Preeclampsia Sera/Hypoxic Trophoblasts Induce HRGECs Hyperpermeability Via The HIF1?/HIF2?-Flt-1/sFlt-1-CAV1 Pathway

ObjectiveTo explore the source of soluble fms-like tyrosine kinase-l(sFlt-l)in preeclampsia serum and hypoxic trophoblast(HTR-8/SVneo)conditioned medium(CM),and to explore the effect of sFlt-1 on the permeability of human renal glomerular endothelial cells(HRGECs)Methods1.Collect the serum and placenta of 20 cases PE and 20 cases normal pregnancies,and use ELISA to detect the sFlt-1 in serum,and use western blot and immunohistochemistryto detect the HIFla/HIF2a and Flt-1 protein in placenta.Cultured the HRGECs and divide into four groups NG serum group,PE serum group,NG+rsFlt-1 group,PE+Axitinib group,use Evens-blue to detect the permeability of HRGECs and use western blot or immunofluorescence to detect the expression of CAVI protein2.Culture the HTR-8/SVneo cells in the hypoxic condition,then use the western blot to detect the HIFla/HIF2a and Flt-1 protein expression,and use the ELISA to detect the sFlt-1 in conditioned medium(CM).Transfect the HIFla/HIF2a plasmid or siRNA into HTR-8/SVneo cells,use western blot to detect the expression of Flt-13.Culture the HRGECs with hypoxic CM,transfection CM,hypoxic CM with Axitinib or MBCD,then use the use Evens-blue to detect the permeability of HRGECs and use western blot or immunofluorescence to detect the expression of CAVI proteinResults1.PE placenta displayed higher intracellular HIFla/HIF2a and Flt-1 protein than normal placenta The sFlt-1 level in the serum of PE was higher than that in normal pregnancy serum.PE serum could increase the permeability and CAVI protein expression in HRGECs,which were inhibited by axitinib and methyl-beta-cyclodextrin(MBCD)2.Hypoxic HTR-8/SVneo cells displayed higher Flt-1 mRNA,intracellular HIFla/HIF2a and Flt-1 protein,and CM sFlt-1 levels than those in normoxic HTR-8/SVneo.Unregulated HIFla/HIF2a expression could increase the mRNA and protein expression levels of Flt-l/sFlt-1 in HTR-8/SVneo.Downregulation of HIFla/HIF2a expression could decrease the mRNA and protein expression levels of Flt-1/sFlt-1 in hypoxic HTR-8/SVneo3.The hypoxic HTR-8/SVneo CM could increase the monolayer permeability and CAVI protein expression in HRGECs,which were inhibited by axitinib.PE serum and recombinant sFlt-1(rsFlt-1)could also increase the endothelial monolayer permeability and CAVI protein expression in HRGECs,which were inhibited by axitinib.The increased HRGECs permeability and rsFlt-1 protein level induced by hypoxic HTR-8/SVneo CM could be inhibited by MBCDConclusionHypoxic trophoblasts play an important role in the mechanism of protein urine in PE by increasing HRGECs monolayer permeability through the HIF1?/HIF2?-Flt-1/sFlt-1-CAV1 pathway.