Trophoblast Cell Function in the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a complex thrombo-inflammatory autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL). Women with APS are at high risk of recurrent early pregnancy loss as well as late obstetrical complications-premature birth due to placental insufficiency or severe preeclampsia. Accumulating evidence implies that vascular thrombosis is not the only pathogenic mechanism in obstetric APS, and that the direct negative effect of aPL on the placental cells, trophoblast, plays a major role. In this review, we summarize the current findings regarding the potential mechanisms involved in aPL-induced trophoblast dysfunction. Introduction on the APS and aPL is followed by an overview of the effects of aPL on trophoblast-survival, cell function and aPL internalization. Finally, the implication of several non-coding RNAs in pathogenesis of obstetric APS is discussed, with special emphasis of their possible role in trophoblast dysfunction and the associated mechanisms.

Macrophage migration inhibitory factor in human early pregnancy events and association with placental pathologies.

Macrophage migration inhibitory factor (MIF) is a versatile cytokine acting as an important regulator of innate and adaptive immunity and implicated in many physiological and pathological processes. It is abundantly expressed at the feto-maternal interface and proposed to have a role in establishing and maintaining a healthy pregnancy. This review presents the current literature data regarding the MIF role in early pregnancy events and its association with some of the placental pathological conditions, including infection, preeclampsia, gestational diabetes mellitus and choriocarcinoma. General information regarding MIF structure and function is followed by an overview of its expression in reproductive tissues and in pregnancy. Futher, we discuss MIF's involvement in the survival of decidual stromal cells, placenta of the first trimester of pregnancy, and in trophoblast cell functions studied in vitro. Current findings associating this cytokine to placental infection, preeclampsia, gestational diabetes mellitus and choriocarcinoma are presented in the final part.

Macrophage migration inhibitory factor modulates cytokine expression in the trophoblast cell line HTR-8/SVneo.

Extravillous trophoblasts are specific placental cells that invade the uterine stroma and spiral arteries modifying and adjusting them to pregnancy. Many pregnancy pathologies are associated with impairment of this process, including preeclampsia and intrauterine growth restriction, among others. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is abundant at the fetomaternal interface. Previous results from our group showed that MIF participates in trophoblast invasion and modulates the expression of molecules known to mediate stromal and endovascular trophoblast invasion. In this study we investigated the possibility that MIF could act as a regulator of cytokines known to modulate trophoblast invasion using the normal extravillous trophoblast-derived cell line HTR-8/SVneo. Expression of trophoblast MIF was attenuated by MIF mRNA-specific small interfering RNAs. Cytokine expression was assessed at the mRNA and protein levels using real-time quantitative polymerase chain reaction and flow cytometry respectively. Knockdown of MIF led to a significant decrease in mRNA for IL-1ß (IL1B) and IL-8 (CXCL8) and interleukin (IL)-8 protein. The addition of recombinant human MIF to cell culture medium increased IL-6 after 24h treatment and IL-6 and IL-8 after 72h treatment. Cell viability was not affected by MIF silencing or rhMIF treatment. The results of this study imply that at least some of the effects of MIF on trophoblast invasion could be mediated through autocrine or paracrine modulation of trophoblast cytokine production.

Macrophage migration inhibitory factor is involved in endovascular trophoblast cell function in vitro.

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine abundantly present at the feto-maternal interface proposed to play a role in establishment of pregnancy. We have previously shown that pharmacological inhibition of enzymatic activity of MIF decreases extravillous trophoblast invasion and migration in vitro. This study aimed to further elucidate potential role of endogenous trophoblast MIF, and to assess its importance for endovascular trophoblast cell function in particular. Attenuation of MIF by siRNA reduced HTR-8/SVneo cell invasion through Matrigel (59 % of control), expression of integrin α1 (86 % of control) and levels of MMP2 and MMP9 (87 % and 57 % of control, respectively). MIF specific siRNA reduced the ability of HTR-8/SVneo to differentiate in to endothelial-like phenotype, as determined by Matrigel tube formation assay. The total tube length was decreased to 68.6 %, while the number of branching points was reduced to 57.8 % of control. HTR-8/SVneo cell capacity to integrate into HUVEC monolayers was reduced by knock-down of MIF. This could be partly caused by reduced N-cadherin expression to 63 % of control, which decreased with knock-down of MIF, as the expression of this protein was recently shown essential for trophoblast-endothelial interaction. These novel findings indicate a novel role for trophoblast MIF in spiral artery remodeling process.

Immunoglobulins from sera of antiphospholipid syndrome patients are internalized in the HTR-8/SVneo cell line and cytotrophoblast in culture.

Women with antiphospholipid syndrome (APS) experience pregnancy complications mostly due to impaired trophoblast cell functions. Antiphospholipid antibodies (aPL) affect extravillous trophoblast in vivo and in culture, but the mechanisms are still poorly understood. Previously, syncytiotrophoblast was shown to bind and internalize aPL, which was not replicated for extravillous cytotrophoblast in short term culture. Here, aPL binding and time dependent internalization was demonstrated with exposure to aPL in the extravillous cell line HTR-8/SVneo and isolated first trimester of pregnancy cytotrophoblast (CT) using immunocytochemistry and flow cytometry. Intracellular aPL were detectable from 2 h of culture, reaching 30.7 ±â€¯3.1% (p < 0.001) positive cells in CT and 24.8 ±â€¯7% (p < 0.01) in HTR-8/SVneo cells at 24 h and 33 ±â€¯4.2% (p < 0.01) at 48 h. The data presented show that extravillous trophoblast cells internalize aPL in a time-dependent manner significantly more than control immunoglobulins after 24 h of exposure.

Integrin ß1 is bound to galectin-1 in human trophoblast.

Interaction of sugar binding proteins-galectins, with glycoconjugates is considered relevant for various reproductive processes. Galectin-1 (gal-1) is a molecule involved in trophoblast cell invasion, which is accomplished through interaction with cell surface and/or extracellular matrix glycoproteins. A possibility of interaction of endogenous gal-1 and trophoblast ß1 integrins, both previously shown relevant for trophoblast invasion, was investigated. Confocal microscopy showed overlap in gal-1 and ß1 integrin localization at the plasma membrane of isolated cytotrophoblast, HTR-8/SVneo extravillous trophoblast cell line and JAr choriocarcinoma cells. Immunoprecipitation confirmed an interaction of gal-1 with integrin ß1, but not with α1 or α5 integrin subunits. Nondenaturing electrophoresis and subcellular fractionation suggested association of gal-1 with ß1 integrin in intracellular and plasma membrane compartments of HTR-8/SVneo cells. Gal-1/ß1 integrin complex was sensitive to chemical and enzyme treatments, indicating carbohydrate dependent interaction. Down-regulation of gal-1 by siRNA, however, had no effect on level or distribution of ß1 integrin, as determined by qPCR and flow cytometry. These results suggest complex lectin type interaction of gal-1 with ß1 integrin at the trophoblast cell membrane, which could influence trophoblast cell adhesion, migration and invasion.

Immunoglobulins from sera of APS patients bind HTR-8/SVneo trophoblast cell line and reduce additional mediators of cell invasion.

Immunoglobulins from sera of patients with antiphospholipid syndrome (APS) decrease trophoblast cell invasion in vitro. This study aimed to extend understanding of cellular effects of immunoglobulins from APS (aPL+) in HTR-8/SVneo cells. aPL+ IgG induced change in effector molecules important for cell invasion was investigated further. After 1h of culture 21% cells bound aPL+ IgG, as opposed to 6% in control (aPL-). This was accompanied by increase in phospho-p38 at 30min. After 24h treatment aPL+IgG decreased protein levels of integrin subunits α1 (78% of control; p<0.01), α4 (65% of control, p<0.01), α5 (76% of control; p<0.01) and ß1 (80% of control; p<0.01), and secreted gal-1 (68% of control; p<0.05). ProMMP-9 was reduced to 70% of control (p<0.001). Treatment with inhibitor of p38 MAPK signaling SB202190 reversed inhibition in integrin ß1 and secreted gal-1. Involvement of p38 MAPK signaling and decrease in integrin subunit α4, proMMP-9, and secreted gal-1 in HTR-8/SVneo cells are novel and extend the list of mediators of trophoblast invasion affected by aPL.

Histological chorioamnionitis in preterm prelabor rupture of the membranes is associated with increased expression of galectin-3 by amniotic epithelium.

PURPOSE: Gal-3, which can regulate immune responses upon infection and inflammation, was not studied so far in intrauterine infection leading to preterm prelabor rupture of the membranes (PPROM), although gal-1 was reported to be implicated in the process. Gal-3 mRNA and protein expression in amnion and its changes during histological chorioamnionitis were studied here. MATERIALS AND METHODS: Fetal membranes were obtained from women with PPROM with (n =15) and without histological chorioamnionitis (n =15) during second and third trimester. Immunohistochemical reactivity was evaluated semiquantitatively and analyzed using t-test. Galectin profile of amniotic epithelia was determined by polymerase chain reaction (PCR) and change assessed in gal-3 in PPROM with (n =5) or without histological chorioamnionitis (n =5) by real-time PCR. RESULTS: Human amniotic epithelium was found to express gal-1, gal-3, gal-7 and gal-8 mRNA. Gal-3 mRNA and protein is increased in fetal membranes and in the amniotic epithelium in patients with chorionamnionitis. CONCLUSION: Histological chorioamnionitis is associated with increased gal-3 expression and strong immunoreactivity of the amnion. Gal-3 may participate in the regulation of the inflammatory responses to chorioamniotic infection and/or direct interaction with pathogens.

Human amniotic membrane grafts in therapy of chronic non-healing wounds.

BACKGROUND: Human amniotic membrane (HAM) has been embraced as a natural wound dressing almost exclusively in ophthalmology. Only recently, emergence of commercial HAM products prompted its use in growing range of indications, especially treatment of chronic non-healing wounds. SOURCES OF DATA: ClinicalTrials.gov database and International Clinical Trials Registry Platform searched with key words 'human amniotic membrane' and 'chronic wounds'. AREAS OF AGREEMENT: HAM can be successfully used as a natural wound dressing to promote healing. AREAS OF CONTROVERSY: It is still unclear, which preparation is more advantageous, cryopreserved HAM or dehydrated HAM. GROWING POINTS: There are an increasing number of commercial HAM products and clinical trials for a variety of dermatological diagnoses. AREAS TIMELY FOR DEVELOPING RESEARCH: In spite of easy procurement and low production costs, to our knowledge, there are currently only a few manufacturers of commercial HAM products tested in clinical trials for cutaneous wounds and all of them are located in the USA.

Interaction of extravillous trophoblast galectin-1 and mucin(s)-Is there a functional relevance?

In the course of embryo implantation extensive interaction of the trophoblast with uterine tissue is crucial for adequate trophoblast invasion. This interaction is highly controlled, and it has been pointed out that a specific glycocode and changes in glycosylation may be important for successful implantation and maintenance of pregnancy. Both uterine and trophoblast cells have been shown to express cell surface glycoconjugates and sugar binding proteins, such as mucins (MUC) and galectins (gals). An increasing number of studies have investigated potential candidates interacting in this process. However, knowledge about the biochemical nature of the interactions and their importance for trophoblast cell function, and, consequently, for pregnancy outcome are still lacking. This review is aimed at deliberating the possibility that mucins, as heavily glycosylated proteins, might be among the functionally relevant galectin ligands in human trophoblast, based on both published data and our original research.

Galectin signature of the choriocarcinoma JAr cells: Galectin-1 as a modulator of invasiveness in vitro.

Our previous findings showed that galectin-1 (LGALS1) plays an important role in the in vitro invasion of normal human trophoblast cells. In the present study, choriocarcinoma JAr cells were found to express LGALS1, -2, -3, -8, -10, and -13 mRNA and at least LGALS1, -3, and -8 protein, as determined by reverse-transcriptase PCR and Western blot, respectively. The galectin mRNA signature of JAr cells thus differed from that of normal first-trimester extravillous trophoblasts. A Matrigel migration assay was also used to investigate and confirm the relevance and effect of LGALS1 on the invasive potential of JAr cells, as observed in other trophoblast models. This modulation in behavior was achieved by specific lectin-glycan binding.

Galectin-1 binds mucin in human trophoblast.

Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.

Monoclonal antibody 26 cross-reactive with ß2-glycoprotein I affects human trophoblast invasion in vitro.

OBJECTIVE: Monoclonal antibody 26 (MAb 26) raised against tetanus toxoid has documented cross-reactivity with ß2-glycoprotein I. Passive introduction of this antibody in mice results in an antiphospholipid syndrome-like condition. We investigated the effects of MAb 26 on first trimester human trophoblast in vitro. STUDY DESIGN: Binding of MAb 26 to placental tissue trophoblast, isolated cytotrophoblast and HTR-8/SVneo cells was analyzed by immunohisto(cyto)chemistry. Possible effects on cell invasion in vitro were assessed by Matrigel assay. Effects on cell viability were assessed by MTT test. A possibility that MAb 26 induces change in levels of effector molecules important for cell invasion was investigated. Integrin subunits α1, α5 and ß1, and galectin-1, were analyzed by qPCR and Western blot. Metalloproteinases -2 and -9 were assessed by gelatin zymography. RESULTS: Immunohisto(cyto)chemistry showed binding of MAb 26 to placental tissue trophoblast, isolated cytotrophoblast and HTR-8/SVneo cells. The antibody had a significant inhibitory effect on cell invasion by both isolated cytotrophoblast and HTR-8/SVneo. The antibody induced significant decrease in protein levels of metalloproteinases, integrin subunit α1 and galectin-1. Cell viability was not affected. CONCLUSION: MAb 26 reduces trophoblast invasion in vitro through decreased levels of metalloproteinases-2 and -9, integrin α1 and galectin-1.

Galectin-1 is part of human trophoblast invasion machinery--a functional study in vitro.

BACKGROUND: Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it. METHODS AND FINDINGS: Function blocking anti-gal-1 antibody was employed to assess participation of endogenous gal-1 in cell adhesion, cell invasion of HTR-8/SVneo cells. When gal-1 was blocked in isolated trophoblast cell invasion was reduced to 75% of control (SEM ± 6.3, P<0.001) and to 66% of control (SEM ± 1.7, P<0.001) in HTR-8/SVneo cell line. Increased availability of gal-1, as two molecular forms of recombinant human gal-1 (CS-gal-1 and Ox-gal-1), resulted in increased cell invasion by cytotrophoblast to 151% (SEM ± 16, P<0.01) with 1 ng/ml of CS-gal-1, and to 192% (SEM ± 51, P<0.05) with 1 µg/ml of Ox-gal-1. Stimulation was also observed in HTR-8/SVneo cells, to 317% (SEM ± 58, P<0.001) by CS-gal-1, and to 200% (SEM ± 24, P<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests. CONCLUSION AND SIGNIFICANCE: These findings qualify gal-1 as a member of human trophoblast cell invasion machinery.

Interleukin-8 (CXCL8) stimulates trophoblast cell migration and invasion by increasing levels of matrix metalloproteinase (MMP)2 and MMP9 and integrins alpha5 and beta1.

Interleukin-8 (IL8/CXCL8) is present in decidua and trophoblast, which also expresses the IL8 receptors, CXCR1 and CXCR2. IL8 was shown to stimulate trophoblast migration. Matrix metalloproteinase (MMP)2, MMP9, and integrins alpha(5)beta(1) and alpha(1)beta(1) were found to play important roles in trophoblast invasion. We hypothesized that IL8 would increase this cell migration and invasion by HTR-8/SVneo cells through the activity of MMPs and integrins. Isolated first trimester of pregnancy cytotrophoblast (CT) and HTR-8/SVneo cell line were used. Migration was studied by monolayer wounding test, and invasion by Matrigel invasion test. The effects of IL8 on MMPs and integrin subunit expression were determined in HTR-8/SVneo cells by gelatin zymography and western blot respectively. The results that were obtained showed that exogenous IL8 stimulated HTR-8/SVneo cell migration and invasion. MMP2 and MMP9 levels were stimulated to 182% (P<0.01) and 134% (P<0.01) respectively. Integrin alpha(5) expression was increased to 119% (P<0.05) and integrin beta(1) expression to 173% (P<0.001) of the control values. The data that were obtained show for the first time the sensitivity of the HTR-8/SVneo cells, in addition to isolated first trimester CT, to IL8. Exogenous IL8/CXCL8 increased trophoblast cell migration and invasion, which may be partly attributable to stimulation of MMP2 and MMP9 levels and an increase in integrins. HTR-8/SVneo cell viability and proliferation were also increased.

Effects of anti-phospholipid antibodies on a human trophoblast cell line (HTR-8/SVneo).

Antibodies to phospholipids (aPL) have been shown to adversely affect trophoblast invasion in vivo and in vitro. HTR-8/SVneo cells derived from first trimester of pregnancy extravillous trophoblast were studied. Matrigel invasion assay, cytochemistry and cell-based enzyme-linked immunosorbant assay (ELISA) with aPL or normal IgG was used. Our data show that aPL at 100 microg/ml decrease invasiveness of HTR-8/SVneo cells to 60% of control (p<0.01), and this was also shown for primary cytotrophoblast (to 15.5% of control, p<0.001). aPL treatment caused a significant decrease in integrin alpha(1), alpha(5), and beta(1) proteins (86%, 84%, and 87%, respectively). We conclude that HTR-8/SVneo cell culture is a suitable model to study mechanisms of action of aPL on trophoblast, which in HTR-8/SVneo cells inhibit invasion by decreasing integrins alpha(5), alpha(1), and beta(1).