Identification of transcriptional drifts in endothelial cells associated with environmental changes

Purpose: Environmental cues, such as shear stress and heterotypic cell interactions play a critical role in endothelial cell function, yet their unique contributions to the endothelial cell transcriptome remain unclear. Methods: Using cell preparations from human umbilical cords (ex vivo), we performed individual sample analysis to assess transcriptional drifts associated with environmental changes but independent of sex or background. Global gene expression profiling by RNA-seq, ATACseq, and protein expression by LC-MS distinguished freshly isolated endothelial cells from genetically matched culture (in vitro) samples. Results: Over 43% of the transcriptome was significantly changed by the in vitro environment. Amongst several signatures, we observed that the expression of TGF-beta and BMP target genes were reduced. In contrast, cytoskeleton-based processes and proliferation-related genes were increased. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes including targets of BMP and Notch signaling known to be sensitive to flow. In contrast, co-culture of endothelial cells with smooth muscle cells normalized networks related to cell growth and differentiation, clathrin-vesicle related genes, and recovered targets downstream TGF-beta, recovering approximately 9% of the original in vivo signature. Conclusions: Our findings highlight specific genes, pathways and functional features of endothelial cells that require contextual information and exposure to physical forces. This transcriptional modulation is important to consider in all paradigms that are focused on understanding the ability of endothelial cells to maintain homeostasis and respond to disease processes. Overall design: HUVEC mRNA profiles under various culture conditions