Transcriptional Regulation of Endothelial Cells after Acute Lung Injury

项目摘要。毛细血管内皮细胞（ECs）在肺泡气体交换中起关键作用， 参与急性肺损伤（ALI）后的肺修复。急性呼吸窘迫综合征（ARDS）是一种生命- 严重并发症，死亡率高。鉴于ALI/ ARDS缺乏重大改善， 临床管理，迫切需要创新的分子方法来补充现有的 ALI/ ARDS治疗。我的实验室以前证明FOXF 1转录因子是一个关键的， 调节小鼠和人类肺血管发育。小鼠Foxf 1基因的全缺失 胚胎致死，而Foxf 1无效等位基因（Foxf 1 +/-）杂合子小鼠的 肺毛细血管和增加新生儿早期的死亡率。中的失活突变 FOXF 1基因位点在>50%的肺泡毛细血管发育不良（ACD）患者中发现，ACD是一种罕见的先天性 由于肺毛细血管严重减少而导致高死亡率的疾病。虽然这些研究表明， FOXF 1在肺血管发育中的重要作用，FOXF 1在成人肺/损伤中的功能 修复仍然未知。在这项提案中，我们提供了初步数据，证明FOXF 1水平是 在小鼠和人类肺损伤后减少。两个Foxf 1等位基因的内皮特异性缺失导致 由于内皮完整性丧失、粘附连接破坏和 降低对EC屏障功能至关重要的基因表达。从EC中仅缺失一个Foxf 1等位基因抑制 肺修复并增加小鼠ALI模型的死亡率。FOXF 1基因在内皮细胞中的敲低延迟细胞凋亡 进入S期，增加细胞周期抑制剂p21 cip 1和p15 ink 4 b的水平，并减少 STAT 3，内皮细胞增殖的关键转录介质。FOXF 1与STAT 3物理结合 FOXF 1蛋白的STAT 3结合区的突变最近在ACD患者中被发现， 强调FOXF 1-STAT 3蛋白相互作用在EC中的重要性。在这份拨款申请中，我们将 使用小鼠和大鼠ALI模型鉴定FOXF 1在内皮修复期间调节的分子机制。 我们将检验FOXF 1促进急性肺损伤后肺修复的假设。在目标1中，我们将使用 培养的EC和具有FOXF 1功能获得和功能丧失的独特小鼠模型，以确定 FOXF 1在EC屏障功能中的作用，并鉴定FOXF 1的直接靶基因。我们还将使用纳米粒子 （特异性靶向体内EC）和小分子FOXF 1激活化合物（最近在 我的实验室），以恢复FOXF 1表达肺损伤后，并确定是否药理学靶向 FOXF 1的表达将促进内皮修复。在目标2中，我们将研究FOXF 1-STAT 3的重要性。 p21 cip 1和p15 ink 4 b的相互作用和FOXF 1介导的对EC增殖的抑制。 我们研究的完成将确定FOXF 1调节肺修复的新分子机制， 确定靶向FOXF 1是否对ALI患者有益。

PROJECT SUMMARY. Capillary endothelial cells (ECs) play a key role in alveolar gas exchange and actively participate in lung repair after acute lung injury (ALI). Acute Respiratory Distress Syndrome (ARDS) is a life- threatening complication of ALI with high mortality rate. Given the lack of major improvements in ALI/ ARDS clinical management, there is a compelling need for innovative molecular approaches to complement existing ALI/ ARDS therapies. My laboratory previously demonstrated that FOXF1 transcription factor is a critical regulator of lung vascular development in mice and humans. Global deletion of Foxf1 gene in mice is embryonic lethal, whereas mice heterozygous for the Foxf1 null allele (Foxf1+/-) had reduced numbers of pulmonary capillaries and increased mortality during the early neonatal period. Inactivating mutations in FOXF1 gene locus were found in >50% of patients with Alveolar Capillary Dysplasia (ACD), a rare congenital disorder with high mortality rate due to severe reduction in pulmonary capillaries. While these studies indicate an important role for FOXF1 in development of pulmonary vasculature, FOXF1 function in adult lung/ injury repair remains unknown. In this proposal, we provide preliminary data demonstrating that FOXF1 levels are reduced after lung injury in mice and humans. Endothelial-specific deletion of both Foxf1 alleles causes respiratory failure and mortality due to loss of endothelial integrity, disruption of adherens junctions and reduced expression of genes critical for EC barrier function. Deletion of only one Foxf1 allele from ECs inhibits lung repair and increases mortality in mouse ALI models. Knockdown of FOXF1 in cultured ECs delays cell entry into S phase, increases levels of cell cycle inhibitors p21cip1 and p15ink4b, and reduces phosphorylation of STAT3, a key transcriptional mediator of endothelial proliferation. FOXF1 physically binds to the STAT3 protein, and mutations in STAT3-binding region of the FOXF1 protein were recently found in ACD patients, emphasizing the importance of FOXF1-STAT3 protein interactions in ECs. In this grant proposal, we will identify molecular mechanisms regulated by FOXF1 during endothelial repair using mouse and rat ALI models. We will test the hypothesis that FOXF1 promotes lung repair after acute lung injury. In Aim 1, we will use cultured ECs and unique mouse models with gain-of-function and loss-of-function of FOXF1 to determine the role of FOXF1 in EC barrier function and identify direct FOXF1 target genes. We will also use nanoparticles (specifically targeting ECs in vivo) and a small molecule FOXF1-activating compound (recently discovered in my laboratory) to restore FOXF1 expression after lung injury and determine whether pharmacological targeting of FOXF1 will promote endothelial repair. In Aim 2, we will examine the importance of FOXF1-STAT3 interactions and FOXF1-mediated repression of p21cip1 and p15ink4b for EC proliferation in vitro and in vivo. Completion of our studies will identify novel molecular mechanisms whereby FOXF1 regulates lung repair and determine whether targeting FOXF1 could be beneficial for ALI patients.