Sveiven, S.; Knutsen, C.; Zanini, F.; Cornfield, D.; Alvira, C.

Objective: To establish a novel strategy that leverages scRNAseq data to map individual pulmonary endothelial cells to vessels of a specific size and location, thereby providing functional and spatial context to transcriptomic identity. Methods and Results: We applied a method of endothelial enrichment and deep single cell RNA sequencing to create a high resolution, transcriptomic dataset from the developing mouse lung. We developed an analytical framework to assign vessel-size scores and categorize individual endothelial cells (EC) and mural cells along a continuum of vessel sizes. We delineated a continuum of proximal arterial through distal venous EC states by uncovering transcriptional signatures associated with vessel size, spanning micro- to macrovascular zones. Our data recapitulated previously established zonally defined signaling axes, including Cxcl12 and Cxcr4 in arterioles, and identified localization of disease relevant markers such as Esr2. This vessel-size informed frameweork was robust across species and revealed how spatial EC heterogeneity underlies key processes in lung development and injury. Conclusions: These findings provide a comprehensive transcriptional map of pulmonary endothelial cells across the vascular continuum, offering valuable insights into spatial inferences and mechanistic insights within single cell RNA sequencing data sets that may help pave the way for targeted therapeutic strategies to treat pulmonary vascular diseases and expansion to tissues outside the lung.