The Role of Placental Maternal Vascular Underperfusion in Neonatal Pulmonary Hypertension

项目摘要 新生儿肺动脉高压（PH）是一种严重的疾病，影响多达三分之一的早产儿 支气管肺发育不良（BPD）。该实体被称为BPD相关PH（BPD-PH），完全 在1-3个月大时发病，死亡风险增加四倍。BPD-PH的幸存者 长期和反复住院，并有慢性心肺和代谢问题的风险。 我们致力于制定有针对性的策略，以预防早产儿的BPD-PH。两个有希望的 治疗靶点是组织学标志物，胎盘母体血管灌注不足（MVU）和 在出生时循环的中间脐带血来源的胎儿单核细胞（iMNC）。我们的核心假设是胎儿 iMNCs被胎盘MVU的慢性胎儿缺氧不利地编程，并有助于早期延迟的MVU。 肺血管生成导致BPD-PH。在目标#1中，我们将阐明MVU导致BPD-PH的机制， 新生儿血管生成延迟。使用胎儿生长受限（FGR，出生体重<第5百分位数）作为 慢性胎儿缺氧，我们将通过流式细胞术分析100例脐带血iMNC VEGFR 1表达， 来自以下婴儿组的样品：1）具有FGR的早产MVU; 2）不具有FGR的早产MVU; 3）具有FGR的早产MVU。 无MVU伴FGR; 4）早产儿无MVU无FGR（早产儿对照）; 5）足月儿无MVU无FGR（足月儿对照）。在 分离的iMNCs，我们将使用PCR阵列比较这些组的基因表达谱， 由MVU驱动的新基因靶点和途径。在目标#2中，我们将确定围产期机制， MVU暴露的胎儿iMNC有助于延迟新生儿肺血管生成：在暴露于MVU的培养iMNC中， 实验性低氧和高氧，我们将评估VEGFA和VEGFR 1基因和蛋白表达。我们 将进行趋化性试验，以比较iMNC迁移对胎盘外源性治疗的反应。 生长因子（PlGF）和粒细胞集落刺激因子（GCSF），我们将比较iMNC表达 气管插管早产儿支气管肺泡灌洗液中VEGFR 1的表达。最后，在目标#3中，我们将移植 将来自5个患者组的iMNC植入新生人GM-CSF敲入（人源化）小鼠中，并追踪肺 慢性高氧暴露后的组织学和PH参数（85%氧气x 14天）。我们将测试 假设来自非MVU、非FGR婴儿（对照）的脐带血衍生的iMNC的移植将有助于 预防BPD-PH。

PROJECT SUMMARY Neonatal pulmonary hypertension (PH) is a serious condition that affects up to one third of premature infants with bronchopulmonary dysplasia (BPD). This entity, known as BPD-associated PH (BPD-PH), is fully established by 1-3 months of age and incurs a four-fold increased risk of death. Survivors of BPD-PH have prolonged and recurrent hospitalizations, and are at risk for chronic cardiopulmonary and metabolic problems. We are committed to developing targeted strategies to prevent BPD-PH in premature infants. Two promising therapeutic targets are the histologic marker, placental maternal vascular underperfusion (MVU) and the intermediate cord blood-derived fetal monocytes (iMNC) that circulate at birth. Our central hypothesis is that fetal iMNCs are adversely programmed by the chronic fetal hypoxia of placental MVU, and contribute to early delayed lung angiogenesis leading to BPD-PH. In Aim #1, we will elucidate the mechanism by which MVU leads to delayed neonatal angiogenesis. Using fetal growth restriction (FGR, birth weight <5th percentile) as the proxy for chronic fetal hypoxia, we will compare iMNC VEGFR1 expression via flow cytometric analysis of 100 cord blood samples from the following groups of infants: 1) preterm MVU with FGR; 2) preterm MVU without FGR; 3) preterm no MVU with FGR; 4) preterm no MVU no FGR (preterm control); 5) term no MVU no FGR (term control). In isolated iMNCs, we will use PCR Array to compare the gene expression profiles of these groups and identify novel gene targets and pathways driven by MVU. In Aim #2 we will identify the perinatal mechanisms by which MVU-exposed fetal iMNCs contribute to delayed neonatal lung angiogenesis: In cultured iMNCs exposed to experimental hypoxia and hyperoxia, we will evaluate VEGFA and VEGFR1 gene and protein expression. We will perform chemotaxis assays to compare iMNC migration in response to exogenous treatment with placental growth factor (PIGF) and granulocyte colony stimulating factor (GCSF), and we will compare iMNC expression of VEGFR1 in bronchoalveolar lavage fluids from intubated preterm infants. Lastly, in Aim #3 we will transplant iMNCs from the 5 patient groups into newborn human GM-CSF knock-in (humanized) mice, and follow the lung histology and PH parameters after chronic hyperoxia exposure (85% oxygen x 14 days). We will test the hypothesis that transplantation of cord blood-derived iMNCs from non-MVU, non-FGR infants (controls) will aid in the prevention of BPD-PH.