our results show that EG-VEGF, but not Bv8/PK2, is expressed in human placenta. EG-VEGF is mainly localized to the syncytiotrophoblast layer with the highest expression detected between the 8th and 10th wk of gestation. EG-VEGF expression within placental villi is different from that of VEGF, which is mainly localized in the cytotrophoblast and extravillous trophoblast cells. In TCs, PKR1 mRNA is about 80 times more abundant than PKR2 mRNA. Both EG-VEGF and PKR1 mRNAs appear to be regulated by hypoxia. These findings suggest that EG-VEGF has a direct effect on TCs via its receptor PKR1 and is likely to play an important role in human placentation. The expression pattern of EG-VEGF, its regulation by oxygen tension, and its complementary localization to that of VEGF suggest that this new factor might also be deregulated in preeclampsia.

Serine Peptidase HTRA3 Is Closely Associated with Human Placental Development and Is Elevated in Pregnancy Serum1

HTRA3 is a newly identified serine peptidase of the mammalian HTRA (high-temperature requirement factor A) family, that is upregulated dramatically during mouse placental development. The current study determined whether HTRA3 was involved in human placentation. During the menstrual cycle, HTRA3 was expressed primarily in the endometrial glands, being significantly upregulated toward the mid- to late secretory phases; prominent expression in the stroma detected only in the decidual cells in the late secretory phase. Thus, overall endometrial HTRA3 expression was highest in the late secretory phase, when the endometrium is prepared for maternal-trophoblast interaction. During the first trimester of pregnancy, both glandular and decidual HTRA3 expression increased further with the decidual upregulation being highly significant. The strong link between HTRA3 expression and endometrial stromal cell decidualization was further established in an in vitro model using primary endometrial stromal cells. HTRA3 was also expressed by certain trophoblast subtypes in the first-trimester placenta: strongly in the villous syncytiotrophoblast, trophoblast shell, and endovascular trophoblast and weakly in the distal portion of the trophoblast cell columns but not in villous cytotrophoblast, the proximal region of the cell columns, or interstitial trophoblast. Upregulation of HTRA3 expression in association with placental development was revealed by a significant elevation of this protein in the maternal serum during the first trimester. We thus propose that HTRA3 is a previously unrecognized factor closely associated with and potentially important for human placentation. This study established crucial groundwork for future investigations toward establishing the physiological roles of HTRA3 in human placentation.

EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation

In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Previously, we have shown that invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. Here, we report that these ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion was accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoided EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration was significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulated chemokine-induced but not growth factor-stimulated migration. We propose that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generated repulsive signals and a reduction in chemokine-mediated responses limit their interaction with veins. When they encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation.

Priming and remodelling of human placental bed spiral arteries during pregnancy – A Review

It is now well known that in order to establish human hemochorial placentation and to provide a progressive increase in blood supply to the growing fetus, the uterine spiral arteries must undergo considerable alterations. This physiological modification is thought to be brought about by the interaction of invasive cytotrophoblast with the spiral artery vessel wall. Despite intensive research our understanding of the mechanisms that control human trophoblast invasion in normal, let alone abnormal pregnancy, are still poorly understood. This is partly due to difficulties in obtaining "true" placental bed biopsies and most investigators have relied on in vitro models of trophoblast invasion. Clearly interpretation of such studies must be tempered with a degree of caution. This review outlines why the placental bed is important, how we can sample and study it, what morphology actually occurs in the placental bed spiral arteries during pregnancy and then briefly summarise the findings on the placental bed in pre-eclampsia.

The fibronectin receptor α5 integrin subunit is upregulated by cell-cell adhesion via a cyclic AMP-dependent mechanism: Implications for human trophoblast migration

Cell adhesion molecules are implicated in the mechanisms regulating trophoblast migration during human embryo implantation and placentation. We investigated the expression and subcellular organization of the fibronectin receptor alpha5 integrin subunit during the differentiation of human trophoblasts in vitro, and the role of cyclic adenosine monophosphate (cAMP) in the process. Human trophoblasts isolated from chorionic villi expressed no alpha5 integrin, but the molecule was upregulated as cells aggregated in vitro. Low levels of expression of alpha5 integrin subunit and a diffuse cellular distribution pattern were seen in migrating mononuclear trophoblasts. Formation of cell aggregates was accompanied by increased expression of the alpha5 integrin, which translocated to the cytoskeleton-bound pool of proteins and clustered within focal adhesion plaques on the cell surface. This coincided with increased binding to fibronectin. In the absence of cell-cell adhesion, trophoblasts did not display an increase in alpha5 integrin messenger RNA or protein and there was no alpha5 integrin in focal adhesion plaques, suggesting that cell-cell contacts specifically trigger the upregulation of alpha5 integrin subunit and its subcellular translocation. Cyclic AMP is the second messenger mediating the aggregation-induced increase in alpha5 integrin: cAMP increased the de novo synthesis of alpha5 integrin protein, particularly in mononuclear cells, whereas the aggregation-induced increase in alpha5 integrin was strongly inhibited by the antagonist Rp-cAMP in aggregating cells. Our data provide evidence that the alpha5 integrin mediates binding of human trophoblasts to fibronectin and is implicated in the regulation of trophoblast migration. This integrin's expression is specifically triggered by cell-cell adhesion and regulated via cAMP-mediated pathway(s). It is hypothesized that these mechanisms may play an important role in the molecular events controlling human placentation.

Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion

Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.

Clinical aspects and conservative management of placenta accreta

Placenta accreta is a rare complication of human placentation with fertility and life‐threatening sequelae. Traditional management has centred upon hysterectomy and its prompt undertaking has been well documented in saving lives. While in many situations hysterectomy will remain appropriate, there are other management options available involving conservative approaches. This article reviews the diagnosis and demographics of this potentially frightening pathology and highlights the important aspects of conservative management if hysterectomy is to be avoided.

Cellular localization of gonadotropin-releasing hormone (GnRH) I and GnRH II in first-trimester human placenta and decidua.

There is increasing evidence to suggest that the classical form of GnRH (GnRH I) and the second mammalian form of this hormone, GnRH II, play regulatory roles in human implantation and placentation. To date, the cellular distribution of these two hormones at the maternal-fetal interface remains poorly characterized. In these studies, we localized GnRH I and GnRH II expression in human placenta and decidua to distinct subpopulations of cells isolated from these tissues and in a chorionic villous/decidual tissue coculture system that mimics many of the cellular events of early placentation. GnRH I and GnRH II mRNA transcripts were detected in first-trimester placenta, whereas only GnRH I was detected in tissues obtained at term. Both hormones were further immunolocalized to the mononucleate villous and distinct subpopulations of extravillous cytotrophoblasts of the placenta in vivo and in vitro. In contrast, GnRH I but not GnRH II was expressed in the outer multinucleated syncytial trophoblast layer of first trimester chorionic villi and in cultures of villous cytotrophoblasts allowed to undergo differentiation and fusion in vitro. GnRH I and GnRH II were also found to be coexpressed in first-trimester decidua and primary cultures of decidual stromal cells. Collectively, these observations demonstrate that GnRH I and GnRH II have both common and discrete cellular distributions in the placenta and decidua and suggest that these two hormones are capable of eliciting their biological actions in an autocrine and/or paracrine manner within and between these maternal and fetal cellular compartments.

Changes in vascular endothelial growth factor production associated with decidualization by human endometrial stromal cells in vitro.

The aim of this study was to clarify changes in the expression of vascular endothelial growth factor (VEGF) during decidualization by endometrial stromal cells (ESCs) in vitro. ESCs were separated by enzymic digestion and filtration, and were cultured with RPMI 1640 in 10% fetal calf serum (FCS) and treated with medroxyprogesterone acetate (MPA) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) to induce decidualization in vitro. The levels of prolactin (PRL) in the culture media were measured by enzyme immunoassay (EIA) and the levels of VEGF were measured by enzyme-linked immunosorbent assay (ELISA). The expression of VEGF mRNA by ESCs and decidualized cells was analyzed by Northern blot analysis. In treated cells, PRL production was significantly increased due to treatment with both db-cAMP (0.5 mmol/L) and MPA (100 nmol/L). VEGF mRNA expression was detected without any stimulation by ESCs. VEGF production was also significantly greater in cells treated with db-cAMP (0.5 mmol/L) and MPA (1 nmol/L or 100 nmol/L) than in control cells. VEGF mRNA was also increased in association with decidualization in vitro. VEGF production increased in association with decidualization in this in vitro study. Decidualization of ESCs may play a role in the induction of growth in the human fetus and/or placentation. The mechanism involved may include influencing the production of angiogenic growth factors such as VEGF.

Heparin-binding EGF-like growth factor regulates human extravillous cytotrophoblast development during conversion to the invasive phenotype

Cytotrophoblasts of the anchoring villi convert during human placentation from a transporting epithelium to an invasive, extravillous phenotype that expresses a distinct repertoire of adhesion molecules. Developing extravillous trophoblasts accumulate heparin-binding EGF-like growth factor (HB-EGF), a multifunctional cytokine, which binds HER1 and HER4 of the human EGF receptor (HER/ErbB) family. HB-EGF is downregulated in placentae of women with preeclampsia, a disorder associated with deficient trophoblast invasion, raising important questions about its physiological impact on cytotrophoblasts. Addition of HB-EGF during explant culture of first-trimester chorionic villi enhanced extravillous trophoblast differentiation and invasive activity. Using a first-trimester human cytotrophoblast line, the potential for autocrine and paracrine regulation of the developing trophoblast was established based on the expression of all four HER isoforms, as well as HB-EGF and related growth factors. HB-EGF did not alter proliferation, but initiated extravillous differentiation, with decreased α6 integrin expression, increased α1, and elevated cell migration. Function-blocking antibodies against EGF family members reduced basal cell motility and antibody inhibition of either HER1 or HER4 ligation prevented HB-EGF-induced integrin switching. We conclude that HER-mediated autocrine and paracrine signaling by HB-EGF or other EGF family members induces cytotrophoblast differentiation to an invasive phenotype.

The effects of tumor necrosis factor-alpha on fetal-placental vascular tone in the ex-vivo human placental cotyledon model

PLACENTAL VASCULAR TONE IN THE EX-VIVO HUMAN PLACENTAL COTYLEDON MODEL ANDREA FISK, BOBBY HOWARD, CHRISTINE KOVAC, PETER NAPOLITANO, DAMIAN PAONESSA, PETER NIELSEN, NATHAN HOELDTKE, Madigan Army Medical Center, Tacoma, WA Tripler Army Medical Center, Honolulu, HI OBJECTIVE: The proinflammatory cytokine, tumor necrosis factoralpha (TNF-a), has been demonstrated to have varying vasoactive effects in different vascular beds. Since TNF-a is increased in a number of obstetric settings, our objective was to determine if TNF-ahas an effect on fetal-placental vascular tone. STUDY DESIGN: The maternal and fetal circulations of two cotyledons from each of six human placentas were perfused for two hours with Hanks’ balanced salt solution. After baseline perfusion pressure was established, 5 ng/ ml of TNF-a was added to the fetoplacental circulation of one cotyledon from each pair (n = 6). Fetoplacental vascular pressures were monitored continuously and recorded at 5 minute intervals. At one and two hours, the vasoconstrictor angiotensin II was bolus injected (0.25 mg) into the fetoplacental circulation of all cotyledons and peak vascular pressures were recorded. RESULTS: In the cotyledons exposed to TNF-a the fetoplacental vascular perfusion pressure decreased by amean of 11.5mmHg compared to the control cotyledons (p < 0.05), and the difference persisted during the two hours of perfusion. There was no difference in relative vasoconstrictive response to angiotensin II between cotyledons exposed to TNF-a and control cotyledons, at either one or two hours. CONCLUSION: In the ex vivo human placental cotyledon, TNF-a has a vasodilatory effect on the baseline fetoplacental circulatory tone, but does not affect the fetoplacental vascular response to angiotensin II.

Angiogenesis during primate placentation in health and disease.

Normal embryonic development is dependent upon a sufficient oxygen, nutrient and waste exchange through the placenta. In primates including humans, this exchange is attained by successful haemochorial placentation which requires the transformation of maternal intramyometrial spiral arterioles by trophoblast invasion to gain uteroplacental circulation, and establishment and maintenance of a competent fetoplacental vasculature. Thus, trophoblast and endothelial cell differentiation, proliferation and invasion occurring during placentation have to be tightly regulated. This review focuses on the diverse developmental steps during haemochorial placentation in humans and other primates and the possible involvement of angiogenic growth factors (vascular endothelial growth factor (VEGF) and angiopoietins (Ang)) in these processes, highlighting the importance of specific actions of angiogenic ligand-receptor pairs. It is hypothesized that VEGF/VEGF-R1 and Ang-1/Tie receptor 2 (Tie-2) may regulate trophoblast differentiation and invasion; VEGF/VEGF-R2 and Ang-1/Tie-2 may promote fetoplacental vascular development and stabilization; and Ang-2/Tie-2 may be involved in maternal vascular remodelling. The importance of a tight regulation of angiogenic factors and their endogenous antagonists for normal development of the placenta is demonstrated by failure of this system, resulting in abnormal placenta vascularization and trophoblast invasion associated with intrauterine growth retardation or pre-eclampsia.

Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation: Receptor Activation Mediates Extravillous Trophoblast Invasion in Vitro

Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation.

Development of the utero-placental circulation: the role of carbon monoxide and nitric oxide in trophoblast invasion and spiral artery transformation.

It is now well known that in order to establish human hemochorial placentation and to provide a progressive increase in blood supply to the growing fetus, the uterine spiral arteries must undergo considerable alterations. This physiological modification is thought to be brought about by the interaction of invasive cytotrophoblast with the spiral artery vessel wall. Despite intensive research our understanding of the mechanisms that control human trophoblast invasion in normal, let alone abnormal pregnancy, are sill poorly understood. This is partly due to difficulties in obtaining "true" placental bed biopsies and most investigators have relied on in vitro models of trophoblast invasion. This article describes the morphological changes that occur within the placental bed throughout human pregnancy along with a review of the various studies which have attempted to sample the placental bed. Thereafter, follows a review of the evidence that invasive trophoblast can release the vasoactive agents nitric oxide and/or carbon monoxide which, in turn, could contribute to early physiological changes in spiral arteries prior to destruction of the smooth muscle within the vessel wall. Current evidence supports the idea that trophoblast-derived carbon monoxide may contribute to spiral artery modification. In contrast there is no evidence for a similar role by nitric oxide.

X-Inactivation Patterns in Human Embryonic and Extra-embryonic Tissues

Mice have skewed X chromosome inactivation (XCI) in extraembryonic tissue while examination of human placentae have yielded conflicting results. We investigated XCI patterns in human embryonic and extra-embryonic tissues. First and early second trimester placental and foetal tissues were collected. Cytotrophoblasts were isolated from the placentae. Female samples were identified and X-inactivation patterns were determined by analysis of androgen receptor (HAR) methylation patterns. Among 55 females heterozygous at the HAR, 37 had random and 18 skewed XCI. In foetal tissues a skewed XCI pattern was only observed in one liver and one intestine sample. A greater incidence of skewed XCI pattern was present in extra-embryonic compared to embryonic tissues (P=0.022). A markedly skewed XCI pattern was only found in one cytotrophoblast sample. Random and skewed XCI patterns were detected in human embryonic and extra-embryonic tissues. The extra-embryonic tissue had a higher proportion of skewed XCI, but marked skewed XCI was uncommon in both tissues. Skewed XCI may not play a role in normal human placentation.

Effects of gonadotropin-releasing hormone (GnRH)-I and -II on regulating the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) in cultured human extravillous cytotrophoblasts

Objective: Implantation in the human is dependent upon the invasion of embryonic trophoblasts into the decidua of the maternal endometrium. The regulated expression of uPA and PAI-1 in the human trophoblasts have been shown to mediate key events in human placenta invasion and placentation. To date, the factors capable of regulating the expression of uPA and PAI-1 in placenta remain poorly characterized. In the present study, we have examined the ability of Gonadotropin-Releasing Hormone (GnRH I) and a novel form of Gonadotropin-Releasing Hormone (GnRH II), that are expressed in the human placenta, to regulate uPA and PAI-1 levels in primary cultures of human extravillous cytotrophoblasts (EVT).

Expression of Matrix Metalloproteinase (MMP)-2 and MMP-9 in Human Placenta and Fetal Membranes in Relation to Preterm and Term Labor

Extensive extracellular matrix (ECM) remodeling is found in many processes during human parturition at term and preterm. These include cervical ripening, fetal membrane rupture, and placental detachment from the maternal uterus. Matrix metalloproteinases (MMPs) are the main mediators of ECM degradation. The present study was designed to investigate the expression of MMP-2 and MMP-9 in human fetal membranes (FMs) and placental (PL) tissues with or without labor at preterm and term parturition. Both zymography and Western blot analysis showed that MMP-9 was significantly (P < 0.01) increased in preterm and term labor FM, compared with nonlabor. Term labor PL also had a much higher (P < 0.05) level of MMP-9 than that of term nonlabor. No significant difference in MMP-2 expression was found between labor and nonlabor tissues. Immunolocalization studies revealed a specific distribution pattern for MMP-2 and MMP-9. MMP-2 was localized to the amnion mesenchyme, chorion laeve trophoblast, decidua parietalis, and blood vessels in PL villi. MMP-9 was localized mainly to amnion epithelia, chorion laeve trophoblast, decidua parietalis, and PL syncytiotrophoblasts. Separate cell culture from different layers of FM and culture of purified PL trophoblast cells showed that PL syncytiotrophoblast and amnion epithelial cells exclusively produced MMP-9; chorion trophoblast cells secreted both MMP-2 and MMP-9, but amnion mesenchymal cells produced only MMP-2. We concluded that MMP-2 and MMP-9 exhibited cell-specific expression in the human PL. An increase in MMP-9 expression may contribute to degradation of the ECM in the FM and PL, thereby facilitating FM rupture and PL detachment from the maternal uterus at labor, both preterm and term.

Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in relation to preterm and term labor.

Extensive extracellular matrix (ECM) remodeling is found in many processes during human parturition at term and preterm. These include cervical ripening, fetal membrane rupture, and placental detachment from the maternal uterus. Matrix metalloproteinases (MMPs) are the main mediators of ECM degradation. The present study was designed to investigate the expression of MMP-2 and MMP-9 in human fetal membranes (FMs) and placental (PL) tissues with or without labor at preterm and term parturition. Both zymography and Western blot analysis showed that MMP-9 was significantly (P < 0.01) increased in preterm and term labor FM, compared with nonlabor. Term labor PL also had a much higher (P < 0.05) level of MMP-9 than that of term nonlabor. No significant difference in MMP-2 expression was found between labor and nonlabor tissues. Immunolocalization studies revealed a specific distribution pattern for MMP-2 and MMP-9. MMP-2 was localized to the amnion mesenchyme, chorion laeve trophoblast, decidua parietalis, and blood vessels in PL villi. MMP-9 was localized mainly to amnion epithelia, chorion laeve trophoblast, decidua parietalis, and PL syncytiotrophoblasts. Separate cell culture from different layers of FM and culture of purified PL trophoblast cells showed that PL syncytiotrophoblast and amnion epithelial cells exclusively produced MMP-9; chorion trophoblast cells secreted both MMP-2 and MMP-9, but amnion mesenchymal cells produced only MMP-2. We concluded that MMP-2 and MMP-9 exhibited cell-specific expression in the human PL. An increase in MMP-9 expression may contribute to degradation of the ECM in the FM and PL, thereby facilitating FM rupture and PL detachment from the maternal uterus at labor, both preterm and term.

The Regulation and Localization of Angiopoietin-1, -2, and Their Receptor Tie2 in Normal and Pathologic Human Placentae

Angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) act on the endothelial cell Tie-2 receptor to regulate vascular integrity and remodeling. The local balance of these factors and the level of other angiogenic factors determine whether blood vessels grow, are maintained or regress. Profound angiogenesis and vascular remodeling occur in the placenta and this is altered in preeclampsia, a major cause of maternal and fetal morbidity and mortality. The mRNAs encoding Ang-1, Ang-2, and Tie-2 were detected and localized in human placentae throughout gestation. The mechanism of regulation angiopoietin mRNAs level was determined by explant culture in ambient and reduced oxygen, and in the presence of actinomycin D. In situ hybridization showed that Ang-2 mRNA was abundant in the syncytiotrophoblast in the first trimester of human pregnancy. Ang-1 mRNA could not be detected by in situ hybridization, but was by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting. Placental vascular structure is altered in preeclampsia and intrauterine growth restriction, conditions where feto-placental oxygenation is perturbed. In villous explant cultures, a reduction in oxygen tension significantly raised the levels of Ang-2 mRNA, and this was dependent on transcription. However, similar experiments showed that the stability of the Ang-1 message was greatly reduced under these conditions. Thus, hypoxia has opposite effects on Ang-1 and Ang-2 mRNA levels. Placentae obtained from women with preeclampsia had reduced levels of Ang-2 mRNA compared to gestationally matched controls. There was no difference in the levels of Ang-1 mRNAs. These data show that the relative levels of Ang-1 and Ang-2 mRNA are regulated by local oxygen tension by different mechanisms and that this may be important during normal human placentation.

Effects of Matrix Proteins on the Expression of Matrix Metalloproteinase-2, -9, and -14 and Tissue Inhibitors of Metalloproteinases in Human Cytotrophoblast Cells During the First Trimester1

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.