15 February 2020 by Rafael Soares Godoy

Single-cell RNA sequencing reveals marked expansion of a discreet cluster of Apelinexpressing endogenous lung endothelial progenitors after induction of widespread endothelial injury

Introduction: Inhibition of VEGF receptors with SU5416 (SU) in rats results in widespread induction of endothelial cell (EC) apoptosis; which, when combined with transient exposure to hypoxia, produces a progressive model of severe pulmonary arterial hypertension (PAH). However, the mechanisms linking EC loss to occlusive arterial remodeling have not been delineated. We hypothesized that the balance between EC injury and repair is critical for theinitiation and progression of PAH.

Methods/Results: We used single-cell RNA sequencing (scRNAseq; 10x Genomics) to identify changes in gene expression profiles in multiple cell populations within the lung before and 1- week after induction of EC jury by a single injection of SU (20mg/kg). Using U-Map cluster analysis, we could identify 23 discrete clusters of cells in the uninjured lung based on transcriptional profiles, including 6 separate EC populations, the largest represented by microvascular (MV) ECs (73%), followed by venous (9%) and arterial ECs (4%) and a distant cluster with overrepresentation of genes related to proliferation and survival (5%), possibly
indicative of an endothelial progenitor cell population. In addition, there was a secondary rare EC group (7%), with abundant expression of proliferation and survival genes, nested close to the MV population, also distinguished by high expression of Apelin, which plays an important role in angiogenesis. One-week after SU alone, a marked shift EC gene expression was observed in the MV cluster while the proportion of arterial ECs was uniquely reduced by 3.9- fold. Interestingly, the secondary progenitor cell cluster was the only population that expanded after SU by more than 2-fold, suggesting a potential role in early lung microvascular repair.

Conclusions: scRNAseq resolved six distinct lung EC populations including two novel clusters that likely represent pools of endothelial progenitors, one of which exhibited high Apelin expression and was uniquely expanded after discrete EC injury.

Key Contributors

Rafael Soares Godoy, Nicholas Cober, Ketul Chaudhary, Yupu Deng, David Cook, Katelynn Rowe, Duncan J Stewart Sinclair Center for Regenerative Medicine, Ottawa Hospital Research Institute, Canada University of Ottawa, Faculty of Medicine, Ottawa, Canada

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