Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension

Abstract

Aims Endothelial cell dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterise endothelial cell (EC) dynamics in PAH at single-cell resolution. Methods and Results We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/Hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature. Conclusions This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

Single-Cell RNA Sequencing of Human Corpus Cavernosum Reveals Cellular Heterogeneity Landscapes in Erectile Dysfunction

“…In the endothelial clusters (C0, C2, C13), focal adhesion and protein localization are mainly enriched functions, especially compact cellular junctions in arterioles; in contrast, their physiological roles in angiogenesis and cell migration have not been apparent in severe ED. The heterogeneous phenotypes of ECs have been investigated in multiple tissues and diseases, showing that vascular ECs have transcriptome similarity across tissues but vary substantially in different pathological states ( 26 – 28 ). Many genes that are specifically expressed in ECs subpopulations other than arterial ECs can regulate ECM organization and the formation of caveolae such as MMRN1 and CAVIN2 ( 29 , 30 ).…”

Effect of invasive mechanical ventilation on the diversity of the pulmonary microbiota

“…Then hypoxia (10% O2 for 3 weeks)/weekly subcutaneous injection of 20 mg/kg Sugen 5416 ( n = 3), or normoxia for 3 weeks. Then sorting TdTomato + mouse lung cells for scRNA-seq TdTomato + mouse lung cells (endothelial cells) NovaSeq S2 (Illumina) 8 clusters Due to the injection of Sugen 5416 in hypoxia group (not in normoxia group), so the study design could not provide hypoxia-exclusive effect on endothelial cells Rodor et al [ 79 ] Rats (Sprague–Dawley) Adult (250 g–350 g) Male Subcutaneous injection of 20 mg/kg Sugen 5416 followed with 10% O2 for 21 days and the normoxia for 14 days ( n = 6); Normoxia control were kept for 28 days ( n = 6) Whole lung HiSeq 4000 (Illumina) 28 clusters Due to the injection of Sugen 5416 in hypoxia group (not in normoxia group), so the study design could not provide hypoxia-exclusive effect on lung cells Hong et al [ 78 ]

Fig. 2 Mouse studies based on single-cell transcriptome analysis suggest that hyperoxia or hypoxia can modulate the local immune microenvironment of the lung.

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“…A series of recent studies have revealed in depth by scRNA-seq techniques that lung cells generate local immune responses to hyperoxia or hypoxia, causing changes in the local immune microenvironment of the lung. A few of these studies identified different patterns of lung cell immune responses that can be triggered by hyperoxia [76,77] and hypoxia [78][79][80], respectively (Table 2, Fig. 2).…”

The Landscape of Noncoding RNA in Pulmonary Hypertension

“…These challenges for bulk RNA-seq have urged the development of single cell RNA-Seq (scRNA-Seq) techniques. Recent studies performed scRNA-Seq techniques on human lung tissues from IPAH patients [ 153 ], rat PH models and lung endothelial cells in a PH mouse model [ 154 ] and identified new dysregulated genes and pathways, and a subpopulation of cells in PH. These single-cell transcriptomic datasets generated from specifical cell types, animal PH models and human patients, have initiated the investigation of species-specific, model-specific, and subtypes of disease-specific variability in the cellular composition of lung and right ventricular in PH disease [ 155 ].…”