Abstract Rationale Pulmonary hypertension (PH) is a frequent complication of interstitial lung diseases (ILDs), leading to increased symptom burden and mortality. Nevertheless, the pathophysiology of ILD-associated PH remains poorly understood compared to rarer PH subtypes, including those classified as WHO Group 1 (G1PH). We analyzed single-cell RNA sequencing (scRNAseq) of lung tissue from individuals with ILD with/without associated PH to identify transcriptional changes that might precede and/or drive ILD-PH, hypothesizing that gene expression would differentiate 1) ILD from non-ILD tissue, and 2) ILD-PH from ILD without PH. Methods Explant lung tissue from 76 patients with ILDs, 26 non-ILD Controls, and 5 patients with G1PH were processed for scRNAseq using 10x Genomics Chromium 5’ system. Analysis of differential gene expression using a cell type-specific, subject-level pseudobulk strategy was performed, including comparisons 1) between ILD or G1PH vs. Control groups, and 2) between ILD-PH vs. ILD without PH. Transcription factor (TF) enrichment was performed using the CollecTRI gene regulatory network. A validation library was generated from tissue collected/sequenced at an independent center (43 ILDs, 36 Controls; 340K cells). Results In the integrated library (800K total cells), comparison of inferred TF enrichment in ILD (with and without PH) vs. Controls demonstrated a pattern of differential expression analogous to that in G1PH, including up-regulation of STAT3, SMAD2/3, and HOXD3 in arterial endothelial cells, and down-regulation of SREBF1/2 in distal epithelial cells. In addition, ILD samples (including without PH) were significantly enriched for venous endothelial cells expressing markers typical of systemic, rather than pulmonary, veins (PLVAP, COL15A1). These findings were confirmed in the independent validation library. In contrast, after merging our investigative and validation libraries, comparison between ILD-PH and ILD without PH yielded a pattern of differential expression distinct from G1PH, including up-regulation of STAT1, MYC, JUN, NFKB, and HIF1A across multiple endothelial populations in ILD-PH. ILD-PH was also associated with FOXO3 up-regulation across several immune-cell populations (CD4+ T cells, alveolar and monocyte-derived macrophages, DC2 cells), which was not seen in G1PH. Conclusions ILD, even without PH, was associated with changes in gene expression that resemble those associated with G1PH, implying an analogous process of endothelial remodeling in ILD that precedes the development of hemodynamic changes that define ILD-PH. In addition, ILD-PH was distinguished from both ILD generally and G1PH by unique changes in endothelial and immune cells, implying a distinct transcriptional program specific to ILD-PH that might underpin the clinical differences between ILD-PH and G1PH. This abstract is funded by: US Federal Government
Linsalata et al. (Fri,) studied this question.