Endothelial Cell Heterogeneity Between Conduit and Resistance Mesenteric Arteries

Abstract

The vascular endothelium contains morphologically similar cells throughout, but individual cells along the length of a single vascular tree or in different regional circulations function quite differently. When observations made in large arteries are extrapolated to explain the function of endothelial cells (EC) in the resistance vasculature/microcirculation, only a fraction of these observations are consistent between artery sizes. To what extent EC from different arteriolar segments of the same tissue are phenotypically different at the single‐cell level remains unknown. Therefore, single‐cell RNA‐seq was performed using a 10X Genomics Chromium system. Cells were enzymatically digested from large (>300 µm) and small (<150 µm) mesenteric arteries to generate single‐cell suspensions from 6 adult male Sprague‐Dawley rats which were pooled to create two samples (3 rats/sample). Cell Ranger software was used for initial data processing, including alignment, filtering, and unique molecular identifier counting (10x Genomics). After initial data processing, Seurat, an R package for single‐cell sequencing analysis, combined datasets and conducted a comparative analysis across the phenotypes. Seurat’s data integration workflow transforms individual datasets into a shared matrix while controlling batch effects and technical differences. After normalized integration, the dataset was integrated and scaled before unsupervised cell clustering and cluster visualization using uniform manifold approximation and projection (UMAP) plots. Differential gene expression (DEG) allowed us to infer the biological identity of individual cells. EC clusters from large and small arteries were further subclustered. Our analysis revealed 601 DEG genes between conduit and resistance arteries. Gene ontology analysis of scRNA‐seq data discovered 449 that were different between large and small arteries. We identified six unique EC subpopulations, with DEG genes and pathways identified for each cluster. The proportion and absolute numbers of EC in each cluster differed between conduit and resistance arteries. These results and this dataset allow the discovery and support novel hypotheses needed to determine the mechanisms that determine the phenotypic heterogeneity between conduit and resistance arteries.