The Expression Of Apelin In Placenta Of Normal Pregnancy And Hypertensive Disorder Complicating Pregnancy And Relationship With Nitric Oxide

Background Hypertensive disorder complicating pregnancy(HDCP) is a common complication during the late pregnancy and the leading cause of maternal and fetal morbidity and the mortality. But the exact cause and pathogenesis of HDCP have not been clear out, there is not any sure or effective treatment for HDCP. Recently, a great many studies about HDCP have been made and some hypotheses were drawn out, such as immune, placenta shallow invasion, blood vessel endothelium damaged, theory of heredity, theory of innutrition, theory of insulin resistance. From plenty of research, we know the key-link for HDCP is the vascular endothelial damage and the damage is wide in body. The imbalance of nitric oxide (NO), endothelin(ET), prostacyclin(PGI2), thromboxane A2(TXA2), plasminogen activation inhibition factor(PAI), tissue plasminogen activation et al, will result in the ET and TXA2 secreting increased, on the other hand NO and PGI2 decreased which leads to all small arteries spasm, then every organ blooding perfuse is deficient. Now, ischemic and hypoxia of placenta is the major reason leading to endothelia cells damaged. To search the factors about placenta ischemic and hypoxia would get a new treatment for HDCP.Apelin is an active cardiovascular peptide which was founded recently. It is a endogenic ligand of G protein couple receptor APJ. In the cardiovascular system, Apelin exerts potent vasodilator and positive inotropic activities. It can promote NO synthesis too. In modulating water-electrolyte metabolism and mediating immuno- regulation. Apelin plays a significant role. There are several isoforms about Apelin and the isoform Apelin-36 and Apelin-13 are common. Now the study about Apelin and APJ is just beginning. There are little reports about Apelin on pregnancy.NO is a relaxing factor of blood vessel endothelium. It has a significant role in regulating fetoplacental circulation. During gestational period, placenta is the principle site for synthesis of NO. Nitric oxide synthase(NOS) is the capital rate-limiting step for synthesizing NO. Studies have confirmed that the reduction of NO in pregnancy period has close relationship to HDCP. But there is no report about the relationship between Apelin and the synthesis NO by placenta villus during pregnancy.HDCP is a special disorder of pregnancy and placenta is a special organ of pregnancy. So to study the physiological and biochemical change of placenta will find the real cause which resulting in uteroplacental ischemia and hypoxia and to blood vessel endothelial cell damage. In our study, placenta was selected as the research object. For the goal of understanding the possible role of Apelin in the pathogenesis of HDCP and the relation with NO. Real-time fluorescence quantitative RT-PCR method (FQ-RT-PCR) was used to detect the expression of Apelin and APJ mRNA, and the expression of Apelin-36 and APJ protein were examined by immunohistochemistry SP and image analysis method in villus and placenta tissue. The stable metabolic end products of NO was measured with nitrate reductase and The NOS activity was assayed by spectrophotometry in placenta tissue. Apelin-36 was used to interfere in placenta villus tissue in vitro, and to observe whether Apelin can exert NO-NOS from placenta villus tissue. So to expect to offer a new find thinking for prevention and treatment measures about HDCP.This study is divided into three parts: PartⅠExpression and localization of Apelin and APJin human placenta and umbilical vesselsObjective To explore the expression and localization of endogenous vasoactive Apelin and its receptor APJ in first trimester placenta, term placenta and umbilical vessels. Methods Ten villus specimens were taken from the pregnant women conceived about 7～9 weeks, and fifteen placenta specimens and umbilical vessels were taken from pregnant women conceived about 37～41 weeks. Real-time fluorescence quantitative RT-PCR method (FQ-RT-PCR) was used to detect the expression of Apelin and APJ mRNA, the expression and localization of Apelin-36 and APJ protein were examined by immunohistochemistry SP and image analysis methods (HPIAS-1000). Results (1) The expression of Apelin mRNA was significantly increased in placentas tissue from term pregnancy compared to first trimester placenta (P <0.05), but the expression of APJ mRNA in two groups had not statistical significant (P>0. 05). (2) The result of immunohistochemistry demonstrated that Apelin-36 was predominantly localized in villous syncytiotrophoblasts and cytotrophoblasts, with weak staining of villous stroma at first trimester. In late trimester, Apelin-36 was was predominantly localized in villous syncytiotrophoblasts, it can be detected in capillaries and a few cells of villous stroma too. APJ predominantly localized in nuclei of villous syncytiotrophoblasts and cytotrophoblasts, secondly, localized in villous endothelium and stroma of placental tissue. Moreover there are stainings of APJ in some vessel muscle cells. (3)There is weak expression of Apelin-36 in villus in first trimester , but its expression increased significantly in placental tissue in the late trimester, and the difference had statistical significant (P <0. 01). The level of APJ expression has no difference between two groups (P >0.05). (4)The results showed that Apelin-36 and APJ appeared in the endothelial cells of human umbilical vessels, APJ staining was detected in some vascular smooth muscle cells of human umbilical vessels. But staining of Apelin-36 and APJ were weaker in umbilical vein than that in umbilical artery. Conclusion Both Apelin and APJ receptor are expressed in placental villus from the first to the third trimester of gestation, suggesting that the autocrine regulation system about Apelin-APJ exists in placenta, and the level of mRNA transcripts for Apelin increased may be associated with the need to increase blood priming volume in placenta in late trimester of gestation. Apelin and and its recptor APJ are positively expressed in the endothelial cells of human umbilical veins. However this expression decreased in umbilical artery. More active compounds may exist in umbilical artery blood to meet the need of fetal growth, and this causes that difference.PartⅡStudy on Apelin and nitric oxide in the pathogenesis of Hypertensive disorder complicating pregnancyObjective To understand the possible role of Apelin and the relationship with nitric oxide (NO) in the pathogenesis and progression of HDCP. The expression of Apelin peptide and its acceptor in the placental tissue of normal pregnancy and HDCP were detected. methods Select 36 cases from the pregnant-woman of HDCP during 36～41 gestational weeks,including 10 cases of Gestational hypertension(GH) and 11 cases of Mild-Preeclampsia(MP) and 15 cases of Severe-preeclampsia(SP). Meanwhile, selected 15 normal pregnant women as the control group. The expression of Apelin-36 and its acceptor APJ-protein was detected with immunohistochemistry technique. The expression of Apelin and its acceptor APJ mRNA was detected with fluorescent quantitation RT-PCR. The level of NO was detected with nitrate reductase technique and the activity of NOS was detected with spectrophotometry. Results (1)The expression site of Apelin-36 and its acceptor APJ was not obviously different between the placental tissue of HDCP and normal pregnancy. The main expression site of Apelin-36 and its acceptor APJ located in the villous syncytiotrophoblasts. (2)The expression of Apelin-36 mRNA and Apelin-36 in the placental tissue of HDCP was the significantly lower than that in the control group (P <0.01), and it decreased gradually according to GH, MP, SP(P <0.01, P <0.05). The levels of expression of Apelin-36 mRNA and Apelin-36 were declined in each group. The levels of Apelin-36 mRNA and Apelin-36 expression in the group of MP and SP were the notable lower than the group of GH(P <0.01, P <0.01). The levels of Apelin-36 mRNA and Apelin-36 expression has statistical significance between the group of MP and SP (P <0.05). (3)The level of APJ mRNA and its protein expression in HDCP showed no statistical significance compared with the control group(P <0. 05). (4) The level of NO from the placenta HDCP was significant lower than that of the control group (P <0.01). and they were decreased gradually according to GH, MP ,SP (P<0.01, P <0.05). The levels of NO in the placenta tissue of MP and SP were lower than the that in GH(P <0.01, P<0.01). There was significant difference between the MP and SP (P <0.05). There was no significant difference between GH and the control group (P >0.05). (5) The level of NOS in HDCP group was notable lower than that in the control(P <0.01). and they were decreased gradually according to GH, MP,SP (P < 0.01, P < 0.05). The levels of NOS in placenta tissue of MP and SP were lower than the that in GH (P <0.05), There was no significant difference between GH and the control group (P >0.05). (6)In HDCP group, the level of NO and the activity of NOS were positive correlation with Apelin mRNA. The coefficient correlation were 0. 823, 0.901 respectively (P <0.01, P <0.01). Conclusoin (1) Both Apelin and APJ receptor are expressed in placental villus of HDCP. In the placenta tissue of HDCP, the expression of Apelin mRNA and Apelin-36 is lower, and they are correlated with the condition of the HDCP. Apelin may play a important role in the pathogenesis of HDCP. (2) The level of NO and the activity of NOS were significant lower in HDCP, which were positive correlated to Apelin mRNA. These hints proved that the level of Apelin in placenta tissue from HDCP was lower, which results in lower activity of NOS, contributed to the synthesis of NO. The lower NO aggravates the placenta ischemic and hypoxia, indicating that this may be involved in the progression of HDCP.PartⅢEffect of Apelin on production of NO, HCG and activity of NOS in human placental villusObjective To investigate the effects of Apelin on nitric oxide synthase (NOS) activity and nitric oxide (NO) production in placental villus. Methods The experiments were performed in human term placental villus in vitro. The cultured villus were treated with Apelin-36 and angiotensin II(Ang II).ELISA was used to detectβ-HCG levels in the medium of the cultures. The amount of NO in placental villus was detected by the nitrate reductase technique. The NOS activity in placental villi was measured with spectrophotometric assay. Resultsβ-HCG levels in the medium of placental villus in each groups were not different with that in control group.(P>0.05). It suggested that the production ofβ-hCG from human term placental villus was not affected by Apelin-36. NO levels in the villus with Apelin-36 were significantly higher than that in control group(P <0.01). In comparison with 10ng/ml Apelin group, the NO levels were significantly higher in 50ng/ml and 250ng/ml Apelin-36 group(P <0.01). There was no significant difference in NO level between 50ng/ml and 250ng/ml Apelin group(P>0.05). Total nitric oxide synthase (tNOS) activities in placental villus in each groups cultured with Apelin were significantly higher than that in control group(P<0.01), and endothelial NOS (eNOS) activities were mainly higher in all concentration groups(P<0. 01), however, the inducible NOS(iNOS) activities were not altered (P>0. 05). the level of NO and the activity of NOS were significantly decreased in the villus induced by AngII(P < 0.01)and Apelin could prevent this decrease(P < 0.05). Conclusion The production ofβ-hCG from human term placental villus was not affected by Apelin-36. Apelin-36 may increase the level of NO by the enhancement of the activity of eNOS in placental villus. AngII can Reduced activity of NOS and production of NO, which could be prevented with Apelin. Apelin may play its possible biological role by inducing production of NO in placenta.