Abstract
<jats:p>Intratracheally delivered bleomycin in mice is the most widely used in vivo model of pulmonary fibrosis, yet key aspects remain poorly defined, including sex-dependent responses and the temporal peak of injury. Standard 2D histology further overlooks regional heterogeneity and cannot resolve the 3D architecture or connectivity of endothelial cells (ECs). Here, we established a multi-scale 3D imaging pipeline integrating precision-cut lung slices from EC lineage-tracing mice, optical clearing, and AI-driven 3D segmentation to map cellular and structural dynamics from whole-lobe tile scans to single-cell resolution. We identified sex as a critical biological variable, with males exhibiting a delayed but more severe fibroproliferative response. Unsupervised K-means clustering identified three distinct tissue microenvironments: healthy parenchyma(KMC1), a myofibroblast-rich fibrotic core (KMC2), and a previously uncharacterized EC-dense perilesional region (KMC3) defined by massively expanded but non-perfused ECs that acquire a pro-inflammatory phenotype. This aberrant endothelial response precedes peak myofibroblast accumulation and persists beyond fibrotic resolution, leaving a 'vascular scar' that extends into the large-vessel hierarchy. Together, this 3D image atlas, made publicly available as an interactive resource [https://mosaic-lung.com/], reveals the activated endothelium as an underexplored therapeutic target in pulmonary fibrosis.</jats:p>