Mapping pulmonary endothelial cell heterogeneity at homeostasis and during tissue regeneration

Pulmonary endothelial cells are an essential component of the lung alveolus, where they assist in integrating this niche with the cardiovascular system to perform gas exchange with the external environment. Despite its importance, the extent and function of endothelial cell heterogeneity within the lung remains incompletely understood. Using single-cell analytics, we show that multiple vascular endothelial cell populations exist in the mouse lung, including macrovascular endothelium (maECs), microvascular endothelium (miECs), and a new population we have termed regulator endothelium (RECs). RECs express a unique gene signature including elevated expression of Cd34 (Cd34high), Vegfr2 (Kdr), Ednrb, and Car4. The location of RECs within the lung is similar to other miECs at homeostasis, but they appear to preferentially invest regions of regenerating lung tissue after acute lung injury. Receptor-ligand analysis indicates that RECs are primed to receive reparative signals from alveolar type I cells through Vegfa-Vegfr2. Interestingly, influenza infection reveals the emergence of a highly proliferative endothelial cell population (PECs) that likely contributes to revascularization of the alveolar space after injury and may arise from Cd34low miECs. These studies map endothelial cell heterogeneity in the adult lung and characterize the preferential response of novel endothelial cell subpopulations required for proper tissue regeneration after acute lung injury. Overall design: All experiments used C57BL/6 female mice aged 6-8 weeks old. Single-cell suspensions were prepared from whole mouse lungs. DAPI-negative, CD45-negative and CD31-positive cells were sorted to obtain heterogeneous pulmonary endothelium. For whole-lung scRNA-seq analysis, DAPI-negative, CD45-negative cells were sorted to obtain all non-immune lung cells. Single-cell barcoded droplets were produced using 10X Single Cell 3' v2 chemistry. Libraries generated were sequenced using the HiSeq Rapid SBS kit. For the heterogeneous pulmonary endothelium samples, the resulting libraries were sequenced across two lanes of an Illumina HiSeq2500 instrument in High-output mode. For the whole lung samples, the resulting libraries were sequenced across a single lane of an Illumina HiSeq2500 instrument in Rapid Run mode. Reads were aligned and gene level unique molecular identifier (UMI) counts were obtained using the Cell Ranger pipeline.