Effect Of Hypoxia On Trophoblast’s Migration Through MAP4 In Pre-eclampsia

Background and Objective:Preeclampsia(PE), a poorly understood disorder, is characterized by maternal hypertension and proteinuria, which affects approximately 5% to 8% of all pregnancies.When untreated,the syndrome can trigger maternal seizures(eclampsia) and result in intrauterine fetal death. The only de?nitive cure is delivery of the fetus and its appendages, which contributes to the morbidity of preterm delivery. Because of poor understanding of preeclampsia’s pathogenesis, the effect of treatment for preeclampsia is not satisfying and the leading theories regarding its etiology implicates the disfunction of trophoblast and improper early placental development.Placentation and implantation are early gestational processes and the most important two determinants of pregnancy outcome.Trophoblast invasion lays the foundations for placentation, as the extent of invasion determines the stability of placenta and depth of engagement between placenta and decidua. Trophoblast invasion of the maternal spiral arteries is important in ensuring adequate fetal oxygen and nutrient supply.Poor invasion can cause obstetric complications including miscarriage, pre-eclampsia, placental abruption and intrauterine death. Excessive invasion leads to occurrance of trophoblastic disease,such as invasive hydatidiform mole, choriocarcinoma with its attendant severe risk to the mother’s life. Thus, trophoblast invasion can be considered as a balance between the demands of the fetus to invade the mother and those of the mother to survive in this invasion. This balance is even more remarkable when it is considered that the majority(90%) of karyotypically abnormal pregnancies miscarry in the ?rst trimester and the majority(93%) of karyotypically normal pregnancies continue. Hence, miscarriage, considered to be failed trophoblast invasion, can be viewed as“Nature’s quality control”. Factors that affect trophoblast invasion of the maternal spiral arteries, will determine the success or failure of pregnancy.Trophoblast’ s invasion is regulated by many intracellular and extracellular signaling pathways and is involved in identifying and digestion of the extracellular matrix, orientation migration,among them, orientation migration of trophoblast is the key step in invasive behavior.The same as the majority of cell’s migration patterns, trophoblast’s directed migration includes cell polarization, pseudopodia formation, matrix adhesion, the tail release, cell body contraction and so on, but the dynamic recombination of cytoskeleton and adhesion structure is a basic link, and also is the important target of a variety of factors regulating trophoblast’s migration.Cytoskeleton mainly includes microtubule and actin filament, our experimental team early found that the epidermal growth factor(EGF) through the microfilament’s structural rearrangement stimulated trophoblast’s migration, while there is less study of the relationship between microtubule and trophoblast’s migration. Microtubule is a hollow tubes with polarity, distribute bunchily and form a rail system to make various vesicles, organelles and other components moving along its direction, at the same time, microtubule also participates in the centriole’s assembling, cell’s morphology maintaining,cell’s movement and splitting process.microtubule skeleton’s stable polymerization rely on a series of microtubule-associated protein(MAP),which combine with the inside and outside of microtubule,among them, MAP4 was expressed in human trophoblast and is a positive adjustment factor for promoting microtubule’s steady extension. Microtubule’s stable polymerization and extending direction accurately point to focal adhesion where cell adhere to extracellular matrix and microtubule moving to focal adhesion relates to the dissolvent of focal adhesion. For cell migration, the dissolvemennt of focal adhesion which rely on microtubule is very important for decomposition of focal adhesion behind the cells.JEG-3 cell lines are derived from human choriocarcinoma tissues and have the same properties with the villous trophoblast,such as morphology, biochemical markers, and functions. So we use JEG-3 to study human trophoblast’s invasion and its possible mechanism.Methods:Part One: Effect of hypoxia on trophoblast’s migration through MAP4 and its mechanism1. We cultivated trophoblast under normoxia and hypoxia, according to the time under hypoxia, cells were divided into 0h,6h, 12 h, 18 h, 24 h group;2. We detected the invadation rate of all cells;3. We observed the change of microtubule and focal adhesion using immunofluorescence;4. We detected the expression of protein using Western-blot;5. We collected the datas, and use the software of Excel and SPSS to analyze datas.Part two: protein expressions of MAP4 and cytoskeleton in placenta tissue with preeclampsia1. We selected the pregnant women who had caesarean deliveries from January,2013 to March,2014 in the third affiliated hospital(Daping hospital) of The Third Military Medical University and collected their placenta tissue. They were divided into two groups which were preeclampsia group(PE) and normal pregnancy group(control);2. We analyzed the difference of villus and vessel between preeclampsia group and normal pregnancy group using HE staining;3. We studied the location and expressions of related proteins in villus using Immunohistochemistry;4. We extracted related proteins,and then analyzed the quantity expressions of these proteins using western-blot.Results:1. Hypoxia inhibited trophoblast’s invasion.Compared to 0h group, the ability of trophoblast’s invasion in 6h group markedly decreased, differences were statistically significant(P<0.05). As the hypoxia time prolonged, the ability of trophoblast ’s invasion further reduced. The 12 h group compared to 0h and 6h groups,the same as 18 h group compared to12 h group, differences were statistically significant(P<0.05);2. As the hypoxia time prolonged, the protein expressions of MAP4 and α-tubulin gradually decreased, the microtubule depolymerized and the focal adhension polymerized gradually, the protein expressions of vinculin gradually increased;3. Compared with control group, placenta tissue presented vasospasm, occlusion and reduced blood flow in PE group;4. Compared with control group, the protein expressions of MAP4 and α-tubulin were lower,but the protein expression of vinculin was higher in PE group,which had the same results compared to trophoblasts cultivated in hypoxia.conclusions:1. The change of MAP4 may regulate trophoblast’s migration by affecting microtubule’s polymerization and focal adhension’s morphology;2. Placenta tissue in PE group presents vasospasm, occlusion, reduced blood flow and villi dysplasia, which is considerd to be associated with local vascular lesions, that provides direct evidence for clinical placental ischemia. At the same time,the abnormal protein expression of MAP4、α-tubulin and vinculin in PE patients’ placenta tissues, may lay a foundation for placental ischemia and provides evidence for the research on etiology of preeclampsia.