Effect Of Rab11 On The Excessive Secretiong Of Syncytiotrophoblast Exosome

Backgroud and Objective:Preeclampsia-PE,a pregnancy disorder characterized by hypertension and proteinuria after 20 th week of gestation.Besides proteinuria,the diagnosis PE characterized by hypertension complicating thrombocytopenia,elevated liver enzymes,renal dysfunction,pulmonary edema,visual disturbances and headaches and other neurological disorders,various body systems.Approximately 3%-10% of all pregnancy are effected by PE,it is the leading cause of maternal and fetal morbidity and mortality.PE can develop in to eclampsia,causing seizuers,there are 2.7-8.2 will develop eclampsia per 10000 pregnannt women.PE or eclampsia complicated with cerebral hemorrhage,liver rupture,pulmonary edema,acute and chronic renal failure,premature birth,intrauterine growth restriction,stillbirth,stillbirth and maternal death.The only effectived treament is termination of pregnancy.The symptoms improved significantly,indicated that placenta is the main factor of PE.After implanatation,the placenta formation process is associated with endometrial microenvironment.The early placenta is formed in a relatively low oxygen enviroment,placenta formation process is a process of changes in the concentration of oxygen.At the time of embryo implantation,intrauterine oxygen content is 3%,while the oxygen content of the decidua and myometrium is in the 8%-12%.This gradient favors to extravillous trophoblast invasion and spiral artery remodeling.Trophoblast cells anchor the parent organization,and invade the spiral arteries,interact with vascular endothelial cells and smooth muscle cells,change the structure of the spiral arteries.At the end of first trimester,the plugged trophoblast cells in the spiral arteries decrease and eventually disappear,the low resistance,high flow spiral arteries formated,maternal blood perfused to placenta villi gap and Maternal-fetal interface is formed.After the remolding of spiral arteries,the oxygen concentration in the placenta elevate to the normal range.The defective trophoblast invasion and the dysfuction of uterine spiral artery remolding cause vasoconstriction of the placenta and reactivity,resulted to hypoxia of placenta and the generation of reactive oxygen species,which active hypoxia-inducible factor-1α（HIF-1α）and lead to oxidative stress.When the placenta subjected to hypoxia and ischemia,uteroplacental blood supply is reduced inadequate perfusion,placental villous structure is destroyed,a large number of vasoactive factors 、 pro-inflammation cytokine and syncytiotrophoblast microvesicles are secreted into maternal circulation.Causing excessive maternal inflammatory response,endothelial cell dysfuction,leading to protenuria,coagulopathy,and other clinical symptoms of PE after the 20 th weeks of pregnancy.The secretion of placenta source sex adverse factors in hypoxia is the key link of PE.Syncytiotrophoblast exosome（STBEX）is one of the syncytiotrophoblast microvesicles.STBEX is the substance of intercelluar communication and a nanoscale vesicles.In normal pregnancy,STBEX play an important role in the signal transduction between villous trophoblast and vascular smooth muscle cells、endothelial cells,promote the remolding of spiral arteries.STBEX signaled between placenta and peripheral immune cells,promoting maternal immune tolerance to fetus.It played an important role in the formation of blood vessels and the success of the maternal-fetal interface during pregnancy.STBEX originally appeared into the circulation in 6 weeks of gestation,and with the increase of gestational age,STBEX levels increased significantly.However,in PE and other pathological pregnancy,STBEX role has not been fully elucidated.Compared with normal pregnancy of the same gestational age,The levels of STBEX increased in PE.Further more,STBEX can activate monocytes to secrete IL-1β,IL-6,TNF-α and other pro-inflammatory cytokines and chemokines,resulting in similar PE severe systemic inflammation.STBEX can also induce T cell proliferation to promote immune responses.HLA-G5、B7-H1 and B7-H3 and other immune molecules in STBEX surface,activate antigen-presenting cells to regulate Th1 / Th2 immune balance.STBEX militate matrix through various channels.STBEX cause local inflammatory response and the whole body systemic immune response.It is considered to be an important regulatory factor between immune balance of maternal and inflammatory cytokines,which plays an important role in the pathogenesis of PE.Rab proteins are the largest branch of Ras superfamily in GTP enzymes.It plays a very important role in vesicular transport and signal transduction process involves a variety of eukaryotic cells.Rab11 expressioned in placenta,regulating the secretion and trafficking of vesicles,but its role in the placenta has not been clearly reported.This article aims to explore the effect of Rab11 on the excessive secretiong of syncytiotrophoblast exsome when subject to hypoxia,and the differentially expressed of Rab11 in placenta betweeen normal pregnancy and PE.Method:Part one:1.Fused Bewo cell with Forskolin,simulating syncytiotrophoblast in late pregnancy placenta;2.Culutere Bewo cell under normoxic conditions or under hypoxia.The culutered Bewo Cell were devied into 4 groups,（1）Bewo cell cultured under normoxic conditions;（2）Bewo cell cultured under hypoxia（92% N2、5%CO₂、3%O₂）;（3）Bewo cell cultured under normoxic conditions after fusioned with Forskolin;（4）Bewo cell cultured under hypoxia（92% N2、5%CO₂、3%O₂）after fusioned with Forskolin;3.Measured the protein concentration of STBEX by BCA;4.The protein expressions of HIF-1α and Rab11 in cells were examined by Western bloting;5.Suppressed HIF-1α by Si RNA,then measured the differentialy expressed of Rab11.Part two:1.We selected the pregnant women who had caesarean deliveries from 10 th,2014 to 10 th,2015 in the Daping hospital of The Third Military Medical University and collected their placenta tissue.They were divided into two groups,preeclampsia group（PE）and normal pregnancy group（control）.2.Culutere placenta tissue under normoxic conditions or under hypoxia.The culutered placenta tissue were devied into 4 groups,（1）The PE placenta tissue cultured under normoxic conditions;（2）The PE placenta tissue cultured under hypoxia（92% N2、5%CO₂、3%O₂）;（3）The Normal placenta tissue cultured under normoxic conditions;（4）The Normal placenta tissue cultured under hypoxia（92% N2、5%CO₂、3%O₂）.Isolated STBEX and measured it’s protein concentration by BCA;3.Measured concentration of HIF-1α in supernatant by ELISA;4.The protein expressions of HIF-1α and Rab11 in placneta were examined with Western bloting method.Rusults:1.Compared with the fused Bewo cells and the Bewo cells cultured under normoxic conditions,the protein concentration of STBEX in Bewo cell and the fused Bewo cell cultured under hypoxia were significantly elevated（P<0.05）;2.The protein expressions of HIF-1α and Rab11 were elevated when subjected to hypoxia;3.The expression of Rab11 in cells decreased when suppressed HIF-1α by Si RNA;4.The concentration of HIF-1α were significantly elevated when placenta tissue cultured under hypoxia（P<0.05）;Compared with the noamal placenta tissue,the concentration of HIF-1α of in PE placenta tissue were significantly elevated（P<0.05）;5.Compared with the noamal placenta tissue,the expressions of HIF-1α and Rab11 in PE placenta tissue were significantly elevated（P<0.05）.Consulation:1.Under hypoxic conditions,expression of Rab11 in PE placenta were significantly elevated,which makes excessive secretiong of STBEX and placental vesicles.It may be associated with shallow implantation in placenta in PE patients.2.The expression of HIF-1α and Rab11 were significantly elevated in the fused Bewo cell when subjected to hypoxia.It illustrate HIF-1α may regulate the excessive secretiong of STBEX through Rab11,causing endothelial cell dysfuction and excessive maternal inflammatory response,which may play a role in the occurrence of PE.