Mapping disease-specific vascular cell populations responsible for obliterative arterial remodeling during development of pulmonary arterial hypertension

Background Pulmonary arterial hypertension (PAH) is a lethal pulmonary vascular disease characterized by arteriolar pruning and occlusive vascular remodeling leading to increased pulmonary vascular resistance and eventually right heart failure. While endothelial cell (EC) injury and apoptosis are known triggers for this disease, the mechanisms by which they lead to complex arterial remodeling remain obscure. Aims We employed multiplexed single-cell RNA sequencing at multiple timepoints during the onset and progression of disease in a model of severe PAH to identify mechanisms involved in the development of occlusive arterial lesions. Methods and Results Single cell transcriptional analysis resolved 44 global lung cell populations, with widespread early transcriptomic changes at 1 week affecting endothelial, stromal and immune cell populations. In particular, two EC clusters were greatly expanded during PAH development and were identified as being disease specific: a relatively dedifferentiated (dD) EC population that was enriched for Cd74 expression while exhibiting a loss of endothelial identity; and an activated arterial EC (aAEC) population that uniquely exhibited persistent differential gene expression throughout PAH development consistent with a growth regulated state. dDECs were primed to undergo endothelial-mesenchymal transition as evidenced by reduced activity of master EC transcription factors, Erg and Fli1, and further supported by RNA velocity analysis showing vectors leading to fibroblast clusters. Of note, aAECs exhibited high expression of Tm4sf1, a gene implicated in cancer cell growth, that was also expressed by a smooth muscle (SM)-like pericyte cluster, and were highly localized to regions of arterial remodeling in both the rat model and PAH patients, contributing to intimal occlusive lesions and SM-like pericytes forming bands of medial muscularization. Conclusions Together these findings implicate disease-specific vascular cells in PAH progression and suggest that TM4SF1 may be a novel therapeutic target for arterial remodeling. Translational Perspectives Using single cell transcriptomic analysis in both human lung samples and a rat model of severe PAH we have identified disease-specific EC populations contributing to complex arterial remodeling, including growth-dysregulated, activated arterial ECs (aAECs) and dedifferentiated ECs (dDECs) that may be primed for endothelial to mesenchymal transition. In particular, aAECs exhibit high expression of a surface marker, TM4SF1, which is essential for their hyper-proliferative phenotype and represents a promising therapeutic target for RNA silencing approaches or as an antigen to guide immune-mediated ablation of this cancer-like EC population in PAH.