Human Aortic Endothelial Cell Shear Experiments

Human aortic endothelial cells (HAECs) were obtained commercially (GIBCO, C0065C or PromoCell, C-12271) and cultured according to the manufacturer’s standard protocol. HAECs were seeded at various density to meet the purposes of different studies, and maintained in endothelial cell medium containing growth supplements (EBM-2, Lonza, CC-3516, CC-4176 or PromoCell, C-22011) with 2% FBS. HAECs between passage 6 and 7 were used for experiments. For shear experiments, confluent HAECs in 100 mm dishes were exposed to steady laminar shear (LS, 15dyn/cm²), or oscillatory shear (OS, ±5dyn/cm²) conditions for 2 days using a cone-and-plate shear device as previously described.^{20 (link), 21 (link)} For shear experiments with gain of function or loss of function components, the commercially available Ibidi pump system (Ibidi, Germany) was used to reduce reagent use, and set up according to manufacturer instructions. Steady laminar shear (LS, 15dyn/cm²) or oscillatory shear (OS, ±5dyn/cm²) was applied to 80% confluent HAECs cultured overnight on microchannel slides (μ-Slide I^0.4 Luer, Ibidi, Germany) coated with 40μg/ml collagen (Collagen Type I, BD Biosciences 35-4236).