Effects of Nitric Oxide on Fetoplacental Angiogenesis

描述（由申请人提供）：本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。我们将使用绵羊胎儿胎盘动脉内皮细胞（OFPAE）和人胎盘微血管（HPME）细胞系用于此目的。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，这对胎儿生长和生存以及新生儿出生体重和生存能力至关重要。血管生成因子bFGF和VEGF是调节血管生成和产生内皮一氧化氮（NO;一种有效的血管扩张剂）的关键因子。bFGF和VEGF的作用由蛋白激酶（即Akt和p38 MAPK）和蛋白磷酸酶（即PP 2A和PP 2B）紧密介导。假设在胎盘中bFGF和VEGF诱导的血管生成部分地通过激活PI 3 K/Akt和p38 MAPK信号通路以及抑制PP 2A和PP 2B来调节，这反过来增加NO的产生，从而促进胎儿胎盘血管生成。3将使用充分表征的OFPAE细胞系解决特定目的。我们将确定：目标一。如果bFGF-和VEGF-刺激的细胞增殖和迁移部分地通过在特异性PI 3 K或p38 MAPK抑制剂的存在下用bFGF或VEGF处理细胞而经由PI 3 K/Akt和p38 MAPK通路的活化介导，使用细胞增殖和迁移测定、蛋白质分析和激酶活性测定; AIM II.如果bFGF和VEGF部分地通过抑制PP 2A和PP 2B活性来激活Akt和p38 MAPK，则通过在特异性蛋白磷酸酶抑制剂存在下用bFGF或VEGF处理细胞，使用Western分析、蛋白磷酸酶和激酶活性测定以及细胞增殖和迁移测定;以及AIM III。bFGF-1和VEGF通过激活PI 3 K/Akt和p38 MAPK以及抑制PP 2A和PP 2B，调节eNOS磷酸化而增加NO的产生。额外的特定目标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。这些研究将为胎盘血管生成的调控机制提供重要信息。

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.