Investigating murine endothelial cell injury in pulmonary arterial hypertension using single-cell RNA sequencing

Endothelial cells (ECs) are highly heterogenous across different organs and within the vascular bed, in both health and disease. However, EC heterogeneity has not been extensively dissected in the context of pulmonary arterial hypertension (PAH). It is a rare and progressive disease characterised by remodelling of the distal pulmonary vasculature. Using an EC lineage-tracing mouse model and single-cell RNA-sequencing (scRNA-seq), I first investigated how enriched mouse ECs respond to Sugen 5416/Hypoxia-induced (SuHx) PAH. Globally, ECs adopt an immunophenotype in PAH, with upregulation of genes relating to the major histocompatibility class II (MHC-II) complex. The strongest upregulation is observed in Artery and one of the two capillary ECs (Capillary A/gCap). The other capillary EC cluster (Capillary B/aerocyte) had a distinct response to PAH, with upregulation of genes regulating apoptosis, migration, and angiogenesis. Comparison with whole-lung scRNA-seq in rat and human PAH identified several candidate genes conserved across all species. Following this, I further examined heterogeneity within Artery and Capillary A ECs, the pulmonary vasculature predominantly affected in PAH. Artery ECs can be subdivided into Proximal and Distal Artery ECs, with both groups having distinct biomarkers. In PAH, a loss of Proximal Artery EC identity and a gain in Distal Artery EC identity is observed. As for Capillary A ECs, three subpopulations were identified. In Control, two of these subclusters (CapillaryA_0, CapillaryA_1) were marked by high expression of Distal Artery markers, and low expression of Proximal Artery markers, whilst the reverse was true for the third CapillaryA_2 subcluster. There was also significantly more CapillaryA_2 ECs in PAH. Differential gene expression analysis found some genes uniquely upregulated in each CapillaryA subcluster: senescence-associated genes in CapillaryA_0 (Cd9, H3f3b, Neat1), signalling genes in CapillaryA_1 (Rab5a, Slc6a6, Tmem252), and both inflammatory (H2-Q4, Irgm1, Tgtp2) and angiogenesis regulatory genes (Cldn5, Cxcl12, Esam, Rhoj) in CapillaryA_2. Overall, this work has revealed in high-resolution, how ECs in different vascular beds distinctly respond to PAH.