Hypoxia Trophoblast-derived SFlt-1 May Contribute To Endothelial Cell Dysfunction By Depriving Of VEGF Activity In Preeclampsia

Objective:To investigate the expression of soluble fims-like tyrosine kinase receptor 1 (sFlt-1) and vascular endothelial growth factor (VEGF) in serum obtained from normal pregnancy and preeclampsia, and the correlation to endothelial cell dysfunction.Methods:1. The level of sFlt-1 and VEGF protein in serum samples of 10 severe preeclampsia females and 10 normotensive females were determined by performing enzyme-linked immunosorbent assay (ELISA).2. The culture of human umbilical vein endothelial cell lines (HUVEC) was interfered with serum samples as described previously. The effect of serum on endothelial cell dysfunction was determined on the basis of following aspects:â‘ monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;â‘¡cell proliferation function was evaluated by performing methl thiazolyl tetrazolium (MTT);â‘¢level of secreted nitric oxide (NO) was estimated by nitrate reductase assay.Result:1. The level of sFlt-1 protein in serum of severe preeclampsia was significantly higher than that of normal pregnancy, whereas the level of VEGF protein was significantly lower. There was obvious negative correlation between the level of sFlt-1 and VEGF in preeclampsia.. 2. Compared to normal pregnancy, the serum of severe preeclampsia could lead to endothelial cell dysfunction, including:â‘ high transfer of fluorescently-labeled BSA across HUVEC monolayer indicated the damage of monolayer barrier function;â‘¡the proliferation ability of HUVEC was markedly decreased;â‘¢the level of secreted NO was low as compared to that of normal pregnancy group.3. There was positive correlation between endothelial cell dysfunction and the level of sFlt-1/VEGF in serum of preeclampsia.Conclusion:The results of our study suggest that endothelial cell dysfunction is closely correlated with disorder of sFlt-1 and VEGF levels in serum of preeclampsia, which may lead to the pathogenesis of preeclampsia.Objective:To investigate the effects of VEGF deficit on endothelial cell dysfunction, in order to study the role of VEGF in pathogenic mechanism of endothelial dysfunction in preeclampsia.Methods:1. VEGF expression was blocked by transfecting HUVEC with VEGF-siRNA. The level of VEGF protein and mRNA in transfected HUVEC was determined by performing reverse transcriptase-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA).2. The effect of VEGF deficit on endothelial cell dysfunction was determined on the basis of following aspects:â‘ monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;â‘¡cell proliferation function was evaluated by performing MTT;â‘¢level of secreted NO was estimated by nitrate reductase assay.Result:1. After transfecting HUVEC with VEGF-siRNA for 48 h, VEGF mRNA expression was significantly reduced as compared to the expression in scramble siRNA-transfected cells (approximately 50.7%). In addition, the concentration of VEGF protein secreted by VEGF-siRNA transfected cells was lower than that of scramble-siRNA treated cells.2. The function of VEGF-siRNA transfected HUVEC was destroyed, including:â‘ significantly high transfer of fluorescently-labeled BSA across HUVEC monolayer indicated the badly damage of monolayer barrier function;â‘¡the proliferation ability of HUVEC was markedly decreased;â‘¢the level of secreted NO was low as compared to that of scramble siRNA-transfected cells.Conclusion:VEGF plays an important role in maintaining the physiological functions of endothelial cells. The deficiency of VEGF can lead to endothelial cell dysfunction, which may induce a series of clinical symptom in preeclampsia.Objective:1. To investigate the effects of hypoxia on sFlt-1 expression respectively in trophoblast cell and endothelial cell, in order to study the reason for abnormal expression of sFlt-1 in preeclampsia.2. To investigate the effect of hypoxia trophoblast-derived sFlt-1 on endothelial cell dysfunction by depriving of VEGF activity, in order to further study the mechanism of trophoblast-endothelial cell dysfunction in preeclampsia.Methods:1. Human first-trimester extravillous trophoblast cell line (TEV-1) and human umbilical vein endothelial cell line (HUVEC) were grown respectively in a hypoxic condition produced by COCl2. The levels of sFlt-1 mRNA and protein in TEV-1 and HUVEC were determined by performing RT-PCR, real-time PCR and ELISA.2. Transwell system was used to perform the cocultivation of hypoxia TEV-1 and HUVEC. The functions of HUVEC were evaluated on the basis of following aspects:â‘ monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;â‘¡cell proliferation function was evaluated by performing MTT;â‘¢level of secreted NO was estimated by nitrate reductase assay. Meanwhile, the levels of sFlt-1 and VEGF mRNA and protein in hypoxia TEV-1 were determined by performing RT-PCR, real-time PCR and ELISA.3. The cocultivation of hypoxia TEV-1 and HUVEC was interfere with VEGF, and the functions of HUVEC were evaluated on the basis of following aspects:â‘ monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;â‘¡cell proliferation function was evaluated by performing MTT;â‘¢level of secreted NO was estimated by nitrate reductase assay.Result:1. COCl2 showed time-dependent promotion on sFlt-1 mRNA (for 72,96 and 120 h treatment, P<0.05) and protein expression (for 96 and 120 h treatment, P<0.05) in TEV-1. However, there were not significant differences in sFlt-1 expression for HUVEC.2. When the cocultivation of hypoxia TEV-1 and HUVEC was performed, the functions of HUVEC was noticeably destroyed, including:â‘ significantly high transfer of fluorescently-labeled BSA across HUVEC monolayer indicated the badly damage of monolayer barrier function;â‘¡the proliferation ability of HUVEC was markedly decreased;â‘¢the level of secreted NO was low as compared to that of normal cocultivation of TEV-1 and HUVEC. 3. Administration of VEGF could protect endothelium cell function from injury, which was determined on the basis of rising of monolayer barrier function, cell proliferation function, and secreted NO levels as compared to that of cocultivation of hypoxia TEV-1 and HUVEC.Conclusion:Chronic hypoxia trophoblast-derived sFlt-1 may be the important source of high expression of sFlt-1 in preeclampsia. Served as toxic factors derived from placenta, sFlt-1 is the key factors to contribute to endothelial cell dysfunction in preeclampsia by depriving of VEGF activity.