Dynamic behavior and lineage plasticity of the pulmonary venous endothelium

Repair of the pulmonary vascular bed and the origin of new vasculature remains underexplored despite the critical necessity to meet oxygen demands after injury. Given their critical role in angiogenesis in other settings, we investigated the role of venous endothelial cells in endothelial regeneration after adult lung injury. Using single cell transcriptomics, we identified the norepinephrine transporter Slc6a2 as a marker of pulmonary venous endothelial cells and targeted that locus to generate a venous-specific, inducible Cre mouse line. Contributions of the venous endothelial cells to angiogenesis were examined during postnatal development, adult viral injury, and adult hyperoxia injury. Remarkably, we observed that venous endothelial cells proliferate into the adjacent capillary bed upon influenza injury and hyperoxia injury, but not during normal postnatal development. Imaging analysis demonstrated that venous endothelial cells exhibit the ability to proliferate and differentiate into general capillary and CAR4 expressing aerocyte capillary endothelial cells after infection, thus contributing to repair of the capillary plexus vital for gas exchange. Single cell transcriptomic analysis of Slc6a2 lineage traced cells confirmed these observations, with progeny exhibiting significant loss of venous identity and gain of capillary marker expression upon injury resolution. Our studies thus establish that venous endothelial cells exhibit demonstrable progenitor capacity upon respiratory viral injury and sterile injury, contributing to repair of the alveolar capillary bed responsible for pulmonary function. Overall design: 10X 3'-biased gene expression (v3.1) scRNA-seq libraries prepared from primary mouse lung tissue post-influenza infection, with fluorescent labelling of venous endothelial cells using Slc6a2-CreERT2 x R26-CAG-LSL-tdTomato-WPRE transgenic mice.