Developmental Heterogeneity of Pulmonary Endothelial Phenotype at Single Cell Resolution

出生后肺在肺泡化过程中的生长显著增加气体交换表面积。快速 肺泡化早期肺血管的生长推动远端肺的生长。作为牙槽骨化 放慢，血管系统从血管生成生长阶段过渡到静止，然而分子 监管这一过渡的机制仍然定义不清。这种知识上的差距妨碍了发展的努力。 靶向疗法治疗血管生成失调和肺泡化受损的疾病，包括 支气管肺发育不良，早产最常见的并发症。我们最近用了单细胞 转录组学研究出生后肺发育过程中内皮细胞的多样性并确定新的 肺血管生成和静息的调控机制。我们的初步数据确定了巨大的 出生后EC多样性增加，表现为出现许多转录上不同的簇。一个 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，指数性肺血管生成的时间。微血管内皮细胞(MEC) 分为Car4表达(Car4)和Car4-MEC。与基因表达的逐渐变化形成对比 在Car4MEC中，随着时间的推移，Car4-MEC中的基因表达发生了戏剧性的变化 群体分为两个转录上截然不同的“早期”(P1-P7)和“晚期”(P21)Car4-MEC簇。高 父系印记基因-3(Peg3)的表达，这是一种由自我更新的祖细胞表达的基因， 区分“早期”和“晚期”的Car4-MEC。PEG3还增强了NFkB信号，这是我们 先前被认为是肺泡化早期肺血管生成的必需物质。值得注意的是，这个表达 受体-配体对的研究表明，来自Car4MEC的串扰可能促进促增殖 以及在Car4-MEC中的促血管生成信号。总而言之，我们的数据表明，总体假设是 早期的Car4-MEC代表了一个特殊的、高度增殖的和血管生成的EC群体，这是快速 在早期肺泡化期间肺血管的生长，这将通过三个特定的目标来测试。 目标1将利用转基因和细胞特异性基因敲除小鼠，先进的成像和初级功能丧失研究 EC探讨Peg3在促进Car4-MEC增殖、血管生成和NFkB活化中的作用。目标2将 使用FACS分选的Car4和Car4-MEC以及一种新的允许靶向Car4 MEC的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了出生后的血管生成。最后，目标3将采用 计算配体-受体分析、ATAC-Seq和EC特异性基因敲除小鼠以确定慢性 高氧通过损害Car4-MEC的增殖、PEG3介导的自我更新和Car4而损害血管生成 和Car4-MEC的相声。这些研究的成功完成将使人们对肺脏的认识更加深刻。 在单细胞分辨率下的血管发育，并确定可能被翻译成新的 在以肺血管生成受损为特征的疾病中促进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.