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.

Public Health Relevance

The formation of the distal air sacs (alveolarization) requires the development of blood vessels by the process of angiogenesis. Disruption of angiogenesis and alveolarization in infants causes bronchopulmonary dysplasia, the most common complication of premature birth. Our studies will explore if lung immune cells regulate angiogenesis in the developing lung, and identify new pathways to allow the development of therapeutic strategies to promote lung growth and regeneration in both infants and adults with lung disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Respiratory Integrative Biology and Translational Research Study Section (RIBT)
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Lin, Sara
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Stanford University
Schools of Medicine
United States
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