Transcriptional Regulation of Endothelial Cells after Neonatal Lung Injury

项目摘要。新生儿重症监护的进步大大提高了早产儿的生存率， 但早产的长期并发症，包括支气管肺发育不良（BPD）， 死亡率和发病率在以后的生活。早产后，肺过渡到非水环境 似乎足以破坏随后的肺泡生长和伴随的血管结构， 有效的气体交换。当早产儿的肺暴露于补充氧气时，这种情况会进一步加剧 和正压通气。氧后肺泡毛细血管的不可逆损失和血管重构 暴露导致肺动脉高压（PH），见于重度BPD患者（BPD-PH）。目前迫切 需要创新的治疗方法来刺激新生儿肺血管生成和保护呼吸系统 BPD-PH婴儿的功能。我的实验室最近创造了PEI 600-MA5/PEG-OA/Cho纳米颗粒， 将具有促血管生成基因的非整合表达质粒递送到肺微血管中 内皮细胞，目的是刺激新生儿肺血管生成。我们还发现了一种 肺内皮祖细胞（EPCs）的亚群，FOXF 1 + EPCs，是最近发现的一个亚群， 发现了一般毛细血管细胞（gCAPs）。FOXF 1 + gCAPs移植增加新生儿肺 在具有先天性肺泡毛细血管缺陷的小鼠中的血管生成和肺泡化。我们建议测试 通过纳米颗粒FOXF 1基因治疗或免疫抑制剂治疗增加新生儿肺血管生成的假设， FOXF 1 + gCAP细胞移植将预防PH并改善BPD小鼠和大鼠模型的肺功能- 博士在目标1中，我们将确定纳米颗粒FOXF 1基因治疗是否具有长期的有益效果。 通过预防PH和右心室（RV）肥大以及加速肺再生对BPH-PH的影响 新生儿高氧肺损伤后。我们还将确定FOXF 1在再生中的新下游靶点， 内皮细胞和测试FOXF 1是否招募STAT 3染色质激活内皮增强剂。 我们的研究将确定FOXF 1-STAT 3蛋白-蛋白相互作用是否是肺再生所必需的 在BPH-PH模型中。在目标2中，我们将确定供体FOXF 1 + gCAP的移植是否具有长时间的 通过预防BPH-PH小鼠模型中的PH和RV肥大，我们还将测试 DLL 4/NOTCH信号传导途径对供体FOXF 1 + gCAP刺激 高氧损伤后肺再生过程中受体内皮细胞增殖和管腔形成。 最后，我们将在体外从胚胎干细胞（ESC）中产生小鼠FOXF 1 + gCAP（通过定向表达）。 ESC分化为FOXF 1 + gCAP）和体内（通过种间小鼠-大鼠嵌合体）。小鼠ESC- 衍生的FOXF 1 + gCAP将用于细胞治疗以预防或延迟小鼠PH和RV肥大 BPD-PH模型总而言之，所提出的临床前研究将直接测试内皮递送的药物组合物是否是有效的。 FOXF 1载体或使用FOXF 1 + gCAP的细胞疗法在BPD-PH中具有治疗潜力。

PROJECT SUMMARY. Advances in neonatal critical care have greatly improved the survival of preterm infants but the long-term complications of prematurity, including Bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. After premature birth, transition of the lung to a non-aqueous environment appears sufficient to disrupt subsequent alveolar growth and the attendant vascular structures required for effective gas exchange. This is further exacerbated when the preterm lung is exposed to supplemental oxygen and positive pressure ventilation. Irreversible loss of alveolar capillaries and vascular remodeling after oxygen exposure cause pulmonary hypertension (PH) seen in patients with severe BPD (BPD-PH). There is an urgent need for innovative therapeutic approaches to stimulate neonatal lung angiogenesis and preserve respiratory function in BPD-PH infants. My laboratory recently created PEI600-MA5/PEG-OA/Cho nanoparticles that can deliver non-integrating expression plasmids with pro-angiogenic genes into pulmonary microvascular endothelial cells with the purpose of stimulating neonatal lung angiogenesis. We also identified a specialized subpopulation of pulmonary endothelial progenitor cells (EPCs), FOXF1+ EPCs, that are a subset of recently discovered general capillary cells (gCAPs). Transplantation of FOXF1+ gCAPs increased neonatal lung angiogenesis and alveolarization in mice with congenital deficiency of alveolar capillaries. We propose to test the hypothesis that increasing neonatal lung angiogenesis via the nanoparticle FOXF1 gene therapy or the FOXF1+ gCAP cell transplantation will prevent PH and improve lung function in mouse and rat models of BPD- PH. In Aim 1, we will determine whether the nanoparticle FOXF1 gene therapy has a long-term beneficial effect in BPH-PH by preventing PH and right ventricular (RV) hypertrophy, and accelerating lung regeneration after neonatal hyperoxic lung injury. We will also identify novel downstream targets of FOXF1 in regenerating endothelial cells and test whether FOXF1 recruits STAT3 to the chromatin to activate endothelial enhancers. Our studies will determine if the FOXF1-STAT3 protein-protein interactions are required for lung regeneration in BPH-PH models. In Aim 2, we will determine whether transplantation of donor FOXF1+ gCAPs has a long- term beneficial effect by preventing PH and RV hypertrophy in mouse model of BPH-PH. We will also test requirements of the DLL4/NOTCH signaling pathway for the ability of donor FOXF1+ gCAPs to stimulate proliferation and tube formation in recipient endothelial cells during lung regeneration after hyperoxic injury. Finally, we will produce mouse FOXF1+ gCAPs from embryonic stem cells (ESCs) in vitro (via directed differentiation of ESCs into FOXF1+ gCAPs) and in vivo (via interspecies mouse-rat chimeras). Mouse ESC- derived FOXF1+ gCAPs will be used for cell therapy to prevent or delay PH and RV hypertrophy in mouse BPD-PH model. Altogether, the proposed preclinical studies will directly test whether endothelial delivery of the FOXF1 vector or cell therapy with FOXF1+ gCAPs have therapeutic potential in BPD-PH.