Apoptotic Receptors and CD107a Expression by NK Cells in an Interaction Model with Trophoblast Cells.

Natural killer cells (NK cells) exert cytotoxicity towards target cells in several ways, including the expression of apoptosis-mediating ligands (TRAIL, FasL). In addition, NK cells themselves may be susceptible to apoptosis due to the expression of TRAIL receptors. These receptors include TRAIL-R1 (DR4), TRAIL-R2 (DR5), capable of inducing apoptosis, and TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), the so-called "decoy receptors", which lack an intracellular domain initiating activation of caspases. Of particular interest is the interaction of uterine NK cells with cells of fetal origin, trophoblasts, which are potential targets for natural killer cells to carry out cytotoxicity. The aim of this work was to evaluate the expression of proapoptotic receptors and their ligands as well as CD107a expression by NK cells in a model of interaction with trophoblast cells. To evaluate NK cells, we used cells of the NK-92 line; cells of the JEG-3 line were used as target cells. The cytokines IL-1ß, IL-15, IL-18, TNFα, IL-10, TGFß and conditioned media (CM) of the first and third trimester chorionic villi explants were used as inducers. We established that cytokines changed the expression of apoptotic receptors by NK cells: in the presence of TNFα, the amount and intensity of Fas expression increased, while in the presence of TGFß, the amount and intensity of expression of the DR5 receptor decreased. Soluble chorionic villi factors alter the expression of TRAIL and FasL by NK-92 cells, which can reflect the suppression of the TRAIL-dependent mechanism of apoptosis in the first trimester and stimulating the Fas-dependent mechanism in the third trimester. In the presence of trophoblast cells, the expression of TRAIL and DcR1 by NK cells was reduced compared to intact cells, indicating an inhibitory effect of trophoblast cells on NK cell cytotoxicity. In the presence of chorionic villi CM and trophoblast cells, a reduced number of NK-92 cells expressing DR4 and DR5 was found. Therefore, soluble factors secreted by chorionic villi cells regulate the resistance of NK cells to death by binding TRAIL, likely maintaining their activity at a certain level in case of contact with trophoblast cells.

Receptor expression by JEG-3 trophoblast cells in the presence of placenta secreted factors.

Preeclampsia still remains one of the most severe pregnancy complications and is an actual problem in the obstetrics practice. At present, the joint impact of cytokines and other placenta secreted factors on trophoblast cell functional activity during preeclampsia complicated pregnancy remains unclear. The aim of the study is to estimate the surface receptors expression by trophoblast cells in the presence of placenta secreted factors during physiological pregnancy and at preeclampsia. Trophoblast cells of the JEG-3 line were incubated in the presence of supernatants obtained by cultivation of placentas from women with physiological pregnancy and with preeclampsia. Surface receptors expression by trophoblast cells was estimated by FACS Canto II flow cytometer. It was established that in the third trimester both under normal and pathological conditions, the placenta secreted factors impact on the cytokine receptor expression by trophoblast differs while the trophoblast response capacity to the migration and proliferation stimulating and inhibiting signals remains stable. JEG-3 line cells enhanced the expression of CD186, CD140a, Integrin ß6, VE-cadherin, CD29, and CD140a in the case of incubation in the presence of placenta supernatants from the third-trimester pregnancy complicated with preeclampsia compared to incubation in the presence of placenta supernatants form the third trimester of physiological pregnancy.

Synergistic Anti-Angiogenic Effects Using Peptide-Based Combinatorial Delivery of siRNAs Targeting VEGFA, VEGFR1, and Endoglin Genes.

Angiogenesis is a process of new blood vessel formation, which plays a significant role in carcinogenesis and the development of diseases associated with pathological neovascularization. An important role in the regulation of angiogenesis belongs to several key pathways such as VEGF-pathways, TGF-ß-pathways, and some others. Introduction of small interfering RNA (siRNA) against genes of pro-angogenic factors is a promising strategy for the therapeutic suppression of angiogenesis. These siRNA molecules need to be specifically delivered into endothelial cells, and non-viral carriers modified with cellular receptor ligands can be proposed as perspective delivery systems for anti-angiogenic therapy purposes. Here we used modular peptide carrier L1, containing a ligand for the CXCR4 receptor, for the delivery of siRNAs targeting expression of VEGFA, VEGFR1 and endoglin genes. Transfection properties of siRNA/L1 polyplexes were studied in CXCR4-positive breast cancer cells MDA-MB-231 and endothelial cells EA.Hy926. We have demonstrated the efficient down-regulation of endothelial cells migration and proliferation by anti-VEGFA, anti-VEGFR1, and anti-endoglin siRNA-induced silencing. It was found that the efficiency of anti-angiogenic treatment can be synergistically improved via the combinatorial delivery of anti-VEGFA and anti-VEGFR1 siRNAs. Thus, this approach can be useful for the development of therapeutic angiogenesis inhibition.

Changes in Functional Activity of JEG-3 Trophoblast Cell Line in the Presence of Factors Secreted by Placenta.

BACKGROUND AND AIMS: Cells in the maternal-fetal interface secrete cytokines that regulate proliferation, migration, and trophoblast invasion during the first trimester of pregnancy and the limitation of these processes during the third trimester. The aim of the study was to evaluate the influence of factors secreted by human placenta during the first and third trimester of pregnancy on cytokine receptor expression and proliferative and migratory activity of JEG-3 trophoblast cells. METHODS: The research was conducted using the explant conditioned media of placentas obtained from healthy women with elective termination of pregnancy at 9-11 weeks and placentas of women whose pregnancy progressed without complications at 38-39 weeks. Assessment of surface molecule expression was performed using FACS Canto II flow cytometer (BD, USA). The proliferative activity of JEG-3 trophoblast cells was evaluated by dyeing with crystal violet vital dye. The migration activity of JEG-3 was evaluated using 24-well insert plates with polycarbonate inserts (pore size 8 microns). RESULTS: Expression of CD116, CD118, CD119, IFNγ-R2, CD120b, CD183, CD192, CD295, EGFR, and TGFß-R2 on JEG-3 was higher when the cells were incubated in the presence of the third trimester placental factors in comparison with the first trimester placental factors. Factors secreted by the placenta during the third trimester of pregnancy had more pronounced stimulatory effect on the proliferation and migration of trophoblast in comparison with baseline levels and with the effect of the first trimester placental factors. CONCLUSIONS: The findings suggest that the behavior of trophoblasts in vitro might not be representative of in vivo behavior in the absence of additional local factors that influence the trophoblast in vivo.