ATF3 promotes endothelial cell response to acute lung injury

Following acute injury, the capillary vascular bed in the lung must be repaired to reestablish gas exchange between pulmonary endothelial cells (ECs) lining these vessels and the alveolar epithelium. However, the factors that control EC stress response and drive regeneration of pulmonary capillaries remain incompletely understood. Viral infections such as influenza and COVID-19 may indirectly damage lung vasculature through loss of epithelial gas exchange partners or through signaling from infiltrating immune cells. To prevent excessive tissue damage and to renew the endothelium, ECs must both withstand cellular stress and proliferate after injury. Here, we show that the transcription factor and immediate early gene Atf3 is essential for both responses in the mouse lung after influenza infection. Atf3 expression defines a subpopulation of capillary ECs enriched in genes involved in cellular response to stress, angiogenesis, and vascular development. Endothelial loss of ATF3 results in defective alveolar regeneration: in the absence of ATF3, ECs exhibit increased apoptosis and decreased proliferation, resulting in an emphysema-like phenotype with enlarged alveolar airspaces lined with regions of lost vasculature. These data implicate ATF3 as an essential component of the vascular response to acute lung injury that is required for successful lung regeneration. This experiment isolated tdTomato+ endothelial cells from Cdh5-CreERT2; ROSA26-tdTomato mice (control or "Atf3-WT" endothelial cells) and Cdh5-CreERT2; ROSA26-tdTomato; Atf3 lox/lox mice (experimental or "Atf3-KO" ECs). Single-cell suspensions were prepared from whole mouse lungs, and tdTomato+ cells were isolated by FACS. Cells were pelleted and lysed for RNA extraction, cDNA preparation, and RNA sequencing library preparation using the NEB Protocol for Low Input RNA kit (NEB#E6420). Sequencing was performed by Genewiz (now Azenta Life Sciences).