In vitro effects of neuropeptide Y on migratory potential and oxidative status of human choriocarcinoma cells

Rezime: Trofoblasti su ćelije placente i karakterišu se visokom sposobnošću proliferacije, migracije i invazije. Njihova konrolisana migracija i invazija u endometrijum, ključni su procesi u implantaciji, kao i u formiranju i razvoju placente. Sposobnost trofoblasta da izvrši migraciju i invaziju na maternalno tkivo leži u osnovi pravilnog odvijanja trudnoće i pravilnog razvoja fetusa. Redukcioni poremećaji trofoblastne migracije uzrokuju aberantnu imlantaciju i plitku placentaciju, što često vodi različitim komplikacijama trudnoće koje su u vezi sa nedovoljnom placentacijom, kao što su preeklampsija i intrauterini zastoj u rastu. U nekoliko patoloških formi trudnoće pokazano je prisustvo značajno povišene koncentracije neuropeptida Y u poređenju sa koncentracijama u zdravoj trudnoći, pa je najveći deo studije fokusiran na ispitivanje potencijalnih patoloških efekata na trofoblaste. Imajući u vidu da je neuropeptid Y prisutan u određenoj koncentraciji i u plazmi zdravih trudnica, uzeti su u razmatranje i efekti ove koncentracije, putem koje je moguće da neuropeptid Y ostvari značajan fiziološki doprinos u optimalnom metabolizmu trofoblasta tokom zdrave trudnoće. Ciljevi ove studije predstavljaju in vitro ispitivanja efekata neuropeptida Y na različite aspekte funkcionisanja humanih trofoblasta. Tokom studije korišćena je ćelijska linija JEG-3 humanih trofoblasta izolovanih iz karcinoma placente, koja je pogodan model sistem za proučavanja procesa migracije i invazije. U eksperimentima su ispitivani efekti neuropeptida Y apliciranog u dve koncentracije (fiziološkoj i patološkoj) u dva vremenska tretmana (24 sata i 72 sata) sa ciljem upoređivanja njegovih fizioloških efekata i efekata koje ostvaruje u koncentracijama koje se javljaju u nekim patološkim stanjima trudnoće. Poseban fokus studije je bio na utvrđivanju potencijalnog doprinosa ovog molekula na promene migratorne i invazivne sposobnosti trofoblasta u njegovim koncentracijama višim od fizioloških. Koncentracije NPY od 0,1 nmol/L i 1 nmol/L korišćene su za testiranje efekata na apoptotski indeks (preživljavanje ćelija), vijabilnost, migracioni kapacitet, oksidativni / antioksidativni status, produkciju metaloproteinaza (MMR-9), kao i za merenje ekspresije gena (iNOS, HIF-1α, VEGF, COX-2 i MMP-9) i distribucije nekih proteina u ćeliji (iNOS, E-kadherin). Kontrolne ćelije su gajene u čistom medijumu za kultivaciju da bi se dobile bazalne vrednosti ispitivanih parametara. Eksperimentalni podaci dobijeni tokom ove studije pokazali su da neuropeptid Y apliciran u fiziološkoj (0,1 nmol/L) i patološkoj (1 nmol/ L) koncentraciji ostvaruje različite efekte na parametre oksidativnog / antioksidativnog metabolizma, migracije, kao i na vijabilnost, apoptotoski indeks i proliferativnu sposobnost humanih trofoblasta linije JEG-3. Najznačajniji efekat fiziološke koncentracije neuropeptida Y je povećavanje vijabilnosti trofoblasta, što ukazuje na značajan doprinos održavanju nivoa oksidtivnog metabolizma u trofoblasima koji prirodno funkcionišu u stanju hipoksije. Fiziološka koncentracija neuropeptida Y ne indukuje značajan oksidativni stres, ukazujući da trofoblasti pri ovoj koncentraciji efikasno mogu neutralisati produkte povišene aktivnosti mitohondrijalnog lanca respiracije. Neuroptpid Y apliciran u koncentraciji od 0,1 nmol/L pokazuje protektivan efekat na preživljavanje trofoblasta, kao i značajan proliferativan efekat. Ovi podaci ukazuju da NPY u fiziološkim koncentracijama može imati doprinos u ostvarivanju funkcija trofoblasta neophodnih za optimalnu implantaciju i placentaciju.Abstract: Trophoblasts cells are placental cell type characterized by high proliferation capacity, migration and invasion. Highly regulated trophoblast migration and invasion of the endometrium, are the essentail process in the implantation and development of the placenta. The migration and invasion of trophoblast cells of maternal tissues are basis of a proper progress of pregnancy and optimal fetal development. Reduced trophoblast migration and invasion cause abberant implantation and shallow placentation, often leading to a various complications of pregnancy that are related with inadequate placentation, such as preeclampsia and intrauterine growth restriction. In several pathological forms of pregnancy the presence of significantly elevated concentration of neuropeptide Y in comparison with its concentrations in healthy pregnancy was recorded, so the largest part of the study were focused on the examination of potential pathological effects of NPY on trophoblast cells. Since neuropeptide Y is also present in a particular concentration in plasma of women with healthy pregnancy, the effects of this physiological concentration by which neuropeptide Y is possible to significantly contribute to the optimal physiological metabolism of trophoblasts during a healthy pregnancy were taken into consideration. The objectives of this study were to investigate in vitro effects of neuropeptide Y on different aspects of the functioning of the human trophoblast. During the study we used the cell line JEG-3 of human trophoblast, isolated from placental cancer, which represents a suitable model system for examinating of the processes of cell migration and invasion. In the experiments we have investigated the effects of neuropeptide Y applied in two concentrations (physiological and pathological) in two different time treatment (24 and 72 hours) with the aim of comparing its physiological effects and the effects achieved by its pathological concentrations present in some pregnancy disorders. A particular focus of the study was to determine the potential contribution of this molecule on changes of the migratory and invasive ability of trophoblast cells induced by concentrations higher than physiological. NPY concentration of 0,1 nmol/L and 1 nmol/L were used to examine the effects on apoptotic index (survival rate of the cells), cell viability, migration capacity, the oxidative / antioxidative status, the production of metalloproteinase (MMP-9), gene expression (iNOS, HIF-1α, VEGF, COX-2 and MMP-9) and the distribution of some proteins in cell (iNOS, E-cadherin). Control cells were grown in pure culture mediim to obtain basal values of the examined parameters. Experimental data obtained in this study showed that neuropeptide Y administered in physiological (0,1 nmol/L) and pathological (1 nmol/L) concentration achieves various effects on the parameters of the oxidative / antioxidative metabolism, migration and the viability index apoptotic index and proliferative rate of human trophoblast cell line JEG -3. The most significant effect of physiological concentrations of neuropeptide Y is increasing trophoblast viability, which indicates a significant contribution to maintaining the levels of oxidative metabolism trofoblasts, naturally operating in a state of hypoxia. Physiological concentrations of neuropeptide Y does not induce significant oxidative stress, indicating that trophoblasts at this concentration can effectively neutralize the products of elevated activity of mitochondrial respiratory chain. Neuropetpide Y administered in a concentration of 0,1 nmol/L also showed protective effect on the survival of the trophoblast, as well as a significant proliferative effect. These data suggest that NPY in physiological concentrations may have a contribution in achieving the function of a trophoblast necessary for optimal implantation and placentation