Diverse Homeostatic Roles for Distinct Macrophages in the Developing Lung Vasculature

肺泡化，肺发育的最后阶段，主要发生在出生后， 气体交换表面积早期肺血管新生引起的肺血管快速生长 肺泡化驱动远端肺生长，并且破坏的血管生成损害肺泡化。在其他器官中， 特化的巨噬细胞通过促进血管形成、提供存活和 迁移线索EC，并促进血管闭塞。然而，巨噬细胞在 肺血管系统的发育仍然完全未知。我们最近开始了一个项目， 单细胞RNA测序确定胚胎晚期和出生后早期肺巨噬细胞多样性 发展巨噬细胞是非常异质的，具有不同的表型，是谱系和组织- 具体的，高度受微环境的影响。本提案中的初步数据表明， 出生后巨噬细胞多样性大幅增加。存在特化的高度增殖的巨噬细胞 在出生后被各种巨噬细胞亚型的复杂和动态混合物所取代 表现出独特的基因特征，基因表达的发育梯度，以及在细胞内的特定位置。 肺表明在组织重塑、血管生成和免疫中的不同功能。有趣的是， 发现胚胎巨噬细胞完全包围小动脉并表达大量基因， 调节肺分支、血管生成和EC表型。出生后，这些细胞转变为一个中间体， 仅在出生后的前几周存在表达额外的组织重塑基因的亚群。 总之，我们的数据表明，不同的巨噬细胞群体支持肺泡化的假设 通过表达影响血管生长的因子来调节肺血管发育 和改造，这将通过三个具体目标进行测试。目标1将联合收割机就地多路复用 杂交、谱系追踪、原发性EC和巨噬细胞研究以及转基因和 基因敲除小鼠，以确定特定巨噬细胞亚群在调节EC表型和调节肺 实质和血管生长。目的2将利用多重原位杂交，条件性基因敲除小鼠 模型，以及来自肺EC和巨噬细胞的单细胞数据集的配体-受体分析，以探测通路 介导巨噬细胞-EC通讯。最后，目标3将确定慢性高氧是否会改变多样性， 表型的肺巨噬细胞在急性损伤和恢复后，并特别损害 肺巨噬细胞的发育和稳态功能。这些研究的成功完成将 提供了一个多方面的看法，不同的功能，肺巨噬细胞在胚胎和出生后早期 开发单细胞分辨率，并确定新的途径，可以直接转化为新的 调节以肺损伤为标志的疾病中血管生长和再生的策略 血管生成

Alveolarization, the final stage of lung development occurring primarily postnatally, markedly increases gas exchange surface area. Rapid growth of the pulmonary vasculature by angiogenesis during early alveolarization drives distal lung growth, and disrupted angiogenesis impairs alveolarization. In other organs, specialized macrophages support angiogenesis by promoting blood vessel formation, providing survival and migratory cues to EC, and facilitating vascular anastomoses. However, the role of macrophages in the developing pulmonary vasculature remains entirely unknown. We recently embarked on a project employing single cell RNA-sequencing to define macrophage diversity during late embryonic and early postnatal lung development. Macrophages are extremely heterogenous with diverse phenotypes that are lineage- and tissue- specific, and highly influenced by the microenvironment. Preliminary data in this proposal demonstrate a tremendous increase in macrophage diversity after birth. Specialized, highly proliferative macrophages present before birth are replaced after birth by a complex and dynamic mixture of diverse macrophage subtypes exhibiting unique gene signatures, developmental gradients in gene expression, and specific locations within the lung suggesting distinct functions in tissue remodeling, angiogenesis, and immunity. Interestingly, a subset of embryonic macrophages was found to completely encircle small arterioles and express numerous genes that regulate lung branching, angiogenesis, and EC phenotype. After birth, these cells transitioned to an intermediate subset present only during the first few weeks of postnatal life that expressed additional tissue remodeling genes. Taken together, our data suggest the hypothesis that distinct macrophage populations support alveolarization by regulating pulmonary vascular development through the expression of factors that influence vascular growth and remodeling, which will be tested through three specific aims. Aim 1 will combine multiplexed in situ hybridization, lineage tracing, studies in primary EC and macrophages, and advanced imaging in transgenic and knock-out mice to define the role of specific macrophage subsets in modulating EC phenotype and regulating lung parenchymal and vascular growth. Aim 2 will utilize multiplexed in situ hybridization, conditional knock out mouse models, and ligand-receptor profiling of single cell datasets from pulmonary EC and macrophages to probe pathways mediating macrophage-EC communication. Finally, Aim 3 will determine if chronic hyperoxia alters diversity and phenotype of the lung macrophages during acute injury and after recovery, and specifically impairs developmental and homeostatic functions of lung macrophages. The successful completion of these studies will provide a multifaceted view of the diverse functions of lung macrophages during embryonic and early postnatal development at single cell resolution, and identify new pathways that could be directly translated into novel strategies to modulate vascular growth and regeneration in diseases marked by impaired pulmonary angiogenesis.