C26-08 Single-cell RNA Sequencing of Pulmonary Artery Endothelial Cell Biopsies Characterizes Endothelial Heterogeneity in Pulmonary Vascular Disease

Abstract Background Pulmonary artery endothelial cell (PAEC) heterogeneity is increasingly described and linked to mechanisms of vasculopathy in pulmonary hypertension (PH). Studies that explore this heterogeneity in PH patients during the course of their disease are lacking. Methods PAECs were harvested from right heart catheter tips, expanded and frozen fresh (passage P0) through P3. Specimens were submitted to Genewiz (South Plainfield, NJ) for single-cell RNA sequencing. Pseudobulk expression profiles were aggregated by specimen and passage, followed by differential expression and pathway analysis (DESeq2 in R). Canonical endothelial, progenitor, and non-endothelial gene programs were quantified using curated marker sets, and pathway enrichment analyses were used to assess biological processes altered with passaging and by PH subtype and disease severity. Associations between gene program scores and clinical or phenotypic variables were assessed using ANOVA, Pearson and Spearman correlation tests, as appropriate. Results Cell biopsies from two idiopathic pulmonary arterial hypertension (PAH), one heritable PAH, two Group 3 PH, two chronic thromboembolic PH, and two control (no hemodynamic PH) participants (female n = 6, male n = 4, total n = 10) were included. P0 samples showed some transcriptomic divergence from later passages. Despite this separation, all samples retained canonical EC markers across passages and by P1 low-EC contamination resolved in four specimens (EC215, EC216, EC217, EC223; Figure, Panel A). Of these, two P0 specimens (EC215P0 and EC223P0; from a control and Group 3 PH participant with idiopathic pulmonary fibrosis, respectively) demonstrated strong non-endothelial signatures (Panel B). As idiopathic PAH-derived PAECs progressed from P0 to P3, genes involved in metabolism and inflammation were increasingly expressed. Compared to controls, idiopathic PAH PAECs upregulated immune and inflammatory pathways, highlighting disease-specific transcriptional activation. Loss of endothelial identity (R2=0.78, p = 0.002) and gain of EC progenitor-like identity (R2=0.58, p = 0.02) was most pronounced in cells exhibiting the greatest transcriptional drift (Panel C) and correlated with higher mean pulmonary arterial pressure (mPAP)(Panel D; r = 0.64, p = 0.09), independent of clinical subtype. Conclusions Cell biopsy from right heart catheter tips retain EC phenotype over serial passaging from a diverse cohort of PH patients and controls. Cells that lose endothelial identity acquire EC progenitor-like traits and associate with more severe hemodynamic impairment, suggesting that endothelial reprogramming and heterogeneity may be a marker of cellular dysfunction in pulmonary vascular disease. This abstract is funded by: R01-HL141268 (CEV), R01-HL174007 (CEV), P20-GM103652 (EOH, CEV), T32-HL134625 (NS, EOH, CEV)