Trophoblasts May Contribute To The Barrier Dysfunction Of Glomerular Endothelial Cells In Preeclampsia

Part I Clinical study on the barrier injury of glomerular filtration in severe preeclampsiaObjective:To investigate glomerular filtration barrier injury and its influence on pregnancy outcomes in severe preeclampsia.Methods:There were 112 cases of severe preeclampsia were analysed retrospectively. Increased serum creatinine and the severity of proteinuria were analyzed. All patients were divided into 2 groups according to their degree of proteinuria.30 patients who had urine dipstick protein values of 1+to 2+ were defined as Group A, and other 82 patients were in the group of 3+ to 4+ proteinuria on dipstick (Group B). The creatinine level, maternal pregnancy outcomes and perineal infant outcomes were compared in two groups. The data was analysised by SPSS 17.0 with t test, X2 test and Fisher's exact probability test.Results:34 cases (30.4%) of the total cases were with increasing serum creatinine. Grade 3-4 proteinuria accounted for 73.2% of severe preeclampsia. The concentration of serum creatinine was elevated obviously, and the number of maternal pregnancy outcomes was significantly increased, and the incidence of fetal distress was higher obviously with the severity of proteinuria.Conclusion:The injury of glomerular filtration barrier and glomerular filtration dysfunction should be common and might induce adverse outcomes. PartⅡTrophoblasts derived sFlt-1 may contribute to the barrier dysfunction of glomerular endothelial cellsObjective:1. To investigate the effect of upregulated soluble fims-like tyrosine kinase receptor 1 (sFlt-1) in trophoblast cells on glomerular endothelial cells barrier function, in order to study the influence of the dysregulation of vascular endothelial growth factor (VEGF) and sFlt-1 on glomerular endothelial cells barrier function in preeclampsia.2. To investigate the effect of VEGF on the barrier dysfunction of glomerular endothelial cells by sFlt-loverexpression induced in trophoblasts, in order to study VEGF playing a role in maintaining glomerular endothelial cells barrier function.Methods:1. The human sFlt-1 expressing plasmid DNA was transfected into human first-trimester extravillous trophoblast cell line (TEV-1) by using Lipofectin method, and the levels of sFlt-1 mRNA and protein in TEV-1 were determined by performing RT-PCR, real-time PCR and ELISA.2. Transwell system was used to perform the coculture of TEV-1 s treated with transfection and human conditionally immortalised glomerular endothelial cell line (ciGEnCs). The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayer3. The cocultivation of TEV-1s treated with transfection and ciGEnCs was interfered with VEGF. The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayerResults:1. The expression of sFlt-1 at both mRNA and protein were enhanced after TEV-1cells were transfected with sFlt-1 plasmid DNA. 2. There was a significant increase of the amount of transferred fluorescently-labeled BSA across ciGEnCs monolayer in coculture of ciGEnCs with TEV-1 cells transfected with sFlt-1 plasmid DNA, and the monolayer barrier function of ciGEnCs was noticeably destroyed.3. Administration of VEGF could protect the monolayer barrier function of ciGEnCs as compared to that of cocultivation of TEV-1s transfected with sFlt-1 plasmid DNA and ciGEnCs.Conclusion:The dysregulation of VEGF and sFlt-1 in trophoblast may induce the barrier dysfunction of glomerular endothelial cells,which may be an important factor to contribute to glomerular endothelial cells barrier dysfunction in preeclampsia.VEGF may play an important role in relieving glomerular endothelial cells barrier dysfunction by sFlt-1 overexpression in trophoblast.PartⅢTrophoblasts induced apoptosis and necrosis may contribute to the barrier dysfunction of glomerular endothelial cellsObjective:1. To investigate the effect of both apoptotic and necrotic trophoblast cells on glomerular endothelial cells barrier function, in order to further study the mechanism of the barrier dysfunction of glomerular endothelial cells in preeclampsia.2. To investigate the effect of VEGF on the barrier dysfunction of glomerular endothelial cells which both apoptotic and necrotic trophoblasts caused, in order to study VEGF playing a role in maintaining glomerular endothelial cells barrier function. Methods:1. TEV-1 cells were induced to undergo apoptotic death by exposure to UV light and induced into necrosis by placing them into a 56℃water bath. Apoptotic and necrotic TEV-1 cells were analyzed by flow cytometry and Annexin V/PI double staining.2. CiGEnCs were inoculated in the upper compartment of the Transwell body until confluent was obtained. Subsequently, TEV-1 cells treated with control medium, apoptosis and necrosis were applied on confluent monolayers of ciGEnCs, respectively.The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayer3. The cocultivation of TEV-1s treated with apoptosis and necrosis and ciGEnCs was interfered with VEGF. The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayer4. TEV-1 cells treated with apoptosis were labeled with Dil cell-labeling solution, and ciGEnCs were labeled with Dil cell-labeling solution. Dil-labeled apoptotic or necrotic trophoblasts were incubated with the confluent monolayers of ciGEnCs for 24 h. Then, the cells were examined under fluorescence microscopeResults:1. More than 80% of the TEV-1 cells were apoptotic by exposure to UV light, and more than 80% of the TEV-1 cells were necrotic by water bath.2. When TEV-1s with medium, apoptosis and necrosis were cultured on confluent monolayers of ciGEnCs, the amounts of transferred fluorescently-labeled BSA across ciGEnCs monolayers were increased significantly.3. Excess of VEGF could protect the monolayer barrier function of ciGEnCs as compared to that of cocultivation of TEV-1s with apoptosis and necrosis and ciGEnCs, but could not restore ciGEnCs barrier function to its normal state.4. Apoptotic TEV-1 cells could be phagocytosed by ciGEnCs.Conclusion:When trophoblasts directly acted on glomerular endothelial cells, VEGF could not relieve completely the barrier dysfunction of glomerular endothelial cells, which may relate to the phagorytosis of ciGEnCs.Deported trophoblasts in the maternal blood phagocytosed by glomerular endothelial cells may be one of the mechanisms of glomerular endothelial cells barrier dysfunction in preeclampsia.