Ljiljana Vićovac

Interleukin-8 (CXCL8) stimulates trophoblast cell migration and invasion by increasing levels of matrix metalloproteinase (MMP)2 and MMP9 and integrins α5 and β1

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.

Interleukin-6 Stimulates Cell Migration, Invasion and Integrin Expression in HTR-8/SVneo Cell Line

Interleukin-6 (IL-6) is present in human endometrium throughout menstrual cycle and in pregnancy. Trophoblast also expresses IL-6. IL-6R and its associated signal transducer gp130 were found in trophoblast as well. IL-6 is generally assumed to be relevant for trophoblast invasion. This study was undertaken to determine influence of endogenous and externally added IL-6 on invasion and migration of first trimester of pregnancy trophoblast in vitro. Integrins alpha(5)beta(1) and alpha(1)beta(1) have been shown to play an important role in trophoblast invasion and the effect of IL-6 on the expression of these integrin subunits was studied. We are showing that in both isolated first trimester of pregnancy cytotrophoblast (CTB) and HTR-8/SVneo cell line IL-6 and IL-6R are present. The effect on migration was studied using cell wounding and migration test on HTR-8/SVneo cells. Effect of IL-6 and function blocking anti-IL-6 antibody in Matrigel invasion tests was studied on both cell types. The effect of IL-6 on integrin subunit expression was determined by cell-based ELISA and Western blot on HTR-8/SVneo cells. The results obtained show that exogenous IL-6 has stimulatory effect on cell migration in HTR-8/SVneo and invasion by both cell types. Function blocking anti-IL-6 inhibited unstimulated invasion by isolated first trimester cytotrophoblast and both cell migration and invasion in unstimulated HTR-8/SVneo. Integrin alpha(5) expression was stimulated by IL-6 to 134% (p<0.05), alpha(1) to 135% (p<0.005), and beta(1) to 134% (p<0.001) of control in cell-based ELISA, but also in Western blot. The data obtained show for the first time sensitivity of extravillous trophoblast cell line HTR-8/SVneo to IL-6, in addition to isolated first trimester cytotrophoblast. We conclude that both exogenous and endogenous IL-6 stimulate trophoblast cell migration and invasion, which may be partly attributable to stimulation of expression of the studied integrin subunits.

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.

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).

Long-term tissue culture of human first-trimester villous trophoblast in collagen gel: Evaluation of its possible use as a model system

Summary To approach the study of hcG production and its regulation, a three-dimensional culture of villous trophoblast in collagen gel is described. Tissue viability has been evaluated by measuring of glucose consumption, LDH discharge. 14 C-leucine incorporation into total proteins and by morphological evaluation of the tissue at the beginning and at the end of the experiment. The obtained results showed that hCG production in vitro can be maintained for seven days in serum supplemented media. Maximal stimulation of hCG synthesis was obtained in the presence of first trimester pregnancy sera. Morphological evaluation of the tissue revealed well perserved villi architecture. Characteristic changes in the structure and syncytiotrophoblast/cytotrophoblast ratio after day 3 in culture coincided with the increase of hCG production in vitro . Further detailed morphological (histo and cytological) evaluation is needed for the interpretation of the obtained data. To conclude, the described three-dimensional culture of trophoblast villi in collagen gel offers a system for the study of hCG production and regulation in vitro .

Tissue interactions in first trimester trophoblast-decidua co-cultures

Summary Understanding of the process of trophoblast differentiation and embryo implantation critically depends on creating a variety of experimental models. In this study, we report results obtained on trophoblast-decidua tissue co-cultures, indicating that under the described conditions, villous trophoblast in vitro forms structures reminiscent of the anchoring villi of the placenta, induces localized degradation of the stroma in contact, and starts to invade the degraded decidual tissue. We believe that this co-culture system might be useful for the understanding of the process of trophoblast differentiation.

Cell Interactions in Trophoblast Invasion

The extent of trophoblastic penetration of the uterine wall during placentation varies widely between species, but nowhere is it more extensive than in the human (1, 2). Certain pathological conditions of pregnancy show either insufficient (preeclampsia and intrauterine growth retardation) or excessive (placenta percreta) trophoblastic penetration of adjacent maternal tissues and have been suggested to be caused by maternal rather than fetal abnormalities (3, 4). In order to diagnose and treat these pathologies, a greater understanding of the normal control of trophoblast invasion is a necessity. This chapter therefore concentrates on interactions between trophoblast and endometrium in the human, but reference is made to other species where comparison is thought to be instructive. This is most often the case in certain primates and rodents that show interstitial implantation.

Monoclonal antibody 26 cross-reactive with β2-glycoprotein I affects human trophoblast invasion in vitro

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.

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. Mol. Reprod. Dev. 82: 765–773, 2015.

Integrin β1 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 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.