Effects of Nitric Oxide on Fetoplacental Angiogenesis

描述(申请人提供)：本申请者的长期目标是确定调节胎盘血管生成的细胞和分子机制，并最终控制胎盘的血流和功能。我们将使用绵羊胎儿胎盘动脉内皮细胞(OFPAE)和人胎盘微血管(HPME)细胞株来实现这一目的。之所以选择这些胎盘内皮细胞，是因为在人和绵羊妊娠期间，胎盘血管的急剧增长与脐血流量的急剧增加有关，而脐血流量对胎儿的生长和存活以及新生儿出生体重和存活率至关重要。血管生成因子bFGF和VEGF是调节血管生成和内皮型一氧化氮(NO；一种有效的血管扩张剂)产生的关键因素。碱性成纤维细胞生长因子和血管内皮生长因子的作用是由蛋白激酶(即Akt和p38MAPK)和蛋白磷酸酶(即PP2A和PP2B)紧密地介导的。该假说认为，在胎盘中，bFGF和VEGF诱导的血管生成部分是通过激活PI3K/Akt和p38MAPK信号通路以及抑制PP2A和PP2B来调节的，这反过来又增加了NO的产生，从而促进了胎儿胎盘的血管生成。3个特定的目标将使用特征良好的OFPAE细胞系来解决。我们将通过细胞增殖和迁移分析、Western分析和激酶活性分析确定bFGF和VEGF刺激的细胞增殖和迁移是否部分通过激活PI3K/Akt和p38 MAPK通路而介导，通过使用特定的PI3K或p38 MAPK抑制剂用bFGF或VEGF处理细胞；If bFGF-和VEGF通过激活PI3K/Akt和p38MAPK，抑制PP2A和PP2B，促进NO的产生，调节eNOS的磷酸化。另一个特定目标IV将使用HPME细胞系来证实从OFPAE细胞中观察到的关键结果。这些研究将收集关于胎盘血管生成调控机制的重要信息。

DESCRIPTION (provided by applicant): The long-term goals of this applicant are to determine the cellular & molecular mechanisms mediating placental angiogenesis & ultimately control placental blood flow and function. We will use Ovine Fetal Placental Artery Endothelial (OFPAE) & Human Placental Microvascular (HPME) cell lines for this purpose. These placental endothelial cells were chosen because dramatic placental vascular growth during human & ovine pregnancy is associated with dramatic increases in umbilical blood flow, which is critical for fetal growth & survival as well as neonatal birth weights and survivability. The angiogenic factors, bFGF & VEGF, are key factors regulating angiogenesis & production of endothelial nitric oxide (NO; a potent vasodilator). Actions of bFGF & VEGF are tightly mediated by proteins kinases (i.e. Akt & p38 MAPK) & protein phosphatases (i.e. PP2A & PP2B). The hypothesis is that in the placenta bFGF- & VEGF-induced angiogenesis is modulated in part via activation of the PI3K/Akt and p38 MAPK signal pathways as well as inhibition of PP2A and PP2B, which in turn increase NO production, so promoting fetal placental angiogenesis. 3 Specific Aims will be addressed using the well-characterized OFPAE cell line. We will determine: AIM I. if bFGF- & VEGF-stimulated cell proliferation & migration are mediated in part via activation of the PI3K/Akt & p38 MAPK pathways by treating cells with bFGF or VEGF in the presence of specific PI3K or p38 MAPK inhibitors, using cell proliferation & migration assays, Western analysis, and kinase activity assays; AIM II. if bFGF & VEGF activate Akt & p38 MAPK in part via inhibiting PP2A & PP2B activities, by treating cells with bFGF or VEGF in the presence of the specific protein phosphatase inhibitors, using Western analysis, protein phosphatase & kinase activity assays, and cell proliferation & migration assays; and AIM III. If bFGF- & VEGF elevate NO production via activation of PI3K/Akt & p38 MAPK as well as inhibition of PP2A & PP2B, regulating eNOS phosphorylation. An additional Specific Aim IV will use the HPME cell line to confirm the key observations made from OFPAE cells. These studies will glean important information on the mechanisms regulating placental angiogenesis.