LML, HY, DJ, JG, and JCS contributed equally

Introduction: Restrictive allograft syndrome (RAS) is a severe, progredient phenotype of Chronic Lung Allograft Dysfunction leading to death in median 6 to 18 months after its onset. Knowledge about the contribution of endothelial cells to its pathophysiology is lacking.

Aims: To define the transcriptional profile of RAS endothelial lung cells and its implication in the pathogenesis of RAS.

Methods: Lung tissue from 15 RAS patients - phenotyped according to ISHLT 2019 - and 17 controls (CTRL) was fixed and enzymatically dissociated with collagenase and liberase. DAPI+ single nuclei were flow sorted, barcoded using the 10x Chromium Single Cell Gene Expression Flex assay, and sequenced on the Illumina NovaSeq platform. Data were analyzed using the R package Seurat. Findings were validated through immunofluorescent microscopy and ImageJ.

Results: Single nuclei endothelial transcriptomes from 15 RAS lungs and 17 CTRL lungs were analyzed. Cluster analysis of vascular endothelial (VE) cells revealed five populations: arterial, venous, two distinctive capillary and a fifth VE cell population, best distinguishable by its expression of COL15A1. In RAS lungs, we observed a substantial increase of COL15A1+ and PLVAP+ and a replacement of normal HPGD+ lung capillary populations. Immunofluorescent microscopy confirmed these changes in the endothelial diversity.

Conclusions: Similar to other fibrotic lung diseases, RAS is characterized by the replacement of alveolar capillaries with COL15A1+ and PLVAP+ blood vessels with potential impact on gas exchange, vascular permeability, and prostaglandin homeostasis.