Abstract Objective Monoclonal antibody 26 (MAb 26) raised against tetanus toxoid has documented cross-reactivity with β2 -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 β1 , 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.
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.
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.
These findings qualify gal-1 as a member of human trophoblast cell invasion machinery.
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.
•MIF is abundantly expressed at feto-maternal interface.•Placental and decidual stromal cell survival are positively regulated by MIF.•Trophoblast invasion and pro-invasive cytokines in trophoblast are increased by MIF.•MIF participates in endovascular trophoblast cell function.•MIF is associated to placental pathological conditions.
Integrin β1 is bound to galectin-1 in human trophoblast Bojic-Trbojevic, Žanka; Jovanovic Krivokuca, Milica; Stefanoska, Ivana ...
Journal of biochemistry (Tokyo),
2018-Jan-01, 2018-01-01, 20180101, Letnik:
163, Številka:
1
Journal Article
Recenzirano
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 β1 integrins, both previously shown relevant for trophoblast invasion, was investigated. Confocal microscopy showed overlap in gal-1 and β1 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 β1, but not with α1 or α5 integrin subunits. Nondenaturing electrophoresis and subcellular fractionation suggested association of gal-1 with β1 integrin in intracellular and plasma membrane compartments of HTR-8/SVneo cells. Gal-1/β1 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 β1 integrin, as determined by qPCR and flow cytometry. These results suggest complex lectin type interaction of gal-1 with β1 integrin at the trophoblast cell membrane, which could influence trophoblast cell adhesion, migration and invasion.
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.
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.
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 ± 3.1% (p < 0.001) positive cells in CT and 24.8 ± 7% (p < 0.01) in HTR-8/SVneo cells at 24 h and 33 ± 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.
Infection is increasingly considered to contribute to pathological conditions
in pregnancy. The placenta acts as a protective immunological fetomaternal
barrier which recognizes microbes by pattern ...recognition receptors on the
trophoblast. Lipopolysaccharide (LPS) is a cell wall constituent of
Gram-negative bacteria that elicits a strong immune response. In this study,
LPS from E. coli was used to treat the HTR-8/SVneo trophoblast cell line and
examine its influence on cytokines IL-6, IL-8 and MIF using real-time PCR,
metalloproteinases (MMP)-2 and -9 by gelatin zymography, and Western analysis
of integrin subunits ?1 and ?1, all known to contribute to migration of human
trophoblasts in vitro. The results described herein for the first time, show
that MIF mRNA and secreted MIF protein were significantly elevated (2.5-3-
and 2-fold, respectively) in LPS-treated cells. MMP-2 and MMP-9 levels were
increased, as well as cell migration, as judged by a wound-healing test,
however, no changes in the studied integrin subunits, cell viability or cell
numbers were observed. The data obtained furthers our understanding of LPS
actions on the trophoblast in vitro, additionally implicate MIF, and suggest
that infection in vivo could indeed alter the functional characteristics of
the trophoblast.
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.
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