Reproductive Phenotype of Adrenomedullin2 (ADM2) knockout mice

摘要 肾上腺髓质素2(Adrenomedullin2，ADM2)是一种新型的促进滋养细胞侵袭的降压肽。较低的ADM2 水平与妊娠并发症有关，如自然流产、胎儿生长受限 (FGR)和先兆子痫(PE)。Pilot数据显示，ADM2基因的去除会引起类似PE的症状 如血压升高和血清可溶性FMS样酪氨酸激酶-1(sFlt-1)水平升高。 1)，以及怀孕ADM2基因敲除(ADM-/-)小鼠的早产和幼崽死亡率增加。这 数据有力地支持了我们发表的研究表明：1)胎盘和血清中ADM2水平较高 人类妊娠胎盘发育的侵袭性阶段，2)ADM2介导的侵袭性增加 妊娠早期绒毛外滋养层细胞(EVCTs)的能力 妊娠中期妊娠妇女胎盘ADM2水平及羊水ADM2水平下降 发展体育。此外，虽然一氧化氮(NO)的产生和合成和分泌增加 胎盘sFlt-1可导致PE的内皮功能障碍，阻断妊娠大鼠的ADM2减少 胎盘NO/基质金属蛋白酶系统伴发FGR和胎盘受损 脉管系统。此外，下调滋养层细胞中ADM2的表达可减少MMP2、MMP9的表达 和eNOS基因的表达。因此，根据上述报道和试点数据，我们的中心假设是 “整体消融ADM2会导致类似于人类先兆子痫的妊娠并发症，而且，它 通过NO/MMPs系统发挥作用。这一假设将通过三个具体目标进行检验。具体目标--1： 评估消融术对胎儿胎盘生长、循环中一氧化氮、17β-雌二醇、 孕酮和怀孕期间的血压调节；并确定是否没有补充剂可以挽救 在ADM2-/-动物中，早期分娩和增加幼崽死亡率，特定目标-2：确定是否消融 小鼠的ADM2增强子宫收缩能力，并增强对以下刺激的收缩反应 催产素和前列腺素-F2α，通过其受体表达增加和特异性靶-3：识别1) 下游信号机制参与了ADM2在EVCT中的侵袭作用，2)评估ADM2是否 补充剂可以增加来自PE的EVCT的侵袭能力，据报道，这些细胞表现出较少的侵袭能力 侵袭性表型与正常妊娠的比较。 由于大多数对人类的研究都受到其内在关联性的限制，我们建议 方法将使我们能够在一系列的研究中描述妊娠期间ADM2的内源性功能 力学实验及其在PE病理生理学中的潜在作用。这些研究可能会填补 胎盘功能调节剂的知识空白可能具有巨大的设计和开发潜力 对高危妇女的病理性妊娠实施有效的预防/治疗。

Abstract Adrenomedullin2 (ADM2) is a novel hypotensive peptide that promotes trophoblast invasion. Lower ADM2 levels are associated with pregnancy complications such as spontaneous abortion, fetal growth restriction (FGR) and preeclampsia (PE).Pilot data shows that ablation of ADM2 gene causes PE-like manifestations such as elevated blood pressure (BP) and increased serum levels of soluble fms-like tyrosine kinase-1 (sFLT- 1), along with early parturition and increased pup mortality in pregnant ADM2 knockout (ADM-/-) mice. This data strongly supports our published studies showing: 1) Higher placental and serum levels of ADM2 during the invasive phase of placental development in human pregnancy, 2) ADM2 mediated increases in the invasive capacity of 1st trimester extravillous trophoblast cells (EVCTs) in human pregnancy, 3) decreased serum and placental ADM2 levels in PE and decreased ADM2 in amniotic fluid of pregnant women in 2nd trimester who develop PE. Further, while impaired nitric oxide (NO) production and elevated synthesis and secretion of placental sFLT-1 is reported to cause endothelial dysfunction in PE, blocking ADM2 in rat pregnancy decrease placental NO/ matrix metallo-proteinase (MMP) system accompanied with FGR and impaired placental vasculature. In addition, knockdown of ADM2 in trophoblast cells decreases the expression of MMP2, MMP-9 and eNOS mRNA. Therefore, based on above mentioned reports and pilot data, our central hypothesis is that “Global ablation of ADM2 results in pregnancy complications mimicking preeclampsia in human and that, it functions through NO/MMP system “. This hypothesis will be tested using three specific aims. Specific Aim-1: Assess the effect of ADM2 ablation on feto-placental growth, circulatory levels of nitric oxide, 17β-estradiol, progesterone and blood pressure regulation during pregnancy; and identify if NO supplementation can rescue early parturition and increased pup mortality, in ADM2-/- animals, Specific Aim-2: Determine if ablation of ADM2 in mice advances uterine contractility and potentiates uterine contractile responses to stimuli such as oxytocin and prostaglandin-F2α, through increase in their receptor expression and, Specific Aim-3: Identify 1) downstream signaling mechanism involved in the invasive function of ADM2 in EVCTs and, 2) assess if ADM2 supplementation can increase the invasive capacity of EVCTs from PE, which are reported to exhibit less invasive phenotype compared to those from normal pregnancy. Since the majority of studies in humans are limited by their inherent correlative nature, our proposed approach will allow us to characterize the endogenous functions of ADM2 in pregnancy in a series of mechanistic experiments and its potential role in the pathophysiology of PE. These studies could potentially fill the gaps in the knowledge of mediators of placental functions that may hold great potential for the design and implementation of an effective prevention/treatment for pathological pregnancies in women at high risk.