Transcriptional profiling of lung cell populations in idiopathic pulmonary arterial hypertension

Abstract

Despite recent improvements in management of idiopathic pulmonary arterial hypertension, mortality remains high. Understanding the alterations in the transcriptome-phenotype of the key lung cells involved could provide insight into the drivers of pathogenesis. In this study, we examined differential gene expression of cell types implicated in idiopathic pulmonary arterial hypertension from lung explants of patients with idiopathic pulmonary arterial hypertension compared to control lungs. After tissue digestion, we analyzed all cells from three idiopathic pulmonary arterial hypertension and six control lungs using droplet-based single cell RNA-sequencing. After dimensional reduction by t-stochastic neighbor embedding, we compared the transcriptomes of endothelial cells, pericyte/smooth muscle cells, fibroblasts, and macrophage clusters, examining differential gene expression and pathways implicated by analysis of Gene Ontology Enrichment. We found that endothelial cells and pericyte/smooth muscle cells had the most differentially expressed gene profile compared to other cell types. Top differentially upregulated genes in endothelial cells included novel genes: ROBO4, APCDD1, NDST1, MMRN2, NOTCH4, and DOCK6, as well as previously reported genes: ENG, ORAI2, TFDP1, KDR, AMOTL2, PDGFB, FGFR1, EDN1, and NOTCH1. Several transcription factors were also found to be upregulated in idiopathic pulmonary arterial hypertension endothelial cells including SOX18, STRA13, LYL1, and ELK, which have known roles in regulating endothelial cell phenotype. In particular, SOX18 was implicated through bioinformatics analyses in regulating the idiopathic pulmonary arterial hypertension endothelial cell transcriptome. Furthermore, idiopathic pulmonary arterial hypertension endothelial cells upregulated expression of FAM60A and HDAC7, potentially affecting epigenetic changes in idiopathic pulmonary arterial hypertension endothelial cells. Pericyte/smooth muscle cells expressed genes implicated in regulation of cellular apoptosis and extracellular matrix organization, and several ligands for genes showing increased expression in endothelial cells. In conclusion, our study represents the first detailed look at the transcriptomic landscape across idiopathic pulmonary arterial hypertension lung cells and provides robust insight into alterations that occur in vivo in idiopathic pulmonary arterial hypertension lungs.

Keywords: endothelial cells; pericytes; pulmonary arterial hypertension; single cell RNA-sequencing.

Fig. 1.

Histopathology of lungs with idiopathic pulmonary arterial hypertension. Histopathology lung explant tissue adjacent to that used for scRNA-seq, showing intimal hyperplasia (arrowhead) and plexiform lesion (arrow) (a–c; magnification 40×, 40 × and 100×, respectively), t-SNE plots showing clusters (d) and origin of cells from control (yellow) and IPAH (purple) lungs (e), proportion of control (yellow) and IPAH (purple) cells in each cluster (f), percentage of proliferating endothelial cells in each control and IPAH lungs (g). IPAH: idiopathic pulmonary arterial hypertension.

Fig. 2.

Regulon and predicted transcription factors regulating endothelial cell transcriptome expression in IPAH endothelial cells. Cluster of transcription factors predicted to be upregulated in IPAH endothelial cells (panel a). T-SNE clustering by regulon (panels b–d), showing the origin of the cells: IPAH (blue) versus control (red) lung explants (panel b). SOX18 expression in regulon t-SNE is indicated by intensity of yellow/brown color (panel c). Level of SOX18 regulon activity in SOX18 regulon t-SNE is indicated by intensity of blue color (panel d). IPAH: idiopathic pulmonary arterial hypertension.

Fig. 3.

Expression of genes associated with hereditary IPAH in different cell types. Violin plots indicate expression of genes associated with hereditary IPAH in each cluster (cluster numbers as in Fig. 1) of cells from both IPAH and control lungs (panels on left). Violin plots showing expression of the cell type with the highest expression in left panels, show expression divided between control and IPAH lungs (panels on the right). For most genes, highest expression is cluster #3 (endothelial cells). For KCNK3, highest expression is in pericytes (cluster #13). For EIF2AK4 highest expression is in dendritic cells (cluster #12).