Development of novel therapeutic approaches for treatment of Alveolar Capillary Dysplasia

项目摘要。肺泡毛细血管发育不良伴肺静脉错位（ACDMPV）是一种 新生儿和婴儿的致命性先天性疾病，由于呼吸功能降低而导致呼吸功能不全 肺泡毛细血管数量的减少，并与肺发育不良、出血、肺动脉错位有关。 静脉和肺动脉高压（PAH）。ACDMPV与Forkhead Box F1中的突变有关 （FOXF 1）基因，对所有可用的治疗和呼吸支持都有抗性，导致发绀， 出生后第一个月出现呼吸衰竭。在极少数情况下，ACDMPV患者存活数个月甚至更长时间。 他们需要在生命早期进行肺移植。由于缺乏对ACDMPV的有效治疗， 迫切需要创新的治疗方法来刺激肺血管生成， 保护ACDMPV婴儿的呼吸功能。在我们的初步数据中，我们已经产生了一个临床上 通过将S52 F FOXF 1突变（在ACDMPV患者中发现）引入ACDMPV的相关模型， 内源性小鼠Foxf 1基因座。Foxf 1 WT/S52 F新生小鼠表现出肺泡毛细血管发育不良， 肺静脉错位、PAH和死亡率增加，这些都是人类ACDMPV的关键特征。 在Foxf 1 WT/S52 F小鼠和人ACDMPV中，内皮增殖和STAT 3信号转导减少 肺在目前的拨款申请中，我们将测试增加新生儿肺血管生成的假设， 在小鼠ACDMPV模型中，可降低PAH，改善存活率并预防肺重塑。目标1： 开发了一种新型纳米颗粒基因递送系统，靶向>85%的肺内皮细胞， 当递送到新生儿血液循环中时，出生后STAT 3 cDNA的纳米颗粒递送 在Foxf 1 WT/S52 F新生儿肺中诱导内皮细胞增殖和肺泡微血管密度增加。 我们将使用ACDMPV的两种小鼠模型（Foxf 1 WT/S52 F和Foxf 1 +/-）来测试纳米颗粒递送ACDMPV是否是有效的。 STAT 3或FOXF 1将减少PAH，改善生存率并防止肺重塑。我们还将确定 FOXF 1/STAT 3/cMYC转录级联是否是新生儿肺血管生成所必需的。在Aim中 2，我们提供的初步数据表明，与肺内皮祖细胞移植， 细胞（EPCs）（FOXF 1 +cKit+ CD 31 + CD 45-）增加Foxf 1 WT/S52 F肺中的毛细血管密度。我们将 研究肺EPCs异质性并测试其在小鼠ACDMPV模型中的治疗潜力。 我们将测试EPCs是否通过BMP-9/ACVRL 1信号通路刺激新生儿肺血管生成。最后， 我们将使用一种新的方法从小鼠和人类胚胎干细胞中体外分化EPCs (ES)并确定用ES衍生的EPCs进行细胞治疗是否对小鼠ACDMPV模型有益。 总而言之，所提出的研究将直接测试是否内皮递送STAT 3或细胞疗法， EPCs在ACDMPV中具有治疗潜力。

PROJECT SUMMARY. Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a fatal congenital disorder of neonates and infants which leads to respiratory insufficiency due to reduced numbers of alveolar capillaries and is associated with lung hypoplasia, hemorrhage, malposition of pulmonary veins and pulmonary arterial hypertension (PAH). ACDMPV is linked to mutations in the Forkhead Box F1 (FOXF1) gene and is resistant to all available therapies and respiratory support, causing cyanosis and respiratory failure in the first month after birth. In rare cases, ACDMPV patients survive several months or even years, but they require lung transplantation early in life. Given the lack of effective treatments for ACDMPV, there is an urgent need for innovative therapeutic approaches to stimulate pulmonary angiogenesis and preserve respiratory function in ACDMPV infants. In our preliminary data, we have generated a clinically relevant model of ACDMPV by introducing the S52F FOXF1 mutation (found in ACDMPV patients) into the endogenous mouse Foxf1 gene locus. Foxf1WT/S52F newborn mice exhibited alveolar capillary dysplasia, misalignment of pulmonary veins, PAH, and increased mortality, all key features of human ACDMPV. Endothelial proliferation and STAT3 signaling were decreased in Foxf1WT/S52F mice and human ACDMPV lungs. In the present grant application, we will test the hypothesis that increasing neonatal lung angiogenesis will decrease PAH, improve survival and prevent lung remodeling in mouse ACDMPV models. In Aim 1, we have developed a novel nanoparticle gene delivery system targeting >85% of pulmonary endothelial cells in vivo when delivered into the neonatal blood circulation. Nanoparticle delivery of STAT3 cDNA after birth induced endothelial proliferation and increased alveolar microvascular density in Foxf1WT/S52F neonatal lungs. We will use two mouse models of ACDMPV (Foxf1WT/S52F and Foxf1+/-) to test whether nanoparticle delivery of STAT3 or FOXF1 will decrease PAH, improve survival and prevent lung remodeling. We will also determine whether the FOXF1/STAT3/cMYC transcriptional cascade is required for neonatal lung angiogenesis. In Aim 2, we provided preliminary data demonstrating that a cell transplantation with pulmonary endothelial progenitor cells (EPCs) (FOXF1+cKit+CD31+CD45-) increases the capillary density in Foxf1WT/S52F lungs. We will investigate heterogeneity of pulmonary EPCs and test their therapeutic potential in mouse ACDMPV models. We will test if EPCs stimulate neonatal lung angiogenesis via the BMP-9/ACVRL1 signaling pathway. Finally, we will use a novel protocol for in vitro differentiation of EPCs from mouse and human embryonic stem cells (ES) and determine if a cell therapy with ES-derived EPCs will be beneficial in mouse ACDMPV models. Altogether, the proposed studies will directly test whether endothelial delivery of STAT3 or cell therapy with EPCs have therapeutic potential in ACDMPV.