Study On The Effect Of TFPI-2 In Preeclampsia

Preeclampsia(PE)is a special type of hypertensive disorders of pregnancy(HDP).PE is a leading cause of maternal and fetal morbidity and mortality,accounting for 10~15% of maternal deaths a year worldwide.Currently,the exact etiologies and pathogenesis for the preeclampsia are unknown.There are several mechanisms that contribute to the development of preeclampsia.In preeclampsia,abnormal placentation and defective spiral artery remodeling by invasive trophoblasts is associated with placental lesions consistent with maternal vascular malperfusion(MVMP)and oxidative stress,ultimately leading to the release of syncytiotrophoblast debris,pro-inflammatory and anti-angiogenic molecules into the maternal circulation.The most efficient therapeutic strategy is acknowledged to be delivery.The tissue factor pathway inhibitor 2(TFPI-2),located on chromosome 7q22,is a 32-k Da Kunitz-type serine proteinase inhibitor.The human TFPI-2 gene,expressed abundantly in full-term placenta and widely in a variety of adult human tissues such as liver,skeletal,muscle,heart,kidney and pancreas,has also been shown to reduce tumor invasion and metastasis and plays an important role in biological behaviors such as cell proliferation,differentiation and apoptosis.Basic and clinical studies have found that trophoblast cell invasion has many similarities with tumor invasion.At present,there is no conclusion about the expression of TFPI-2 in preeclampsia,and it is not clear whether it is involved in the regulation of the trophoblast cell biological function.Therefore,we conducted a further study on the expression and biological effects of TFPI-2 in preeclampsia.Part One The expression level of TFPI-2 in peripheral blood and pla-cental tissues of preeclampsiaObjective: To study the expression level of TFPI-2 expression in peripheral blood and placenta of preeclampsia.Methods: 1.We recruited 60 pregnant women with normal pregnancy,60 pregnant women with early-onset PE,and 60 pregnant women with late-onset PE to obtain the maternal peripheral blood samples and placental tissue.2.The expression of TFPI-2 in maternal peripheral bloods were determined by ELISA.3.The localization of TFPI-2 in placental tissues were determined by immunohistochemistry.4.RT-PCR and western blotting were used to detect the expression of TFPI-2 in placental tissues.Results: 1.The protein levels of TFPI-2 in serum were significantly increased in both the early-onset and late-onset PE compared with the NC group(P<0.05).However,TFPI-2 levels were higher in patients with early-onset pre-eclampsia,but there is no obvious statistical difference between the two groups(P>0.05).2.TFPI-2 is mainly expressed in syncytiotrophoblasts.TFPI-2 was highly expressed in both the early-onset and late-onset PE compared with the NC group.3.The levels of TFPI-2 protein and m RNA in placenta of preeclampsia were significantly higher than those in normal pregnancy(P<0.05).However,TFPI-2 levels were higher in patients with early-onset pre-eclampsia,but there is no obvious statistical difference between the two groups(P>0.05).Summary: The levels of TFPI-2 in the serum and placenta tissues were significantly increased in pregnant women with PE compared with that in normal pregnancies,and it plays an important role in the development of preeclampsia.Part Two TFPI-2 is involved in the regulation of the biological function of trophoblast cellsObjective: To investigate the effects of TFPI-2 on migration,proliferation and apoptosis of trophoblast cells under Hypoxia/reoxygenation(H/R)conditions.Methods: 1.RT-PCR and western blotting were used to study the expression of TFPI-2 in trophoblast cells under hypoxic conditions.2.MTT assay was used to estimate cell growth ability in HTR-8/SVneo cells.3.Annexin V/Propidium Iodide(PI)Apoptosis Assay was used for the function of TFPI-2 in cell apoptosis of in HTR-8/SVneo cells in H/R condition.4.The function of TFPI-2 in cell migration was examined by transwell assay.Results: 1.Hypoxia/reoxygenation(H/R)promotes the expression of TFPI-2 in HTR-8/SVneo cell line 2.H/R culture significantly suppressed the HTR-8/SVneo cell proliferation.whereas downregulation of TFPI-2 by si TFPI-2 reversed this trend.In addition,the decreased cell proliferation ability by H/R culture was enhanced by overexpression of TFPI-2 in HTR-8/SVneo cells.3.In H/R condition,the increased cell apoptosis in HTR-8/SVneo cells was enhanced by the overexpression of TFPI-2 and suppressed by the downregulation of TFPI-2 expression.4.H/R culture significantly inhibited the cell migration in HTR-8/SVneo cells,whereas downregulation of TFPI-2 by si TFPI-2 reversed this trend.In addition,the decreased cell migration ability by H/R culture was enhanced by overexpression of TFPI-2 in HTR-8/SVneo cells.Summary: 1.TFPI-2 plays an important role in trophoblast proliferation,apoptosis and migration.2.TFPI-2 participates in the pathogenesis of preeclampsia by affecting trophoblast cell function.Part Three The mechanism of TFPI-2 and Vitexin in preeclampsiaObjective: TFPI-2 plays an important role in preeclampsia through HIF-1/VEGF pathway.Methods: 1.we applied a PE rat model induced by nitro-L-arginine methyl ester(L-NAME).2.RT-PCR and western blotting were used to study the expression of TFPI-2,HIF-1 and VEGF in PE rats.3.Different doses of Vitexin(VI)were given to PE rats to study the changes of TFPI-2,HIF-1 and VEGF expression levels.Results: 1.The m RNA and protein expression levels of TFPI-2,HIF-1 and VEGF were significantly increased in PE rats(p < 0.01).2.VI treatment decreased L-NAME-induced high SBP dose and time dependently in PE rats.3.VI reduced the expression of TFPI-2、HIF-1 and VEGF in placenta in L-NAME-induced PE rat models.4.VI treatment with higher dosages(45 and 60 mg/kg)corrected the L-NAME-induced reduction in pups weight.Summary: 1.L-NAME can be used to establish a rat model of preeclampsia.2.TFPI-2 may play a role in preeclampsia rat model through the TFPI-2/HIF-1/VEGF pathway.3.VI treatment can improve the pregnancy outcome of in L-NAMEinduced PE rat model.Conclusions: 1.The levels of TFPI-2 in the serum and placenta tissues were significantly increased in pregnant women with PE compared with that in normal pregnancies.2.TFPI-2 plays an important role in trophoblast proliferation,apoptosis and migration.3.TFPI-2 may play a role in preeclampsia rat model through the TFPI-2/HIF-1/VEGF pathway.4.VI treatment can improve the pregnancy outcome of in L-NAMEinduced PE rat model.