Investigating the Role of Venous Endothelial Cells in Lung Injury and Fibrosis

Abstract Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by excessive matrix deposition and vascular dysfunction. Vascular abnormalities, including aberrant angiogenesis and vascular leak have been shown to accompany IPF progression. Recent findings from our group and others have revealed a marked heterogeneity within the lung vasculature, yet the specific contribution of distinct endothelial cell (EC) subpopulations to IPF pathogenesis remains unclear. We reported the expansion of venous ECs (VECs) expressing atypical chemokine receptor 1 (ACKR1) in mouse lungs with progressive fibrosis and in human IPF lungs. Here, we sought to investigate the role of ACKR1+VEC remodeling following lung injury and explore ACKR1-associated signaling pathways in lung fibrosis pathogenesis. Methods: We analyzed scRNA-seq datasets from bleomycin-treated mouse lungs and human IPF lungs. Additionally, we carried out spatial transcriptomic analysis on human IPF lungs exhibiting different degree of fibrosis. Immunohistochemistry was performed to validate gene expression at the protein level. ACKR1+VECs were isolated using MACS and characterized by qPCR and Western blot. Finally, co-cultures of ACKR1+VECs with lung fibroblasts were carried out to assess the paracrine effect of ACKR1+VECs. Results: scRNA-seq analysis showed that ACKR1 was selectively expressed in an expanding population of VECs in bleomycin-treated and human IPF lungs. Transcriptional analysis revealed that ACKR1+VECs expressed high levels of genes associated with glycolysis, hypoxia, and immune cell recruitment. Spatial transcriptomics coupled with immunohistochemistry analysis of IPF lungs demonstrated that ACKR1+VECs were surrounded by CD45+ immune cells, suggesting a role of these VECs in immune cell recruitment. ACKR1+VECs were primarily located around airways as well as in distal alveolar regions exhibiting fibroblastic foci and aberrant basaloid cell accumulation, indicating cross communications between these pathogenic cells. In vitro analysis of freshly isolated IPF-derived ACKR1+VECs showed elevated expression of pro-fibrotic genes compared to ACKR1 negative (ACKR1-) ECs. Strikingly, co-culture of ACKR1+VECs with lung fibroblasts resulted in a strong upregulation of profibrotic genes and enhanced contractility relative to ACKR1-ECs. Conclusion: ACKR1+VECs are spatially restricted to lung areas exhibiting immune cell aggregation, fibroblastic foci, and aberrant basaloid cells in fibrotic lungs supporting their profibrotic function. IPF-derived ACKR1+VECs promote fibroblast activation in vitro, suggesting that ACKR1+VEC expansion contributes to fibrosis progression through paracrine signaling-mediated fibroblast activation. Taken together, our data offer a distinctive picture of the VEC dynamics associated with pulmonary fibrosis and suggest that aberrant expansion of ACKR1+VECs in IPF lungs may play a critical role in lung fibroblast activation and fibrosis progression.