Investigation On The Role And Mechanism Of Transcription Factor TWIST1 In Preeclampsia

BACKGROUNDHypertension during pregnancy is a worldwide pregnancy-related complication that seriously threatens the health of mothers and infants,the incidence of which is about 5%-12%.Preeclampsia happens after 20 weeks of pregnancy.The preeclampsia with serious clinical symptoms is severe preeclampsia.Severe preeclampsia that happens before the 34th week of pregnancy is called early-onset severe preeclampsia(EOSP).The earlier the patient relapses and the more severe the condition,the worse the maternal prognosis.The basic pathophysiological changes in preeclampsia are manifested in systemic small blood vessel spasm,vascular endothelial injury,and reduced organ blood perfusion,which induces ischemic and hypoxic damage to multiple organs.At the same time,the insufficient placental perfusion can cause fetal injury.It can be seen that the shallow placental implantation is the basis of preeclampsia.Previous studies have confirmed that EOSP is one type of the preeclampsia most closely related to the defects of placental development.Since the placental trophoblastic cells are the executors of placental infiltration into the endometrium and myometrium,exploring the factors that regulate the activity and function of trophoblasts and the corresponding intervention methods are the key points to elucidate the pathogenesis of preeclampsia and guide its prevention and treatment.TWIST1 has functions in regulating cell differentiation,inhibiting apoptosis and enhancing invasion in a variety of human tissues and tumors.TWIST1 is widely expressed in placental trophoblasts,and its expression level shows a gestational-dependent increase tendency.It has been reported that TWIST1 has the ability to promote the differentiation and invasion of trophoblasts,so we speculate that it possibly plays an important role in the placental-related disease during pregnancy,including the pathogenesis of preeclampsia,but the relationship between them required being confirmed.In addition,it is still unclear whether TWIST1 plays a role in the mediating trophoblasts apoptosis.Low molecular weight heparin(LMWH)and Aspirin are currently clinically recognized as effective drugs to reduce the risk of preeclampsia.Recent studies have shown that the above two drugs have the effects of directly regulating the activities and functions of trophoblasts,however,the specific regulation mechanism has not been fully clarified.PART I:TROPHOBLAST APOPTOSIS RATE AND TWIST1EXPRESSION IN EOSP PATIENTS AND IN VITRO HYPOXIA MODELBackground And Objective:In early pregnancy,placental trophoblasts are in a physiologically hypoxic state,and then the trophoblasts gradually infiltrate the endometrium until the inner third of the muscle layer and enter into the uterine spiral arteriolar lumen to replace the vascular endothelium,leading to the enlargement of lumen and the increase of elasticity.The placental villous area gradually expands,thus the uterine-placental blood supply resistance decreases,and the blood volume increases,which makes the hypoxic state of placental trophoblasts improved.The increased placental trophoblast apoptosis rate and decreased invasion ability will cause insufficient placental infiltration and uterine revascularization obstruction,resulting in insufficient placental blood supply and trophoblast oxidative stress,and eventually induce the occurrence of preeclampsia.The role of TWIST1 in promoting trophoblast differentiation and invasion has been confirmed,but its expression in trophoblasts of patients with preeclampsia has not been reported.In part 1,the weights and scores of neonates as well as the placental weights delivered by EOSP patients and non-hypertensive gravida with matched gestational weeks are firstly compared.The differences in placental TWIST1 expression levels and trophoblast apoptosis rates between the two groups are examined.Then the hypoxia status of placental trophoblasts in early pregnancy and preeclampsia patients is simulated in vitro,from which the changes in TWIST1 expression level and cell apoptosis rate are detected.Methods:1.Using a retrospective study,statistical analysis is performed on the differences in neonatal body weights,Apgar scores,and placental weights of 27 EOSP patients and 31 non-hypertensive gravidas with matched gestational weeks who delivered between 2016.12 and 2018.10.2.Western blot,PCR and immunohistochemistry are used to detect the TWIST1 expression levels in placenta of EOSP patients and non-hypertensive gravidas with matching gestational weeks.Tunel staining and western blot are employed to detect and compare the apoptosis of placental trophoblasts between the two groups.3.In in vitro hypoxia model,the TWIST1 expression level in trophoblasts are detected by using Western blot and the cell proliferation and apoptosis are detected by Hoechst 33258 staining and Annexin V.Results:1.Compared with those of non-hypertensive gravidas who delivered during the same pregnancy,neonatal body weights,Apgar scores,and placental weights of EOSP patients are found significantly reduced.2.Compared with those of non-hypertensive gravidas who delivered during the same pregnancy,the TWIST1 expression levels in the placenta of EOSP patients have been found to decrease,and the trophoblast apoptosis rates increase.3.Hypoxia will reduce the expression level of TWIST1 in trophoblasts cultured in vitro,and increase the apoptosis rate,which shows time dependence.Conclusions:The incidence rate of adverse neonatal outcomes in EOSP patients is high.The decrease of TWIST1 expression level in placental trophoblasts and the increase of cell apoptosis rate are positively correlated with the incidence of EOSP.PART II:ROLES OF TWIST1 IN HYPOXIC TROPHOBLASTSBackground And Objective:In female reproductive function,TWIST1 can regulate decidualization of uterine stromal cells,embryonic development and organ differentiation.The development of placenta and the growth of tumors have extremely similar characteristics in many aspects,so it could be speculated that the regulation mechanism of TWIST1 on tumor cell apoptosis also acts similarly on trophoblasts.In this chapter,focusing on the changes in the activity and function of trophoblasts caused by hypoxia,and by regulating the expression of TWIST1,the changes in the proliferation,apoptosis,invasion,and angiogenesis of anatrophic trophoblasts are detected and the roles of TWIST1 in placental development and pathogenesis of preeclampsia are investigated.Methods:1.By transfecting plasmids with overexpression of TWIST1,the cell lines with overexpressed TWIST1 are firstly obtained.Then stable cell lines that can interrupt TWIST1 expression are achieved by using a lentiviral vector.The above cell lines are cultured under hypoxia.2.Edu staining,Hochst33342 staining,Annexin V and Western blot are employed to detect the influences of over-/low-expression TWIST1 in the hypoxic model on the proliferation and apoptosis of trophoblasts,as well as the expressions of apoptosis-related proteins(Bcl-2,Bax,Caspase-3,Caspase-9)and invasion-related proteins(MMP-2,MMP-9).3.The effects of over/low expressed TWIST1 on the clone ability,cell migration and invasion,and angiogenesis abilities of trophoblasts in hypoxia model are detected by plate cloning,Transwell and angiogenesis experiments.4.Flow cytometry is used to detect the effects of over/low expressed TWIST1 on ROS and mitochondrial membrane potential in trophoblasts.Results:1.Overexpression of TWIST1 inhibits the apoptosis of trophoblasts induced by hypoxia,promotes cell proliferation,and the apoptosis-related proteins also changes correspondingly,while low expression of TWIST1 promotes cell apoptosis and inhibits cell proliferation.2.Overexpression of TWIST1 can reverse the reduction of trophoblastic cloning,angiogenesis,cell migration and invasion abilities of trophoblasts caused by hypoxia,which is accompanied by increased expression of invasion-related proteins.However,low expression of TWIST1 further exacerbates the reduction of the invasiveness of trophoblasts induced by hypoxia.3.Overexpression of TWIST1 decreases the ROS level of trophoblasts and increases the mitochondrial membrane potential of cells,while low expression of TWIST1 further exacerbates the hypoxia induced increase of ROS and mitochondrial membrane potentials of trophoblasts.Conclusions:TWIST1 participates in regulating the proliferation,apoptosis,angiogenesis,and cell migration abilities of trophoblasts under hypoxic conditions,which inhibits cell apoptosis,and promotes cell invasion ability.PART III:EFFECTS OF APOPTOTIC ACTIVATORS/INHIBITORS ON TWIST1 REGULATING HYPOXICTROPHOBLAST APOPTOSISBackground And Objective:Apoptosis is a genetically controlled autonomous and orderly dying process of cells.Up to date,three apoptosis pathways have been identified:the mitochondrial pathway,the death receptor pathway,and the endoplasmic reticulum pathway.The mitochondria1 pathway is the most important apoptosis process in mammals.After the mitochondrial pathway is activated,the mitochondrial membrane potential and the apoptosis-related protein Bcl-2/Bax ratio decrease,which in turn initiates the Caspase family cascading reaction and leads to apoptosis.Caspase-9 is a key connecting link factor in the mitochondrial apoptotic pathway,while Caspase-3 is a common substrate of the three apoptotic pathways,acting as an executor of apoptosis.Our previous results have confirmed that TWIST1 can regulate the apoptosis of hypoxic trophoblasts,accompanied by changes in apoptosis-related proteins and mitochondrial membrane potentials in the mitochondrial pathway,which suggests that TWIST1 may affect the mitochondrial apoptotic pathway of trophoblasts.In this chapter,by adding Caspase-3 activator and Caspase-9 activator/inhibitor into the hypoxic trophoblast model,the mechanism of TWIST1 in regulating the trophoblast apoptosis is further explored.Methods:1.Over-expressing TWIST1 in hypoxic trophoblast model,and adding Caspase-3 activator(PAC-1),Caspase-9 activator(YC137)or inhibitor(Z-LEHD-FMK),the cell apoptosis is detected and analyzed by Hoechst staining and Annexin V.2.Western blot is used to detect the expressions of mitochondrial apoptosis-related proteins(Bcl-2,Bax,Caspase-3,Caspase-9)and invasion-related proteins(MMP-2,MMP-9)in hypoxic trophoblasts with Caspase-3 activator,Caspase-9 activator or inhibitor added.Results:1.Overexpression of TWIST1 inhibits the hypoxia-induced trophoblast apoptosis.The addition of Caspase-3 and Caspase-9 activators can reverse the protective effect of TWIST1 on apoptosis,while the addition of Caspase-9 inhibitors enhances the role of TWIST1 in inhibiting apoptosis.2.Overexpression of TWIST1 inhibits the effects of hypoxia on the expressions of trophoblast apoptosis-related and invasion-related proteins,which is reversed by the injection of Caspase-9 activator,however,enhanced by the addition of Caspase-9 inhibitor.Conclusions:TWIST1 regulates the hypoxia induced apoptosis of trophoblasts through the apoptotic pathway of mitochondria.PART IV:MECHANISM OF TWIST1 IN REGULATING EFFECTS OF ASPIRIN AND LMWH ON TROPHOBLAST ACTIVITY AND FUNCTIONBackground And Objective:The role of Aspirin and LMWH in reducing the risk of preeclampsia has been well recognized.Studies have confirmed that besides the improvement of placental circulation by the anticoagulant effect,aspirin and LMWH can also directly act on placental trophoblasts to regulate their activity and function.However,the mechanism of action has not been fully clarified.In this chapter,Aspirin and LMWH are added to trophoblasts cultured under hypoxia condition to explore the effect of drugs on trophoblast apoptosis and invasion ability.In addition,the influence mechanism of Aspirin and LMWH on the trophoblasts is further explored by interrupting the expression of TWIST1.Methods:1.The hypoxic trophoblasts are treated with different doses of Aspirin and LMWH,and the changes of the apoptosis rate are detected by using Annexin V.Then the drug dose that has the most significant effect on apoptosis and the lowest concentration is chosen.2.Edu staining and transwell are used to detect the effects of Aspirin and LMWH on the proliferation and invasion abilities of hypoxic trophoblasts.The Real-time PCR,Western blot,and immunofluorescence are used to detect the influences of Aspirin and LMWH on the expressions of TWIST1 and apoptosis-related proteins,and invasion-related protein of the hypoxic trophoblasts.The flow cytometry is employed to detect the effects of Aspirin and LMWH on ROS and mitochondrial membrane potential of hypoxic trophoblasts.3.Lowly expressing the TWIST1 in hypoxic trophoblast cell model,the effects of Aspirin and LMWH on the proliferation,apoptosis,migration and invasion abilities of trophoblasts are detected by using Edu staining,Annexin V,Transwell and Western blot methods.Flow cytometry is used to detect the influences of Aspirin and LMWH on the ROS and mitochondrial membrane potential of hypoxic trophoblasts after interrupting the TWIST1 expression.Results:1.Aspirin and LMWH can reduce the apoptosis rate of hypoxic trophoblasts and increase their abilities of proliferation and invasion.Aspirin and LMWH can reverse the hypoxia induced changes of TWIST1 expression,apoptosis and invasion-related protein.Aspirin and LMWH can reverse the hypoxia induced increase of ROS and decrease of mitochondrial membrane potential of the trophoblasts.2.Low expression of TWIST1 can reverse the protective effects of Aspirin and LMWH on the proliferation,migration and apoptosis of hypoxic trophoblasts,and weaken the effects of Aspirin and LMWH on ROS and mitochondrial membrane potential of hypoxic trophoblasts.Conclusions:Aspirin and LMWH can inhibit the apoptosis of hypoxic trophoblasts by improving the expression of TWIST1,and promote cell invasion,playing a protective role in the processes of placental development and uterine revascularization.The clarification of this mechanism provides a theoretical basis for the early prevention of preeclampsia by using Aspirin and LMWH.SUMMARYIn this dissertation,the differences in neonatal outcomes and placental weights between EOSP patients and non-hypertensive gravidas,as well as the differences in placental TWIST1 expression and apoptotic rates between the two groups have been firstly compared.The results show that poor fetal prognosis,decreased expression of TWIST1 in trophoblasts and increased apoptosis happen to EOSP patients.Then,a cell model has been established to simulate the hypoxic state of trophoblasts in early pregnancy and preeclampsia patients,and the effects of TWIST1 on trophoblast apoptosis and invasion abilities have been investigated.The mechanism by which TWIST1 can regulate apoptosis of trophoblasts has been explored.It is found that TWIST1 can reduce the hypoxia induced apoptosis and promote cell invasion through the mitochondrial apoptotic pathway.Finally,Aspirin and LMWH have been added to trophoblasts cultured in hypoxia,and the changes in cell activity and function have been detected.By interrupting the expression of TWIST 1,the action mechanism of drugs has been investigated,which well shows that Aspirin and LMWH can reduce the apoptosis of trophoblasts and enhance its invasion ability by promoting the expression of TWIST1.