Longitudinal Transcriptional Response of Endothelial Cells to Shear Stress

The goal of this study was to find longitudinal transcriptional response of Human Umbilical Vein Endothelial Cells (HUVECs) to pulsatile shear (PS) and oscillatory shear (OS). PS is associated with an atheroprotective endothelial phenotype, while OS is associated with an atheroprone endothelial phenotype. Using RNASeq method (single-ended 50-bp sequencing on Illumina Hi-seq 2000 instrument), we measured the transcriptional response at 10 time-points (1, 2, 3, 4, 6, 9, 12, 16, 20, 24 hr) under PS and OS conditions. Low flow scenario was used as static condition. Two replicates were used for each condition/time-point. Results: Through combining the temporal data on differentially expressed transcription factors and their targets with existing knowledge on relevant functional pathways, we infer the causal relationships between disparate endothelial functions through common transcriptional regulation mechanisms. Our study presents the first comprehensive temporally longitudinal experimental study and mechanistic model of shear stress response. By comparing the relative endothelial expressions of genes between OS and PS, we provide novel insights and an integrated perspective into endothelial cell function in response to differential shear. Overall design: HUVECs were cultured in medium M199 (Gibco) supplemented with 15% FBS (Omega), 3 ng/mL ß-EC growth factor (Sigma), 4 U/mL heparin (Sigma), and 100 U/mL penicillin-streptomycin. Pulsatile shear ?ow (PS) or oscillatory shear ?ow (OS) was applied to ECs with a shear stress of 12 ± 5 dyn/cm2 or 0.5 ± 5 dyn/cm2, respectively. Samples for RNA sequencing analysis were collected at 1, 2, 3, 4, 6, 9, 12, 16, 20, and 24 hours after exposure to shear. Endothelial cells (ECs) were also exposed to a “low flow” condition, i.e. 0.5 dyn/cm2 laminar flow, and designated as a “static” flow condition (ST). Samples for RNA sequencing analysis were collected at hour 1 for the ST condition. Two biological replicates were collected for each time point and for each shear condition. Total RNA was extracted from ECs, processed and sequenced on the Illumina HiSeq 2000 50-bp single-end reads platform. More details are provided in Ajami et al. (Systems Biology Analysis of Longitudinal Functional Response of Endothelial Cells to Shear Stress, Proc Natl Acad Sci U S A., 2017).