Abstract
Pulmonary arterial hypertension (PAH) is an insidious disease characterized by severe remodeling of the pulmonary vasculature caused in part by pathologic changes of endothelial cell functions. Although heterogeneity of endothelial cells across various vascular beds is well known, the diversity among endothelial cells in the healthy pulmonary vascular bed and the pathologic diversity among pulmonary arterial endothelial cells (PAEC) in PAH is unknown and previously unexplored. Here single-cell RNA sequencing technology was used to decipher the cellular heterogeneity among PAEC in the human pulmonary arteries isolated from explanted lungs from three patients with PAH undergoing lung transplantation and three healthy donor lungs not utilized for transplantation. Datasets of 36,368 PAH individual endothelial cells and 36,086 healthy cells were analyzed using the SeqGeq bioinformatics program. Total population differential gene expression analyses identified 629 differentially expressed genes between PAH and controls. Gene Ontology and Canonical Ingenuity analysis revealed pathways that are known to be involved in pathogenesis, as well as unique new pathways. At the individual cell level, dimensionality reduction followed by density based clustering revealed the presence of eight unique PAEC clusters that were typified by proliferative, angiogenic or quiescent phenotypes. While control and PAH harbored many similar subgroups of endothelial cells, PAH had greater proportions of angiogenic and proliferative subsets. These findings identify that only specific subgroups of PAH PAEC have gene expression different than healthy PAEC, and suggest these subpopulations lead to the pathologic functions leading to remodeling.
Highlights
Pulmonary arterial hypertension (PAH) is an insidious disease characterized by severe remodeling of the pulmonary vasculature caused in part by pathologic changes of endothelial cell functions
In order to correct for this, Mutual Nearest Neighbors (MNN) batch effect correction was applied to the data matrix using the Batchelor algorithm[13], followed by a fresh round of Principal Component Analysis (PCA), and TriMap dimensionality reduction on MNN batch effect corrected data. (Supplemental Fig. S2)
Several pathways known to play a role in PAH were seen in these analyses, confirming that our cell populations were representative of PAH and healthy pulmonary vascular beds as previously published[5,11,14,15,16,17]
Summary
Pulmonary arterial hypertension (PAH) is an insidious disease characterized by severe remodeling of the pulmonary vasculature caused in part by pathologic changes of endothelial cell functions. While control and PAH harbored many similar subgroups of endothelial cells, PAH had greater proportions of angiogenic and proliferative subsets These findings identify that only specific subgroups of PAH PAEC have gene expression different than healthy PAEC, and suggest these subpopulations lead to the pathologic functions leading to remodeling. Because endothelial remodeling in PAH is p atchy[11], PAEC isolated from explanted lung contain a mixture of cells from healthy and affected areas. Analysis of these cells in bulk cultures may mask phenotypically altered PAEC subsets. Transcriptomic profiles identified expansion of clusters with proliferative and angiogenic gene signatures in PAH
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