Altered Heterogeneity of Ageing Lung Endothelium is a Hallmark of Idiopathic Pulmonary Fibrosis

Background Older age is the main risk factor for chronic lung diseases including idiopathic pulmonary fibrosis (IPF). Halting or reversing progression of IPF remains an unmet clinical need due to limited knowledge of underlying mechanisms. The lung circulatory system, composed of blood (pulmonary and bronchial) and lymphatic vessels networks, has been implicated in IPF pathophysiology in elderly people, based solely on reports of altered density and increased permeability of vessels. Aim We aimed to define heterogeneity and IPF-associated changes of lung endothelial cells (EC or endothelium) by comparing gene expression in tissues from elderly people - transplant donors and recipients with IPF. Methods Single-cell RNA sequencing (scRNAseq) datasets of “ageing lung” tissues were selected only from those publicly available sources that contain age-matching samples for both groups (49- 77 years old donors and IPF patients; nine pairs in total), integrated and compared. Findings were validated by immunohistochemistry using EC-specific markers. Results The generation of integrated single-cell maps of ageing lung tissues revealed 17 subpopulations of endothelium (12 for blood and 5 for lymphatic vessels, including 9 novel), with distinct transcriptional profiles. In IPF lung, the heterogeneity of ageing lung endothelium was significantly altered - both in terms of cell numbers (linked to disease- related changes in tissue composition) and differentially expressed genes (associated with fibrosis, inflammation, differentiation and vasodilation) in individual pulmonary, bronchial and lymphatic EC subpopulations. Conclusions These findings reveal underappreciated extent of heterogeneity and IPF-associated changes of ageing lung endothelium. Our data suggest direct involvement of specific subpopulations of ageing lung endothelium in IPF pathophysiology, uncovering cellular and molecular targets which may have potential diagnostic, prognostic and therapeutic relevance. This study creates a conceptual framework for appreciating the disease-specific heterogeneity of ageing lung endothelium as a hallmark of IPF. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The work is funded by Kate Garthwaite Pulmonary Fibrosis Research Fund, the University of Hull Endothelial Cell Research Fund, PhD Scholarships Fund for 'Health Global Data Pipeline (Health*GDP) for biomedical research and clinical applications' cluster and The Anatomical Society. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IPF samples and age-matched donor samples in cohort 1 were selected from the gene expression omnibus (GEO) GSE122960\_RAW and in cohort 2 - from GSE136831\_RAW I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors