Transcriptional Regulation of Endothelial Cells after Acute Lung Injury

急性肺损伤后内皮细胞的转录调控

• 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
• 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

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.

项目总结。毛细血管内皮细胞(ECs)在肺泡气体交换中起着关键作用 参与急性肺损伤(ALI)后的肺修复。急性呼吸窘迫综合征(ARDS)是一种生活- 严重的ALI并发症，死亡率高。鉴于ALI/ARDS缺乏重大改进 临床管理，迫切需要创新的分子方法来补充现有的 ALI/ARDS疗法。我的实验室以前证明了FOXF1转录因子是一个关键的 小鼠和人类肺血管发育的调节剂。Foxf1基因在小鼠体内的全局缺失 胚胎致死，而Foxf1零等位基因杂合子(Foxf1+/-)的小鼠减少了 肺毛细血管和新生儿早期死亡率增加。失活的突变在 一种罕见的先天性肺泡毛细血管发育不良(ACD)患者中有50%存在FOXF1基因座 由于肺毛细血管严重减少，导致高死亡率的疾病。虽然这些研究表明 FOXF1在肺血管发育中的重要作用及FOXF1在成人肺/损伤中的作用 修复工作仍不得而知。在这项提案中，我们提供了初步数据，证明FOXF1水平是 在小鼠和人类的肺损伤后减少。两个Foxf1等位基因的内皮特异性缺失导致 因内皮完整性丧失、黏附连接破坏和 对EC屏障功能至关重要的基因表达减少。内皮细胞中只有一个Foxf1等位基因的缺失抑制了 肺修复和增加小鼠ALI模型的死亡率。FOXF1基因在培养内皮细胞中的敲除延迟细胞 进入S期，细胞周期抑制因子p21cip1和p15INK4b水平升高，细胞周期蛋白磷酸化水平降低。 STAT3，内皮细胞增殖的关键转录调节因子。FOXF1物理绑定到STAT3 最近在ACD患者中发现了FOXF1蛋白的STAT3结合区突变， 强调FOXF1-STAT3蛋白在内皮细胞中相互作用的重要性。在这项拨款建议中，我们将 利用小鼠和大鼠ALI模型确定FOXF1在内皮修复过程中调控的分子机制。 我们将验证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 培养的内皮细胞和独特的具有FOXF1功能获得和功能丧失的小鼠模型 FOXF1在EC屏障功能中的作用及FOXF1直接靶基因的筛选我们还将使用纳米颗粒 (特别针对活体内皮细胞)和一种小分子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 FOXF1具有促进内皮修复的作用。在目标2中，我们将研究FOXF1-STAT3的重要性 P21cip1和p15INK4b在体内外相互作用及FOXF1对EC增殖的抑制作用 我们研究的完成将确定FOXF1调节肺修复和 确定靶向FOXF1是否对ALI患者有利。