Chronic hypoxia, AMPK activation and uterine artery blood flow

项目摘要 高海拔居住（HA，>2500 m）增加先兆子痫（PreE）和宫内生长的频率 限制性生长（IUGR）3倍。由于缺氧是一种常见的原因，HA研究具有独特的定位，以评估 缺氧的机械作用，发现新的治疗方法，并缓解“几乎完全缺乏药物” PAR-13-389中指出的“产科适应症的发展”。腺苷一磷酸激酶（AMPK）是一种 在缺氧条件下影响血管生长和功能的代谢平衡调节剂。我们有 表明AMPK在调节人子宫动脉（UtA）血流和胎儿发育中起重要作用。 在HA生长，AMPK活化在分离的鼠UtA中具有血管舒张作用， 缺氧我们的中心假设是AMPK激活促进子宫胎盘循环中的血管舒张 以提高UtA血流量并改善HA处的胎儿生长。因为缺氧会导致 表观遗传变化改变了AMPK活性和胎儿生长调节基因的表达，我们提出 DNA甲基化会影响AMPK介导的过程。我们提出了新的小鼠和人类数据， 表明缺氧a）相对于总AMPK水平升高胎盘磷酸化（P）-， AMPK靶向胸主动脉和胎盘中的总蛋白水平; B）增加关键蛋白的表达， 在UtA和胎盘中激活AMPK的酶;和c）改变AMPK的甲基化-表达关系。 对胎儿生长很重要的信号基因。我们将通过确定以下参数来检验我们的中心假设： 1.目的1：探讨HA妊娠对AMPK信号的影响及其与UtA血流和胎儿生长发育的关系。 人类的成长。我们将招募102名低海拔（LA，1600 m，n=53）或HA（3000 m， n=49）;纵向测量UtA血流和胎儿生物统计学;并确定AMPK的激活， 上游调控因子和下游靶点，相关基因的表达水平和DNA甲基化 外周血单核细胞（PBMC）中的基因和AMPK调节剂的血浆水平。 2.目的2：研究缺氧对人子宫肌层动脉（MA）和胎盘AMPK活化的影响，以及缺氧对人子宫肌层动脉（MA）和胎盘AMPK活化的影响。 MA血管反应性。在参与目标1的择期剖腹产女性（n=19/海拔）中，我们将 获得子宫肌层活组织检查和胎盘以确定a）活化、表达和DNA甲基化 AMPK的状态、其完善的调节剂和下游靶点; B）HA妊娠对AMPK的影响; MA血管反应性;和c）缺氧是否增强MA中AMPK激活的血管舒张作用。 3.目的3：AMPK在缺氧时对子宫胎盘血流和胎儿生长的调节作用 对小鼠将用AMPK激活剂（AICAR）处理饲养在海平面（SL）或HA的怀孕小鼠， 抑制剂（化合物C）或媒介物（对照）用于测定缺氧的单独和组合作用 和AMPK激活对UtA、胎盘和胎儿组织中AMPK信号传导的影响; UtA血管反应性和 血流和胎儿生长。

Project Summary High altitude residence (HA, >2500 m) increases the frequency of preeclampsia (PreE) and intrauterine growth restriction (IUGR) 3-fold. Since hypoxia is a common cause, HA studies are uniquely positioned to evaluate the mechanistic role of hypoxia, discover new treatments, and alleviate the “almost complete lack of drug development for obstetric indications” noted in PAR-13-389. Adenosine monophosphate kinase (AMPK) is a regulator of metabolic homeostasis that also affects vascular growth and function under hypoxia. We have shown that AMPK plays important roles in the regulation of human uterine artery (UtA) blood flow and fetal growth at HA, and that AMPK activation has vasodilator effects in isolated murine UtA that are potentiated by hypoxia. Our central hypothesis is that AMPK activation promotes vasodilation in the uteroplacental circulation in response to hypoxia to raise UtA blood flow and improve fetal growth at HA. Because hypoxia leads to epigenetic changes that alter the expression of genes regulating of AMPK activity and fetal growth, we propose that DNA methylation influences these AMPK-mediated processes. We present new murine and human data to show that hypoxia a) raises placental phosphorylated (P)- relative to total AMPK levels and the P- as well as the total protein levels of AMPK targets in thoracic aorta and placenta; b) increases the expression of key enzymes activating AMPK in UtA and placenta; and c) alters methylation-expression relationships of AMPK- signaling genes important for fetal growth. We will test our central hypothesis by determining: 1. In Aim 1, the effect of HA pregnancy on AMPK signaling and its relationship to UtA blood flow and fetal growth in humans. We will recruit 102 healthy residents of low altitude (LA, 1600 m, n=53) or HA (3000 m, n=49); measure UtA blood flow and fetal biometry longitudinally; and determine the activation of AMPK, its upstream regulators and downstream targets, the expression levels and DNA methylation of relevant genes in peripheral blood mononuclear cells (PBMCs), and plasma levels of AMPK regulators. 2. In Aim 2, the effects of hypoxia on AMPK activation in human myometrial artery (MA) and placenta, and on MA vasoreactivity. In women participating in Aim 1 who deliver by elective C-section (n=19/altitude), we will obtain myometrial biopsies and placentas to determine a) the activation, expression and DNA methylation status of AMPK, its well-established regulators and downstream targets; b) the effect of HA pregnancy on MA vasoreactivity; and c) whether hypoxia potentiates vasodilator effects of AMPK activation in MA. 3. In Aim 3, the role of AMPK in regulating uteroplacental blood flow and fetal growth in response to hypoxia in mice. Pregnant mice housed at sea level (SL) or HA will be treated with the AMPK activator (AICAR), inhibitor (Compound C), or vehicle (control) for determining the separate and combined effects of hypoxia and AMPK activation on AMPK signaling in UtA, placental and fetal tissues; UtA vascular reactivity and blood flow; and fetal growth.