Transcriptional Regulation of Endothelial Cells after Acute Lung Injury

急性肺损伤后内皮细胞的转录调控

基本信息

批准号：
9900064
负责人：
Vladimir Kalinichenko
金额：
$ 52.43万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9900064
关键词：
Acute Lung Injury Adherens Junction Adult Adult Respiratory Distress Syndrome Alleles Alveolar Applications Grants Binding Binding Sites Blood Vessels Blood capillaries Capillary Endothelial Cell Cell Cycle Cell Proliferation Cells Cessation of life ChIP-seq Clinical Management Complement Complication Congenital Disorders Congenital alveolar dysplasia Data Development Elements Embryo Endothelial Cells Endothelium FOXF1 gene Gases Gene Activation Gene Expression Genes Genetic Transcription Homeostasis Human Hyperoxia Impairment In Vitro Inflammation Injury Kinetics Laboratories Life Lung Lung Inflammation MAPK8 gene Mediating Mediator of activation protein Modeling Molecular Mus Mutation Patients Permeability Pharmacology Phosphorylation Play Proteins Pulmonary Edema Rattus Regulation Regulatory Element Repression Respiratory Failure Role S Phase STAT3 gene Severities Signal Transduction Site Site-Directed Mutagenesis Small Interfering RNA Stat3 protein Structure Tamoxifen Testing Transcriptional Regulation Transgenes Transgenic Mice Vascular Permeabilities Vinculin alpha catenin cadherin 5 gain of function gene repression genetic approach genomic locus improved in vivo inhibitor/antagonist injury and repair innovation knock-down loss of function lung development lung injury lung repair mortality mouse model nanoparticle neonatal period novel plakoglobin prevent promoter protein protein interaction repaired small molecule transcription factor transcriptome sequencing

项目摘要

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.

项目摘要。毛细血管内皮细胞（EC）在肺泡气体交换中发挥着关键作用，并积极参与参与急性肺损伤（ALI）后的肺修复。急性呼吸窘迫综合征 (ARDS) 是一种致命的疾病 ALI 的威胁性并发症，死亡率高。鉴于 ALI/ARDS 缺乏重大改进临床管理中，迫切需要创新的分子方法来补充现有的 ALI/ARDS 治疗。我的实验室之前证明 FOXF1 转录因子是一个关键的转录因子小鼠和人类肺血管发育的调节剂。小鼠中 Foxf1 基因的整体缺失胚胎致死，而 Foxf1 无效等位基因 (Foxf1+/-) 杂合的小鼠的胚胎致死数量减少肺毛细血管损伤和新生儿早期死亡率增加。失活突变在超过 50% 的肺泡毛细血管发育不良 (ACD) 患者中发现了 FOXF1 基因位点，这是一种罕见的先天性疾病由于肺毛细血管严重减少而导致死亡率高的疾病。虽然这些研究表明 FOXF1 在肺血管发育中发挥重要作用，FOXF1 在成人肺/损伤中的功能修复情况仍未知。在本提案中，我们提供了初步数据，证明 FOXF1 水平小鼠和人类肺损伤后减少。两个 Foxf1 等位基因的内皮特异性删除导致由于内皮完整性丧失、粘附连接破坏而导致呼吸衰竭和死亡对 EC 屏障功能至关重要的基因表达减少。仅从 EC 中删除一个 Foxf1 等位基因会抑制小鼠 ALI 模型中的肺修复并增加死亡率。培养 EC 中 FOXF1 的敲低可延迟细胞生长进入 S 期，增加细胞周期抑制剂 p21cip1 和 p15ink4b 的水平，并减少 STAT3，内皮增殖的关键转录介质。 FOXF1 与 STAT3 物理结合最近在 ACD 患者中发现了 FOXF1 蛋白 STAT3 结合区的突变，强调 FOXF1-STAT3 蛋白相互作用在 EC 中的重要性。在此拨款提案中，我们将使用小鼠和大鼠 ALI 模型确定内皮修复过程中 FOXF1 调节的分子机制。我们将检验 FOXF1 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 FOXF1 功能获得和功能丧失的独特小鼠模型，以确定 FOXF1 在 EC 屏障功能中的作用并确定 FOXF1 的直接靶基因。我们还将使用纳米粒子（专门针对体内 EC）和小分子 FOXF1 激活化合物（最近在我的实验室）恢复肺损伤后 FOXF1 的表达并确定药物靶向是否有效 FOXF1 会促进内皮修复。在目标 2 中，我们将研究 FOXF1-STAT3 的重要性相互作用以及 FOXF1 介导的 p21cip1 和 p15ink4b 抑制对体外和体内 EC 增殖的影响。完成我们的研究将确定 FOXF1 调节肺修复的新分子机制确定靶向 FOXF1 是否对 ALI 患者有益。