| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 91 === Angiopoietin-1 (Ang-1), an angiogenic factor, existed in the placental tissues including cytotrophoblasts and syncytiotrophoblasts. In endothelium, angiopoietin-1, an anti-permeability factor, stabilizes the vessels and facilitate endothelial survival and vascular maturation. Therefore, Ang-1 may facilitate placental development by promote trophoblast growth and support fetoplacental vascular development and stabilization in placental tissue, but the functional roles of Ang-1 in placental formation are still unclear. Vascular endothelial-cadherin (ve-cad), a 130 kD Ca2+ -depend adhesion molecules, exclusively and constitutively expressed at interendothelial junctions. Ve-cad also can be detected in the placental tissues including intermediate and terminal villi. The increased expression of ve-cad on intercellular junctions decreases capillary permeability by increasing vessel integrity. In addition to ve-cad, Ang-1 and its receptor Tie-2 are also detected in human and primate placental tissues including syncytiotrophoblasts, cytotrophoblast, and perivascular cells. Even though ve-cad, Ang-1 and its receptor Tie-2 exist in cytotroblasts of human term placenta, no current studies further examine whether Ang-1 can modulate the expression of ve-cad. Therefore, we examined whether Ang-1 could increase the expression of ve-cad. First of all, we characterized the expression of Ang-1 and it receptor (Tie-2) throughout gestation and examined the dose-effect of Ang-1 on ve-cad expression. As our data indicated, its receptor was highly expressed in the G18 placenta. Ang-1 at lower concentrations (1-10 ng/ml) increased the expression of ve-cad but that at higher concentrations did not. Accordind our data indicate that the ve-cad expression may regulate by Ang-1 in placental tissues. Secondly, we further investigate how Ang-1 increased ve-cad. It has been reported that Ang-1 can increase NO production by activation of PI3K/AKT pathway. In smooth muscle, the most important target for NO is soluble guanylyl cyclase (sGC). Activation of sGC by NO increases the production of cGMP. Binding of cGMP to cGMP-dependent protein kinase (PKG) type Ⅰα causes PKG autophosphorylation and triggers PKG-mediated cellular responses such as sooth muscle relaxation. The induction of cGMP-dependent protein kinase (Type 1α PKG) phosphorylation by cGMP also could modulate gene expression through cellular signal transduction pathway, foe example, the ERK/MAP kinase pathway. In recent studies, the NO-cGMP-PKG system also considered that may regulate capillary permeability by modulating intercellular junctions. Therefore, to examine whether the increased expression of ve-cad by Ang-1 was through a NO-cGMP-dependent pathway, we first examined the influences of Ang-1 on NO production and then the expression of ve-cad. As our data indicated that Ang-1 at 1.0 ng/ml increases NO production and facilitate ve-cad expression. The increased NO and ve-cad by Ang-1 were reversed by L-NAME. These data suggest that Ang-1 can modulate the expression of ve-cad by change the NO production. To comfirm cGMP is involved in Ang-1 induced ve-cad expression signal transduction pathway, we measure the PKG phosphorylation. The phosphorylated Type 1 PKG was concentrated by immunoprecipitation and confirmed by Western blot analysis. PKG phosphorylation was measured by immobilized affinity chromatography (IAC). Ang-1 at 1.0 ng/ml increased the abundance of phospho-PKG but at higher concentrations (>1.0 ng/ml) did not. IAC data was supported our findings from western blotting analysis. We also use MS/MS assay to find out other signal pathway which are involved in Ang-1 induced ve-cad expression through a NO-cGMP dependent pathway. In conclusion, Ang-1 increased the expression of ve-cad may through a NO-cGMP dependent signal pathway. And then Ang-1 may play an important role in placental formation by regulate the expression of ve-cad.
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