Development of novel therapeutic approaches for treatment of Alveolar Capillary Dysplasia

项目总结。肺泡毛细血管发育不良伴肺静脉错位(ACDMPV)是 新生儿和婴儿的致命性先天性疾病，因减少而导致呼吸功能不全 肺泡毛细血管数量增多，并与肺发育不良、出血、肺错位有关 静脉和肺动脉高压(PAH)。ACDMPV与叉头盒F1的突变有关 (FOXF1)基因，对所有可用的治疗和呼吸支持都具有抵抗力，可导致青紫和 出生后第一个月呼吸衰竭。在极少数情况下，ACDMPV患者可以存活几个月甚至 数年，但他们需要在生命早期进行肺移植。鉴于ACDMPV缺乏有效的治疗方法， 迫切需要创新的治疗方法来刺激肺血管生成和 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的 将S52F FOXF1突变(在ACDMPV患者中发现)导入ACDMPV相关模型 内源性小鼠Foxf1基因座。Foxf1WT/S52F新生小鼠表现为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。 Foxf1WT/S52F小鼠和人ACDMPV内皮细胞增殖和STAT3信号转导降低 肺部。在目前的赠款申请中，我们将检验增加新生儿肺血管生成的假设 能降低PAH，提高小鼠存活率，防止小鼠ACDMPV模型肺重塑。在目标1中，我们 已经开发出一种新的针对肺内皮细胞85%的纳米颗粒基因递送系统 活体时进入新生儿的血液循环。生后STAT3基因的纳米粒递送 诱导Foxf1WT/S52F新生大鼠肺内皮细胞增殖，增加肺泡微血管密度。 我们将使用ACDMPV的两个小鼠模型(Foxf1WT/S52F和Foxf1+/-)来测试纳米粒传递 STAT3或FOXF1可降低PAH，提高存活率，防止肺重塑。我们还将确定 新生肺血管生成是否需要FOXF1/STAT3/cMYC转录级联反应。在AIM 2、我们提供的初步数据表明，肺内皮祖细胞移植 细胞(EPC)(FOXF1+cKit+CD31+CD45-)可增加Foxf1WT/S52F肺毛细血管密度。我们会 研究肺内皮祖细胞的异质性并检测其在小鼠ACDMPV模型中的治疗潜力。 我们将测试内皮祖细胞是否通过BMP-9/ACVRL1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体外分化小鼠和人类胚胎干细胞的内皮祖细胞 (ES)并确定ES来源的EPC的细胞疗法是否对小鼠ACDMPV模型有益。 总之，拟议的研究将直接测试STAT3的内皮递送或细胞治疗 内皮祖细胞对ACDMPV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.