Abstract 12456: Apelin Expressing Endothelial Stem-Like Cells Orchestrate Lung Microvascular Repair in a Model of Lung-Targeted Endothelial Cell Ablation

Abstract

Introduction: Endothelial damage plays a central role in acute lung injury (ALI) contributing to alveolar leak and inflammatory cell trafficking. In preclinical models of ALI, repair and regeneration of lung vascular endothelium is required for its resolution. Therefore, we sought to define the cellular and molecular mechanisms underlying lung microvascular regeneration in acute lung injury induced by lung endothelial cell (EC) ablation. Methods and Results: Transgenic mice were created expressing endothelial-targeted human diphtheria toxin receptor. Intratracheal instillation of diphtheria toxin (DT) resulted in the ablation of ~70% of lung endothelial cells, producing severe acute lung injury, with complete resolution by 7 days. Changes in global lung cell populations and gene expression profiles were determined using single-cell RNA sequencing of dissociated lung cells (10x Genomics) at baseline (day 0), 3, 5 and 7 days after lung EC ablation. Single cell analysis revealed 8 distinct EC clusters, including type-A capillary ECs which were characterized by the unique expression of apelin at baseline. DT-induced EC ablation resulted in the emergence of novel endothelial stem-like cells within a transient population of ‘general’ capillary ECs, characterized by the de novo expression of apelin at day 3 post injury. As well, this population exhibited unique expression of the vascular endothelial stem cell marker, protein C receptor, together with the progenitor cell marker, CD34. This was followed by the appearance of proliferative ECs at day 5 expressing apelin receptor and the transcription factor, FoxM1, and these cells were responsible for replenishment of all depleted EC populations. Finally, treatment with an apelin receptor antagonist, ML221, prevented lung microvascular recovery and resulted in death at 4-5 days post DT. Conclusion: Targeted EC ablation revealed a remarkable regenerative capacity of the lung microvasculature. Using single cell transcriptomics, we have demonstrated that lung microvascular repair was orchestrated by newly emergent apelin-expressing endothelial stem-like cells.