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：
10211048
负责人：
Cristina Maria Alvira
金额：
$ 70.82万
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/10211048
关键词：
APLN gene ATAC-seq Adult Affect Air Air Sacs Alveolar Alveolar Cell Type I Appearance Area Automobile Driving Birth Blood Vessels Bronchopulmonary Dysplasia Cell Proliferation 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 Natural regeneration Neonatal Hyperoxic Injury Organ Pathway interactions Perinatal Phase Phenotype Population Premature Birth Process Resolution Role Signal Transduction Stimulus Surface Testing Time Transgenic Organisms Translating 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.

出生后肺泡化过程中的肺部生长显着增加了气体交换表面积。早期肺泡化过程中肺血管的生长驱动远端肺的生长。减慢，脉管系统从血管生成阶段过渡到静止阶段，但是分子监管这一转变的机制仍然不明确，这种知识差距阻碍了发展努力。治疗血管生成失调和肺泡化受损疾病的靶向疗法，包括支气管肺发育不良是早产最常见的并发症，我们最近采用了单细胞。转录组学可定义出生后肺发育过程中的内皮细胞 (EC) 多样性并识别新的我们的初步数据发现了巨大的调节肺血管生成和静止的机制。出生后 EC 多样性增加，以大量转录上不同的 A A 簇的出现为标志。高度增殖的 EC 簇在出生前大量存在，出生后几乎消失，但在出生时再次达到峰值早期肺泡化，即指数肺血管生成的时期。与基因表达的逐渐变化相反，分为Car4表达（Car4+）和Car4-MEC。随着时间的推移，在 Car4+ MEC 中，Car4- MEC 中的基因表达发生了巨大变化，并且与此分离群体分为两个转录上不同的簇：“早期”（P1-P7）和“晚期”（P21）Car4-MEC。父系印记基因 3 (Peg3) 的表达，这是一种由自我更新祖细胞表达的基因，区分“早期”和“晚期”Car4-MEC 还增强 NFkB 信号传导，这是我们的一个途径。先前被认为对于早期肺泡化过程中的肺血管生成至关重要，值得注意的是，该表达。受体-配体对的研究表明，Car4+ MEC 产生的串扰可能促进促增殖综上所述，我们的数据表明总体假设是：早期 Car4-MEC 代表了快速增殖所需的专门的、高度增殖和血管生成的 EC 群体。早期肺泡化过程中肺血管系统的生长，将通过三个特定目标进行测试。目标 1 将利用转基因和细胞特异性敲除小鼠、先进成像和初级功能丧失研究 EC 探究 Peg3 在 Car4-MEC 中促进增殖、血管生成和 NFkB 激活的作用。使用 FACS 分选的 Car4+ 和 Car4- MEC 以及允许靶向 Car4+ MEC 的新型小鼠模型来测试是否这两种不同的 MEC 之间的相互作用促进出生后血管生成。最后，目标 3 将采用。计算配体-受体分析、ATAC-Seq 和 EC 特异性敲除小鼠以确定是否患有慢性高氧通过损害 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提供对肺部的深入了解。以单细胞分辨率观察血管发育，并确定可转化为新途径的新途径在以肺血管生成受损为特征的疾病中增强肺生长和再生的策略。