Study On The Mechanisms Of Urocortin Vasodilation In Placental Vessel And The Regulation Of Urocortin In Human Trophoblast Cells

During pregnancy, fetal growth is dependent on placenta as a media by which nutrition and oxygen can be transported from mother to fetus. Successful blood exchange in placenta requires adequate fetal - placental circulation. As the tetal vessels of the human placenta were not innervated, control of blood flow in this vascular bed is partly dependant on locally produced and circulating vasoactive factors. Previous studies have demonstrated that placental vasculature appears to be almost maximally dilated under conditions of basal pressure. Once mother or fetal perfused condition was changed, placental function was damaged. These would induce preeclampsia and/or fetal intrauterine growth restriction (FGR) that lead to harmful outcome of mother and fetus.Urocortin (UCN) is a relatively new member of corticotropin - releasing hormone (CRH) family, which has been found in the human placenta and de-cidua. In vitro, exogenous UCN is a potent vasodilator of the fetal -placental vasculature, and its effect was 25 times more potent than CRH and mediated by activation of CRH 2β receptors. Florio et al. have shown that CRH - R2β is strategically located in villous blood vessels. These results indicate a possible physiological role for UCN in the modulation of human fetal placental vascular tone by causing vasodilatation. The mechanism by which UCN causes vasodilatation in the placenta is presently unknown. Many studies on the mechanism of UCN - induced vasodilatation has been reported, but the results are inconsistent, including endothelial independent - or/and depedent - pathways. These data suggest that endothelium may have different role in various vessels. It isknown that the placental vasodilatation of CRH was mediated by nitric oxide (NO) cGMP. The mechanism of UCN dilating placental vessels is under investigation.Untill now, very little is known regarding regulation of expression of UCN in intrauterine tissue. NO is an important regulator of neuronal and endocrine function, in particular hormone release. It plays a role in both stimulation and inhibition of hormone release, depending upon the cell and systems. The human placenta and its syncytiotrophoblast (STB) cells are known to contain and release NO and NOS, and UCN has been localized to the STB layer of the placental villus. It was reported that NO and glucocoticoid were the important reg(?)ators of CRH expression in placenta. Whether NO and glucocoticoid may be involved in the regulation of UCN release in the placenta has not previously been reported. Therefore, the mechanisms are of considerable interests.Preeclampsia affects about 5-8% of pregnancies in the northemeast of China, requires intense monitoring and clinical supervision, and is potentially threatening to mother and fetus. The pathophysiology of preeclampsia is much more than the increased blood pressure and altered renal function that facilitale diagnosis. Preeclampsia is associated with umbilical - placenta! vascular insufficiency and locally ischemia and hypoxia, so that mother and fetus are in stress situation. Although numerous studies have been carried out on placental CRH expression in preeclampsia, the expression of UCN is unknown. UCN is found so modulate placental vessel and be protective against ischemia /hypoxia in cardiac myocytes. Therefore, we hypothesize that UCN may have some role in preeclampsia. The purpose of this study was to investigate and compare expression levels of UCN and its receptor CRH - R2β in placentas from women with preeclampsia.Accordingly, the purposes of this study are: (1) To determine whether UCN can affect the release of nitric oxide and expression of nitric oxide synthase in the human umbilcial vein endothelial cells; (2) To investigate the effects of nitric oxide and glucocoticoid on UCN expression of syncytiotrophoblasts; (3) To examine the level of UCN and corticotrophin - releasing hormone receptor type 2β in the placentas of preeclampsia.Methods1. The study on the mechanisms of the vasodilatation of UCN in the fetal -placental circulation.We established the method of primary cultured human umbilical vein endothelial cells ( HUVEC). After cocultured with three kinds concentrations of UCN (0. lmmoI/L - 10nmol/L) , We evaluated NO production and iNOS and eNOS mRNA and protein expressions. NOS mRNA expression was detected by reverse transcription polymerase chain reaction (RT - PCR) , and the protein expression was determined by western blotting. The concentrations of NO secreted were detected by the means of nitrate reductase. The medians of 3 replicates were used for two - tailed paired - samples T test or one - way ANOVA. A P < 0.05 was considered statistically significant.2. The study of nitric oxide and glucocoticoid regulating the UCN expression in the trophoblast cells,We isolated and cultured trophoblasts and placenta explants in vitro. More than 90% trophoblasts were obtained by improved means. The trophoblasts were cocultured with sodium nitroprusside as nitric oxide donor at 3 kinds of concentration (1 - 100μmol/L), endotrogene nitric oxide synthase inhibitor L -NAME and a manmade synthesis glucocoticoid (dexamethasone) for 24h. Im-munoreactive UCN were measured by immunocytochemistry, and UCN mRNA was determined by RT - PCR. The results were analyzed by t test and Kruskal -Wallis test. A P<0.05 was considered statistically significant.3. The level of UCN and corticotropin - releasing hormone receptor type 2βin the placentas of preeclampsia.A case - control study. Thirty women with preeclampsia (10 mild, 20 severe cases) and 20 women with normal pregnancy (NP) were enrolled as case group and control group, respectively. Their placentas were collected immediately after the neonate were deliveried. The level of UCN mRNA and CRH - R2β mRNA were determined by semi - quantitative reverse transciptase polymerase chain reaction (RT -PCR) , and the UCN peptide were detected by immunohis-tochemistry. The results were analyzed by t test and Mann - Whitney test. A P <0.05 was considered statistically significant.Results1. The study on the mechanisms of the vasodilatation of UCN in the fetal -placental vessels.(1) NO production were induced by stimulation with UCN and CRH in HUVEC, and presented after 4 -8h, with the feature of time - and concentration - dependence,(2) UCN and CRH increased iNOS mRNA and protein expression, but no effects on eNOS expression,(3) The effects of CRH on HUVEC NO production and iNOS expression were reduced in comparison with equimolar UCN.(4) The effects of UCN and CRH were decreased by CRH receptor inhibitor.2. The study of nitric oxide and glucocoticoid on regulating the UCN expression in the trophoblast cells.(1) SNP, NO donor significantly reduced UCN expression.(2) Production of UCN was augumented by the NO synthase competitive inhibitor L-NAME.(3) The expression of UCN in syncytiotrophoblast was down -regulated by dexamethasone.3. The level of UCN and corticotrophin - releasing hormone receptor type 2β in the placentas of preeclampsia.(1) The UCN mRNA expression was significantly higher in preeclampsia when compared with normal pregnancies.(2) There was no significantly difference between the expression of CRH ~ R2β mRNA of preeclamsia and of normal pregnancies,(3) The result of immuriohistochemistry demonstrated thai UCN was predominantly localized in syntrophoblast with weak staining of cyntrophoblast and capillaries. UCN of preeclampsia in syntrophoblast was significantly higher than...