Effects of Nitric Oxide on Fetoplacental Angiogenesis

描述（申请人提供）：该申请人的长期目标是确定介导胎盘血管生成的细胞和分子机制，并最终控制胎盘血流和功能。我们将使用羊胎胎盘动脉内皮（OFPAE）和人胎盘微血管（HPME）细胞系进行研究。选择这些胎盘内皮细胞是因为人类和绵羊怀孕期间胎盘血管的急剧生长与脐血流量的急剧增加有关，这对胎儿的生长和生存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性一氧化氮（NO，一种有效的血管舒张剂）生成的关键因子。bFGF和VEGF的作用由蛋白激酶（如Akt和p38 MAPK）和蛋白磷酸酶（如PP2A和PP2B）紧密介导。假设在胎盘中，bFGF-和vegf诱导的血管生成部分通过激活PI3K/Akt和p38 MAPK信号通路以及抑制PP2A和PP2B来调节，从而增加NO的产生，从而促进胎儿胎盘血管生成。将使用特性良好的OFPAE细胞系来解决特定目的。我们将确定：AIM 1 .如果bFGF和VEGF刺激的细胞增殖和迁移在一定程度上是通过PI3K/Akt和p38 MAPK通路的激活来介导的，在特定PI3K或p38 MAPK抑制剂存在的情况下，使用bFGF或VEGF处理细胞，使用细胞增殖和迁移试验，Western分析和激酶活性试验；目的二世。如果bFGF和VEGF部分通过抑制PP2A和PP2B活性激活Akt和p38 MAPK，则在特定蛋白磷酸酶抑制剂存在的情况下，使用Western分析、蛋白磷酸酶和激酶活性测定、细胞增殖和迁移测定，用bFGF或VEGF处理细胞；和AIM III。如果bFGF-和VEGF通过激活PI3K/Akt和p38 MAPK以及抑制PP2A和PP2B来提高NO的产生，调节eNOS磷酸化。一个额外的特异性目标IV将使用HPME细胞系来确认从OFPAE细胞中获得的关键观察结果。这些研究将收集有关胎盘血管生成机制的重要信息。

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.