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

Abstract

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.

Single Cell Transcriptome Data Analysis Defines the Heterogeneity of Peripheral Nerve Cells in Homeostasis and Regeneration

“…In the intact nerve, in addition to the lymphatic EC cluster (cluster 13, Figure 1A ) identified by marker genes for lymphatic ECs such as Lyve1, Mmrn1, Prox1, and Flt4 ( Supplementary Figure 1 ) (Engelbrecht et al, 2020 ; Fujimoto et al, 2020 ; Wolbert et al, 2020 ), our analysis shows three distinct sub-clusters of blood vessel ECs in the intact nerve not described in the previous studies (cluster 3, 4, and 5, Figure 1A ). All three new EC sub-clusters express classic EC marker genes such as Pecam1/Cd31, Tie1, and Emcn ( Supplementary Figure 2 ) (Zhao et al, 2018 ; Carr et al, 2019 ; Kalluri et al, 2019 ; Toma et al, 2020 ; Wolbert et al, 2020 ).…”

“…Previous studies using scRNA-seq to analyse endothelial cell profiles of specific structures of the blood supply system such as the aorta, the hematopoietic niche, the lymph nodes and the heart, often resulted in the identification of 2–4 sub-clusters of endothelial cells. These studies showed that it can be very difficult to name individual sub-clusters of EC cells because of overlapping expression patterns of key endothelial cell genes (Kenswil et al, 2018 ; Feng et al, 2019 ; Kalluri et al, 2019 ; Engelbrecht et al, 2020 ; Fujimoto et al, 2020 ).…”

“…Recently, Wolbert et al also identified lymphatic ECs in the intact nerves in their scRNA-seq data analysis using lymphatic EC marker genes Lyve1 and Prox1 (Wolbert et al, 2020 ). Other scRNA-seq data analysis have shown that lymphatic ECs have distinct gene expression profiles including the expression of Lyve1, Mmrn1, Prox1, and Flt4 (Engelbrecht et al, 2020 ; Fujimoto et al, 2020 ). Therefore, we used these marker genes to identify the cluster containing lymphatic ECs.…”

High-Density Lipoproteins in Kidney Disease

“…In addition to regulating glomerular and tubular cells, apoA-I/HDL modulates the phenotype of lymphatic endothelial cells and growth of the lymphatic vascular network [ 76 , 77 ]. S1P/S1PR1 signaling is involved in the regulation of lymphangiogenic and inflammation-related gene expression in lymphatic endothelial cell (LECs) [ 78 ]. We found that IsoLG-modified apoA-I stimulated higher expression of inflammation markers (IL-6, IL-23, CCL21) and S1P related genes (SPHK2, SPNS2) in cultured LECs than unmodified apoA-I.…”

Endothelial ontogeny and the establishment of vascular heterogeneity

“…[66] This trend is also observed when restricting analyses to a large artery; scRNA-seq analysis of the adult aorta identified multiple BEC clusters, with expression patterns reflecting specific anatomical locations (i.e., branch points) and suggesting distinct roles in adhesion, signaling, and immune function. [67] Although largescale comparisons across different organs have failed to capture significant heterogeneity among LECs, [64] targeted scRNA-seq of lymph node lymphatic vessels revealed multiple subpopulations that may play distinct roles in neutrophil homing. [68] More examples of EC transcriptional heterogeneity emerge on a frequent basis with new scRNA-seq datasets.…”