The Expression Of MMP9/TIMP1 In Hypoxic Cytotrophoblast And The Relationship With Preeclampsia

Background Preeclampsia(PE) is a specific complication during the late pregnancy and the exact cause and pathogenesis of PE have not been clear out. A currently accepted hypothese is the placenta shallow implantation that the uterine spiral arteries remodeling has been obstacled by defective invasion of cytotrophoblasts. It might cause the ischemia and hypoxia of trophoblasts and increase local cellular immune response to placental oxidative stress with manifesting as lipid peroxidation and releasing of oxygen free radicals. Then released inflammatory factors and activated neutrophils directly or indirectly might cause maternal vascular endothelial damaged and accompany systemic small artery spasm, ultimately developing hypertension, proteinuria, edema, and eclampsia. Therefore, It may be a key factor in the pathogenesis of preeclampsia that defective invasion of cytotrophoblasts would hinder placental bed vascular remodeling.Placental angiogenesis and the physiological rebuilding of uterine spiral arteries depend on the proliferation and differentiation of cytotrophoblastes and the invasion of cytotrophoblastes to the uterine decidua and spiral arteries. The process includes the trophoblast adhesion, matrix degradation and cell migration and involves the related proteases, adhesion molecules, extracellular matrix and decidua inhibitory factor etc. The degradation of extracellular matrix is an important part of the process. Matrix metalloproteinase family (MMPs) is one of the most important group of proteases, which can degrade almost all of the matrix elements. Tissue inhibitor of metalloproteinases(TIMPs) is a natural inhibitor of MMPs and both of MMPs and TIMPs play an important role in the maintenance of ECM homeostasis and structural integrity. In recent years,MMP9 which is one of currently known species of more than 20 MMPs is paid more attention to the cytotrophoblastic invasion and placental vascular remodeling. Experiments have shown that TIMP1 and MMP9 specific antibodies can completely inhibit the invasion of trophoblast. Therefore, The balance of MMP9/TIMP1 is particularly important in the invasive processes of cytotrophoblast.The imbalance of MMP9/TIMP1 may lead to the placenta shallow implantation that might initiate preeclampsia, but the study is mainly in the level of the organization now. This experiment simulated the process of cytotrophoblast at the stage of early pregnancy when the pregnancy was physiological hypoxia condition and at the stage of late pregnancy when the damaged vascular endothelial was due to pathological hypoxia condition. We will further research the role of MMP9 gene in the level of the cell and also investigat the effect of the damaged vascular endothelial cells on the invasion of cytotrophoblasts.we hope to provide new ideas for a model in vitro of preeclampsia to study the pathogenesis.This study is divided into three parts as follows:Part I Inhibitory effects of siRNA-silencing MMP9 gene on CytotrophoblastObjective To confirm the relationship between mmp9 and plancenta-shallow implanation of preeclampsia by means of siRAN-silencing mmp9 gene that inhibit the invasion of cytotrophoblast. Methods Effective siRNA of mmp9 gene was synthesized in vitro.The siRNA was transfected into cytotrophoblast by lipofectamine TM 2000.Then mmp9mRNA and protein expressions in the transfected cytotrophoblast were detected by fluorescence quantitative reverse transcriptase polymerase chain reaction (FQ-PCR) and Western blotting. The biological effects of the cytotrophoblast were evaluated through the detection of their anchorage-in-dependent growth and in vitro invasion by cell migration assay and transwell chamber assay.Results The expression of mmp9 in transfected cytotrophoblasts was markedly depressed at both mRNA and protein levels as compared with the controlled group Anchorage-in-dependent growth assay showed the growth of transfected cells was retarded. Transwell chamber assay showed the invasion ability of transfected cells was inhibited significantly (p<0.01).Conclusion Effective siRNA can be successfully transfected into Cytotrophoblast and can effectively inhibit the expression of mmp9mRNA and protein. Therefore, the proliferation and invasion of transfected cells are inhibited. Increasing the expression of mmp9 may have a potential role in prevention and treatment of pregnancy-induced hypertension.PartⅡHypoxia Alters Matrix metalloproteinases9/Tissue inhibitor of metalloproteinases1 and promotes invasion of cytotrophoblast in VitroObjective To investigate the effection of Hypoxia on expression of matrix metallo-proteinases9/Tissue inhibitor of metalloproteinasesl and invasion activity in vitro cytotrophoblast.Methods immortalized human extravillous trophoblast cell line were induced in hypoxia circumstance by 150μmol/L CoCL2. Immunocytochemistry,fluorescence quantitative real-time PCR and Western blotting were used to determine the gene expression of MMP9/TIMP1 in cytotrophoblast. The invasion of hypoxia-induced cytotrophoblast were assessed by Transwell chamber assay.Results The mRNA of MMP9 and TIMP1 and the protein expression of both were up-regulated in hypoxia group(P<0.05),and the ratio of MMP9/TIMP1 was increased with prolonged time. The invasion of hypoxia group was more enhanced than the controlled group(P<0.01).Conclusion Physiological hypoxia could enhance cytotrophoblast invasion during early pregnancy with the increase of the ratio of MMP9/TIMP1,and TIMP1 may promote cytotrophoblast proliferation and differentiation besides regulating MMP9.PartⅢHypoxia induces FGF2 production by co-cultured vascular endothelial cells and alters MMP9 and TIMP1 expression in extravillous trophoblasts and their invasivenessObjective To investigate the role of fibroblast growth factor 2 (FGF2) secretion by vascular endothelial cells during co-cultured hypoxic cytotrophoblast and the effection of FGF2 on expression of matrix metallo-proteinases9/Tissue inhibitor of metalloproteinasesl and invasion activity in vitro cytotrophoblast.Methods The human extravillous cytotrophoblast cell line, TEV-1, and umbilical vein endothelial cell line, HUVE-12, were co-cultured under normal and hypoxic(150μmol/L CoCL2)conditions. FGF2 expression in HUVE-12 cells and matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in TEV-1 cells was analyzed using fluorescence quantitative RT-PCR and Western blotting analyses. TEV-1 cell invasion was also examined by Transwell chamber assay.Results The mRNA and the protein expression of FGF2 in HUVE-12 cells co-cultured with TEV-1 cells was significantly increased under hypoxic conditions (P < 0.05). In TEV-1 cells co-cultured with HUVE-12, hypoxia reduced the mRNA and the protein expression of MMP9 (P<0.01)and increased the mRNA and the protein expression of TIMP1(P< 0.05); The invasion of co-cultured hypoxic group was more reduced than the controlled group(P<0.01). Conclusion FGF2 release by stressed endothelial cells of uterine spiral arteries might decrease MMP9 and increase TIMP1 production in extravillous cytotrophoblast in response to stress, resulting in reduced extravillous cytotrophoblast invasion and possibly shallow implantation of the placenta, but further study need be continued as the exact regulatory mechanism is unclear.