Developmental Heterogeneity of Pulmonary Endothelial Phenotype at Single Cell Resolution

出生后肺泡化过程中的肺生长显著增加了气体交换表面积。快速 在早期肺泡化过程中肺脉管系统的生长驱动远端肺生长。肺泡化 当血管生长减慢时，血管系统从血管生成生长的阶段过渡到静止，然而， 管理这一过渡的机制仍然不明确。这种知识上的差距阻碍了发展 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，早产最常见的并发症。我们最近采用了单细胞 转录组学来确定出生后肺发育期间内皮细胞（EC）的多样性， 调节肺血管生成和静止的机制。我们的初步数据显示 出生后EC多样性的增加，其标志是许多转录上不同的簇的出现。一 高度增殖的EC簇在出生前大量存在，出生后几乎消失，但在出生后再次达到峰值。 早期肺泡化，指数肺血管生成的时间。微血管EC（MEC）广泛 分离为表达Car 4的（Car 4+）和Car 4- MEC。与基因表达的逐渐变化相反， 在Car 4 + MEC中，随着时间的推移，Car 4- MEC中的基因表达发生了显著变化， 群体分成两个转录上不同的簇“早期”（P1-P7）和“晚期”（P21）Car 4- MEC。高 父系印记基因-3（Peg 3）的表达，该基因由自我更新祖细胞表达， 区分“早期”和“晚期”的Car 4- MEC。Peg 3还增强NFkB信号传导， 以前被认为是早期肺泡化过程中肺血管生成所必需的。值得注意的是， 受体-配体对的研究表明，来自Car 4 + MEC的串扰可能促进促增殖 和促血管生成信号。综上所述，我们的数据表明， 早期Car 4- MEC代表了一种特化的、高度增殖的和血管生成的EC群体， 在早期肺泡化过程中肺血管的生长，这将通过三个特定的目标进行测试。 目的1将利用转基因和细胞特异性敲除小鼠，先进的成像，并在初级功能丧失的研究 EC以探测Peg 3在促进Car 4- MEC中的增殖、血管生成和NFkB活化中的作用。目标2将 使用FACS分选的Car 4+和Car 4- MEC以及允许靶向Car 4 + MEC的新型小鼠模型来测试 这两种不同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，以确定慢性 高氧通过损害Car 4- MEC增殖、Peg 3介导的自我更新和Car 4+而损害血管生成 和Car 4- MEC串扰。这些研究的成功完成将提供一个深入了解肺 血管发展在单细胞分辨率，并确定新的途径，可能会转化为新的 在以肺血管生成受损为标志的疾病中增强肺生长和再生的策略。

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.