Ivana Stefanoska

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.

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.

The edible mushroom Laetiporus sulphureus as potential source of natural antioxidants

Hot water extract (LN), partially purified polysaccharides (LP) and hot alkali extracted polysaccharides (LNa) obtained from fruiting bodies of the wild basidiomycete Laetiporus sulphureus were examined for their antioxidant activities. LNa was the most active antioxidant, as shown by the median effective concentrations (EC50 values) of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity (0.5 ± 0.2 mg/ml), reducing power (4.0 ± 0.3 mg/ml) and ferrous ion-chelating ability (1.5 ± 0.1 mg/ml). LNa contained the highest level of α-glucan (17.3 ± 1.2 g/100 g dw), whereas LP contained mostly β-glucans (66.8 ± 1.3 g/100 g dw). The prevalent monosaccharide in all extracts was glucose. The EC50 values of all three antioxidant activity assays were well-correlated with the α-glucan content. Strong and significant correlation was found between total phenolic compounds and DPPH scavenging ability and also reducing power. The three investigated extracts (at concentrations of 0.1–10 mg/ml) were not toxic to HTR-8/SVneo trophoblast cell line.

Pharmacological inhibition of MIF interferes with trophoblast cell migration and invasiveness.

INTRODUCTION Macrophage migration inhibitory factor (MIF) is expressed by villous and extravillous cytotrophoblast. This study was aimed to investigate functional relevance of MIF for human trophoblast. METHODS MIF mRNA and protein were documented in cytotrophoblast (CT) and extravillous trophoblast cell line HTR-8/SVneo by RT-PCR, Western blot (WB), and immunocytochemistry. Recombinant human MIF (rhMIF), or its specific inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) were used in Wound healing migration and Matrigel invasion tests. Potential effectors, integrin subunits and matrix metalloproteinases (MMP) were studied using WB and gelatin zymography, respectively. RESULTS Blocking endogenous MIF by ISO-1 decreased HTR-8/SVneo cell migration dose dependently, most significantly with 200 μg/ml to 65% of control. Supplementation with rhMIF induced a significant stimulation to 129% of control with 200 ng/ml. In CT cell invasion test, ISO-1 at 200 μg/ml reduced invasion to 59% of control, while rhMIF (200 ng/ml) induced stimulation to 159% of control. In HTR-8/SVneo cells, invasion was significantly inhibited by ISO-1 to 40%, and increased to 150% of control by rhMIF (200 ng/ml). Integrin α1 was reduced by ISO-1 in both cell types, while integrins α5 and β1 were not changed. Addition of rhMIF increased integrin α1. In the presence of ISO-1, levels of MMP-2 and MMP-9 were reduced in CT and HTR-8/SVneo, while rhMIF stimulated MMP-2 in CT and MMP-9 in HTR-8/SVneo cells. CONCLUSION Reported findings provide the first insight into the cellular effects of MIF in human trophoblast, which acts to promote cell migration and invasion.

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.

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 β1 (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 β1 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.

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 ± 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.

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

Abstract 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.