Developmental Heterogeneity of Pulmonary Endothelial Phenotype at Single Cell Resolution

单细胞分辨率肺内皮表型的发育异质性

基本信息

批准号：
10678976
负责人：
Cristina Maria Alvira
金额：
$ 69.5万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678976
关键词：
ATAC-seq Adult Affect Air Air Sacs Alveolar Alveolar Cell Type I Angiogenesis Inhibition Appearance Area Automobile Driving Birth Blood Vessels Bronchopulmonary Dysplasia Cell Proliferation Cell Separation Cells Chronic Complication Cues Data Data Set Development Disease Distal Endothelial Cells Endothelium Experimental Models Gases Gene Expression Gene Expression Profile Genes Genetic Transcription Growth Heterogeneity Hyperoxia Image Impairment In Vitro Infant KITLG gene Knockout Mice Knowledge Life Ligands Lung Mediating Microcirculation Molecular Mus NF-kappa B Natural regeneration Neonatal Hyperoxic Injury Organ Pathway interactions Perinatal Phase Phenotype Population Premature Birth Process Proliferating Resolution Role Signal Transduction Sorting Stimulus Surface Testing Time Transgenic Organisms Translating Vascular Endothelial Cell Vascular regeneration Work angiogenesis blood vessel development in vivo loss of function lung development lung injury migration mouse model novel novel strategies novel therapeutic intervention paternal imprint postnatal postnatal development premature progenitor rapid growth receptor receptor expression response self-renewal single-cell RNA sequencing stem stem cells targeted treatment transcription factor transcriptomics virtual

项目摘要

Postnatal lung growth during alveolarization markedly increases gas exchange surface area. Rapid growth of the pulmonary vasculature during early alveolarization drives distal lung growth. As alveolarization slows, the vasculature transitions from a phase of angiogenic growth to quiescence, however the molecular mechanisms regulating this transition remain poorly defined. This gap in knowledge confounds efforts to develop targeted therapies to treat diseases of dysregulated angiogenesis and impaired alveolarization, including bronchopulmonary dysplasia, the most common complication of preterm birth. We recently employed single cell transcriptomics to define endothelial cell (EC) diversity during postnatal lung development and to identify novel mechanisms regulating pulmonary angiogenesis and quiescence. Our preliminary data identified a tremendous increase in EC diversity after birth, marked by the appearance of numerous transcriptionally distinct clusters. A highly proliferative EC cluster is abundant before birth, virtually disappears just after birth, but peaks again at early alveolarization, a time of exponential pulmonary angiogenesis. The microvascular EC (MEC) broadly separated into Car4 expressing (Car4+) and Car4- MEC. In contrast with gradual changes in gene expression in the Car4+ MEC over time, gene expression changed dramatically in the Car4- MEC, with separation of this population into two transcriptionally distinct clusters of “early” (P1-P7) and “late” (P21) Car4- MEC. High expression of the paternally imprinted gene-3 (Peg3), a gene expressed by self-renewing progenitor cells, distinguished the “early” from the “late” Car4- MEC. Peg3 also enhances NFkB signaling, a pathway we previously identified as essential for pulmonary angiogenesis during early alveolarization. Of note, the expression of receptor-ligand pairs suggested that cross-talk stemming from the Car4+ MEC may promote pro-proliferative and pro-angiogenic signaling in the Car4- MEC. Taken together, our data suggest the overall hypothesis that the early Car4- MEC represent a specialized, highly proliferative and angiogenic EC population required for the rapid growth of the pulmonary vasculature during early alveolarization, which will be tested through three specific aims. Aim 1 will utilize transgenic and cell-specific knock out mice, advanced imaging, and loss of function studies in primary EC to probe the role of Peg3 in promoting proliferation, angiogenesis and NFkB activation in Car4- MEC. Aim 2 will use FACS sorted Car4+ and Car4- MEC and a novel mouse model permitting targeting of Car4+ MEC to test if interaction between these two distinct MEC promotes postnatal angiogenesis. Finally, Aim 3 will employ computational ligand-receptor analysis, ATAC-Seq, and EC-specific knock out mice to determine if chronic hyperoxia impairs angiogenesis by impairing Car4- MEC proliferation, Peg3-mediated self-renewal and Car4+ and Car4- MEC cross-talk. The successful completion of these studies will provide a deep view of pulmonary vascular development at single cell resolution, and identify new pathways that may be translated into novel strategies to enhance lung growth and regeneration in diseases marked by impaired pulmonary angiogenesis.

肺泡后产后肺的生长显着增加了气体交换表面积。迅速的早期肺泡化期间肺血管的生长驱动肺部远端生长。作为肺泡化放慢速度，脉管系统从血管生长的阶段转变为静止，但是分子调查此转变的机制仍然很差。知识的差距使发展努力针对治疗失调血管生成和肺泡不足的疾病的靶向疗法，包括支气管肺发育不良，最常见的早产并发症。我们最近工作了单元转录组学以定义产后发育过程中内皮细胞（EC）多样性并识别新型调节肺血管生成和静止的机制。我们的初步数据确定了巨大的出生后EC多样性的增加，标志着许多转录不同的簇的出现。一个高度扩散剂EC簇在出生前很丰富，实际上在出生后消失，但再次达到顶峰早期牙槽化，指数型肺血管生成的时间。微血管EC（MEC）广泛分成CAR4表达（CAR4+）和CAR4- MEC。与基因表达的等级变化相反在CAR4+ MEC随着时间的推移中，基因表达在CAR4-MEC中发生了巨大变化，分开人口分为“早期”（P1-P7）和“晚期”（P21）CAR4- MEC的两个转录不同的簇。高的亲子印迹基因-3（PEG3）的表达，一种由自我更新祖细胞表达的基因，将“早期”与“晚” CAR4-MEC区分开来。 PEG3还增强了NFKB信号传导，我们先前在早期肺泡化期间被认为是肺血管生成必不可少的。值得注意的是，表达受体配体对表明，来自CAR4+ MEC的交叉言论可能会促进促销和CAR4-MEC中的促血管生成信号传导。综上所述，我们的数据表明了总体假设早期CAR4- MEC代表了快速需要的专业，高度增殖和血管生成的EC种群早期肺泡化期间肺脉管系统的生长将通过三个特定目的进行测试。 AIM 1将利用转基因和细胞特异性的敲除小鼠，高级成像以及初级功能研究的丧失 EC探测PEG3在CAR4-MEC中促进增殖，血管生成和NFKB激活中的作用。 AIM 2意志使用FACS排序的CAR4+和CAR4- MEC以及一种新型的鼠标模型，允许针对CAR4+ MEC的FACS进行测试。这两个不同的MEC之间的相互作用促进了产后血管生成。最后，AIM 3将使用计算配体受体分析，ATAC-SEQ和EC特异性敲除小鼠，以确定是否慢性高氧通过损害CAR4- MEC增殖，PEG3介导的自我更新和CAR4+来损害血管生成和CAR4- MEC串扰。这些研究的成功完成将为肺部提供深刻的视野单细胞分辨率下的血管发育，并确定可以翻译成新颖的新途径增强肺部血管生成受损的疾病中肺部生长和再生的策略。