Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta

Abstract

26Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular 27 heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. 28We report the comprehensive characterization of transcriptomes (bulk and single-cell) and 29 chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse 30 aortic endothelial cells. First, S1PR1 regulates NFkB and nuclear glucocorticoid receptor 31 pathways to suppress inflammation-related mRNAs. Second, spatially distinct S1PR1 signaling in 32 the aorta is associated with heterogenous endothelial cell (EC) subtypes. For example, a 33 transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-34 independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin 35 coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory signature. 36Moreover, an adventitial lymphatic EC (LEC) population shows suppression of lymphangiogenic 37 and inflammation-related transcripts in a S1P/S1PR1-dependent manner. These insights add 38 resolution to existing concepts of GPCR signaling and S1P biology. 39 40 41 et al., 2014). Adapted from the "Tango" system (Barnea et al., 2008), S1PR1-GFP signaling 65 (S1PR1-GS) mice convert ß-arrestin recruitment to the GPCR to H2B-GFP expression (Kono et 66 al., 2014). We previously used the S1PR1-GS mouse and showed that high levels of endothelial 67 GFP expression (i.e. S1PR1/ß-arrestin coupling) are prominent at the lesser curvature of the aortic 68 arch and the orifices of intercostal branch points (Galvani et al., 2015). In addition, inflammatory 69 stimuli (e.g. lipopolysaccharide) induced rapid coupling of S1PR1 to ß-arrestin and GFP 70 expression in endothelium in an S1P-dependent manner (Kono et al., 2014). These data suggest 71 that the S1PR1-GS mouse is a valid model to study GPCR activation in vascular ECs in vivo. 72To gain insights into the molecular mechanisms of S1PR1 regulation of endothelial 73 transcription and the heterogenous nature of S1PR1 signaling in vivo, we performed bulk 74 transcriptome and open chromatin profiling of GFP high and GFP low aortic ECs from S1PR1-GS 75 mice. We also performed transcriptome and open chromatin profiling of aortic ECs in which S1pr1 76 was genetically ablated (S1pr1-ECKO) (Galvani et al., 2015). In addition, we conducted single-77 cell (sc) RNA-seq of GFP low and GFP high aortic ECs. Our results show that S1PR1 suppresses the 78 expression of inflammation-related mRNAs by inhibiting the NFkB pathway. Second, the high 79 S1PR1 signaling ECs (GFP high cells) are more similar to S1pr1-ECKO ECs at the level of the 80 transcriptome. Third, scRNA-seq revealed eight distinct aorta-associated EC populations 81 including six arterial EC subtypes, adventitial lymphatic ECs, and venous ECs, the latter likely 82 from the vasa vasorum. S1PR1 signaling was highly heterogenous within these EC subtypes but 83 was most frequent in adventitial LECs an...

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