Contact with smooth muscle cells dictates the endothelial transcriptome

The phenotypes of endothelial cells (ECs) in conduit and resistance arteries differ in part due to the presence of myoendothelial junctions in resistance arteries. These are direct contact points between ECs and smooth muscle cells (SMCs) that form through holes in the inner elastic lamina. To investigate the consequences of SCM contact on endothelial phenotypes, we used transwells to culture ECs in three conditions: cultured without SMCs (monolayer ECs), co-cultured without direct contact with SMCs (no-contact ECs), and co-cultured with SMCs such that myoendothelial junctions could form (contact ECs). We then collected the ECs and performed bulk RNA sequencing. These data demonstrate the impact of SMC-derived signals on endothelial phenotypes. Overall design: To model resistance arteries, endothelial cells were cultured following the vascular cell co-culture model as previously reported. Briefly, human coronary artery smooth muscle cells (SMCs) were plated (75,000 cells/well) on the bottom of a 6-well plate or on the underside of the transwell. The following day, human coronary artery ECs were seeded on the top side of the transwell. An additional group of ECs were plated in a transwell dish without smooth muscle cells. In all three conditions, endothelial cell growth media MV (Promocell) was placed on the top of the transwell and SMC media (Lonza) was placed in the bottom chamber of the transwell. Two days after plating ECs, the media was changed, and ECs were collected the following day. For transcriptomics, RNA was isolated using Quick-RNA MiniPrep (Zymo Research). PE150 reads were generated with the Illumina NovoSeq platform. The paired-end reads were mapped to the hg38 reference genome using STAR (v2.7.9a). Gene counts were generated with featureCounts in the Subread package. For all datasets, DESeq2 (v1.34.0) was used to identify differentially expressed genes (DEGs) (Padj < 0.05). Ensemble IDs were converted to gene symbols using the org.Hs.eg.db package.