Lung injury shifts pulmonary capillary endothelial cells towards regeneration-associated Lrg1+ subpopulations with delayed dynamics in aged mice

Introduction and main objectives Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and irreversible interstitial lung disease (ILD), that increases dramatically in incidence and prevalence with age. While successful alveolar regeneration after injury depends on pulmonary capillary endothelial cells (PCEC) reprogramming, the steps involving PCEC during lung injury and resolution as well as the influence of aging are unknown. Methods We used single-cell RNA-seq (scRNA-seq) and spatial transcriptomics to compare the transcriptome of bleomycin-induced fibrotic lungs of young (7 weeks) and aged (18 months) mice, at 3 time points corresponding to the peak of fibrosis (14 days), regeneration (28 days) and resolution (60 days). Results Among the 44541 sequenced and annotated cells, we confirmed the transcriptomic dynamics of several cell types including macrophages, in which conversion is conserved between young and aged mice. We also found that lung injury shifts the transcriptomic profiles of recently described PCEC cell types, with 4 prominent signatures. These signatures are characterized by the overexpression of Lrg1 and are associated with pro-angiogenic signaling, potentially supported by adjacent cell types into the alveolar niche. These signatures were not equally maintained through the resolution process and between young and old animals. Moreover, part of this set of resolution-associated markers was also detected in pulmonary endothelial cells (ECs) from IPF samples. Finally, we found that aging also altered the transcriptome of general capillary cells (gCap) which display typical pro-fibrotic and pro-inflammatory features. Conclusions We provide a detailed characterization of the cellular dynamics associated with fibrosis development and resolution in young and aged lungs and propose that age-associated alterations in specific PCEC subpopulations may interfere with the process of lung progenitor differentiation contributing to the persistent fibrotic process typical of human pathology. ### Competing Interest Statement The authors have declared no competing interest.