Tight Junction Dysfunction Induced By CoCl₂-Mimic Hypoxia In Human Trophoblast Cell Through HIF-1α-VEGF Axis

Objective:Placenta is a critical organ that protects the fetus and sustain pregnancy. Placenta hypoxia is related with many pregnancy complications. Tight junction (TJ) barrier is a critical structure of placenta barrier and located at the cell junction of trophoblast cells as well as endothelial cells. TJ structure plays a main part in implementation of placental transportation. Oxygen is one of the key factors in placentation and uterus homeostasis. Hypoxia leads to tight junction dysfunction which is correlated with many pregnancy diseases [like preeclampsia (PE), intrauterine growth retardation (IUGR), hydatidiform mole and so on]. However, until now, the tight junction alternation of trophblast cells in hypoxia and it’s possible mechanism is barly investigated. Herein, in this study, we detected how the structure and junction of tight junction changes undergoing hypoxia in trophoblast cell and explored it’s possible mechanism. The study would provide further knowledge of placental TJ barrier at terms of structure, function as well as it’s regulation. It would give a new insight in pregnnacy complications and promote the diagnosis and therapy.Materials and Methods:(1) Placental samples derived from normal pregnant women were collected and detected by transmission electron microscope (TEM) for tight junction constructure. Immunohistochemistry was conducted to compare the location and expression of tight junction-related cytokines (ZO1and CLDN8) and HIF-la between the two group. Real time PCR and Western Blot were carried out to measure the gene and protein level expression of tight junction cytokines ZO1, CLDN4, CLDN8and OCLN, as well as protein level expression of HIF-1α between the two groups.(2) In vitro culturing cell monolayer model of human trophoblast derived cell line Be Wo cells, we detected the tight junction location by TEM. Immunofluorescence (IF) tech was utilized to investigate the exact location of tight junction proteins ZO1, CLDN4, CLDN8and OCLN in BeWo cells.(3) Culturing in a millicell chambers system in vitro, BeWo cell monolayer was exposed to a hypoxia environment induced by cobalt chloride (C0Cl2) with a series of concentration (0μmol/L,125μmol/L,250μmol/L and500μmol/L) for different time (Oh,6h,12h,24h). At the indicating time points, the cell vibility was detected by a MTT method. And a suitable concentration of250umol/L CoCl2was chosed for no cell cytotoxicity was observed at this dose. Then a cell chemical hypoxia model was established using250μmol/L CoCl2in vitro, and more functional and mechanistic experiments were carried out.(4) To investigate how tight junction function changes undergoing CoCl2-mimic hypoxia in vitro culturing monolayer, transepithelial electrical resistance (TER) and paracellular permeability (CPP) was detected by a MILLICELL ESR apparatus and fluorescein isothiocyanat (FITC-detran) transportation between the upper and lower chambers. Further, Real time PCR and Western Blot were performed to analyze the expression of tight junction factors ZO1, CLDN4, CLDN8and OCLN of BeWo cells exposed to hypoxia both at mRNA and protein level, respectively. IF was carried out to detected the alternation of tight junction proteins.(5) To further study the possible mechanism behind the TJ alternation under hypoxia, we measured whether hypoxia induced factor (HIF)-la and vascular endothelial growth factor (VEGF) were involved in the TJ regulation. Western Blot and ELISA were performed to analyze the protein expression of HIF-la and VEGF between hypoxia group nad normal. A HIF-la selective inhibitor3-(5’-hydroxymethyl-2’-furyl)-l-benzylindazole (YC-1) was used to determine whether TJ alternation was ameliorated when HIF-la protein expression was enormously inhibited.Results:(1) From the results of TEM, we confirmed that tight junction structure was localized in the apical part of the syncytium and also between the cell-cell contacts of fetal blood vessel endothelial. IHC shows that ZO1located in both the trophoblast cells and endothelial cells of placentae, CLDN8located in the trophoblast cells, and their expression were changed in preeclamptic placentae. ZO1was down regulated and CLDN8upregulated (P<0.05). HIF-1α was located in the placental trophoblast cells with a increasing expression in preeclamptic placentae (P<0.05).(2) TEM shows TJ-like strcture in the junction of adjacent BeWo cells. IF experiment was conducted to detect the location and expression tight junction-related proteins. Results shows that ZO1is principally located in the cell junction, most of OCLN protein was located in the cytoplasm with a small part in the cell junction and endonuclear,while CLDN4and CLDN8mainly in the cytoplasm(3) There was no significant difference in viability after125μmol/L and250μmol/L CoCl2treatment (P>0.05). However, treatment of500μmol/L CoCl2shows tremendous cytotoxicity on Be Wo cell and it’s viability was cut down overwhelmingly. So we chose250μmol/L CoCl2as a suitable and appropriate dose and established a cellular chemical hypoxia model for posterior study.(4) Results of TER and CPP showed a decrease in TER and a increase in CPP under CoCl2-induced hypoxia in a time-and concentration-dependent manner. Results of real time PCR and Western Blot showed a decreasing expression of ZO1and CLDN4(P<0.05), increasing protein expression of CLDN8(P<0.05), and no significant differences in OCLN expression. (5) We further explored a significantly increased in HIF-1α expression and VEGF production in hypoxia cells. Pretreated with YC-1can ameliorate the tight junction alternation induced by CoCl2-mimicking hypoxia in BeWo cells, as well as blunt the enhancement of VEGF secretion.CONCLUSION:(1) We detected the location of tight junction construction in placenta tissues, and investigated a differential expression some tight junction cytokines and HIF-1α in PE placenta. This indicated a tight junction dysfunction in PE placenta. Further reseach of hypoxia and tight junction, might put forward a new thought in generation and evolution in PE.(2) A vitro chemical hypoxia model induced by250μmol/L CoCl2was established in culturing trophoblast cell monolayer. This provides a suitable and steady research model for study of trophoblast cell in future.(3) Our datas for the first time demonstrated that hypoxia induced a alternation of tight junction cytokines expression and location, in turn leaded to a tight junction dysfunction, mediated by upregulated HIF-1α-VEGF axis. Our study offers a new pointcut and target spot for pregnancy disease.