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Single cell RNA-seq profiling of murine endothelial cells in response to pulmonary hypertension

ABSTRACT: (1) Rationale: Endothelial cell dysfunction plays a critical role in the development and pathogenesis of pulmonary arterial hypertension (PAH). (2) Objectives: We aimed to characterise the endothelial cell dynamics in PAH at a single cell resolution. (3) Methods: We carried out single-cell RNA sequencing of lung endothelial cells isolated from an endothelial cell lineage tracing mouse model in control and SU5416/Hypoxia-induced PAH conditions. (4) Measurements and Main Results: Endothelial cell populations corresponding to the different lung vessel types could be identified in both control and PAH mice. Differential gene expression analysis revealed novel global and vessel-type specific responses in endothelial cells due to PAH. Global changes included the up-regulation of the major histocompatibility complex class II pathway, supporting a role for endothelial cells in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population, with the up-regulation of genes involved in cell localization and angiogenesis. Comparison with human genetics and transcriptomics data revealed the regulation of four genes with variants associated to PAH and five genes up-regulated in endothelial cells in both human and mouse scRNA-seq. Among them, Aqp1 and Adam15 genes represent promising new candidates to target endothelial dysfunction. Finally, we identified zonation-dependent changes across the arteriovenous axis in PAH using an in-silico cell ordering approach and showed the up-regulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature. (5) Conclusions: This study uncovers the murine endothelial cell transcriptomics changes in PAH at a high resolution, revealing novel candidates relevant to PAH.

ORGANISM(S): Mus musculus

PROVIDER: GSE154959 | GEO | 2021/09/24

REPOSITORIES: GEO

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Project description:Bmpr2 mutations are critical risk factors for hereditary pulmonary arterial hypertension (hPAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-lipoxygenase (5-LO) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4 (LTB4), are candidates for the ‘second hit’. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically-silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to one year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus (AdAlox5), monocrotaline, SU5416 or chronic hypoxia and analyzed. Bmpr2-mutant hPAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells (PAECs) were cultured with lentivirus expressing short hairpin RNA (shRNA) targeting Bmpr2 (shBmpr2) to model the impaired BMPR2 signaling, and were then exposed to 5-LO-expressing adenovirus (AdAlox5), and were assessed for phenotypic and transcriptomic changes. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative PAECs with mesenchymal characteristics. These transformed cells expressed nuclear envelop-localized 5-LO consistent with induced LTB4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated NF-kB, IL-6 and TGF-β signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation. Thus, in a new two-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced LTB4 production. This study offers one explanation of how an environmental injury unleashes the destructive potential of an otherwise-silent genetic mutation.

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Single cell RNA-seq profiling of murine endothelial cells in response to pulmonary hypertension

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