Human coronary artery endothelial cells

Primary human coronary artery endothelial cells (HCAEC) were obtained from Promocell (Germany). The HCAEC were isolated from the left and right coronary arteries from a single donor. Cells were cryopreserved immediately after isolation, shipped and stored in liquid nitrogen until use. Cells were then defrosted and cultured at 37 °C, 5% CO₂ in specialised endothelial growth cell medium (MV2, Promocell, Germany) until confluent. Primary cultures were dissociated using DetachKit (Promocell, Germany) and used for experiments at passage 4–6. Endothelial cell preconditioning was performed using the Ibidi pump system (Ibidi, Germany). HCAECs (1.7 × 10⁵) were seeded on to gelatin coated, 0.4 µm deep flow chambers (Ibidi µ-slide I^0.4 Leur, Ibidi, Germany) and incubated for 2 h at 37 °C and 5% CO₂ to allow adhesion. All materials, including slides, perfusion sets, and Ibidi units were autoclaved prior to being placed in an incubator at 37 °C and 5% CO₂ to equilibrate for at least 4 h prior to commencing the experiment. HCAECs were cultured under high unidirectional shear stress (HSS; 13 dyne cm⁻²) or low oscillatory shear stress (OSS; 4 dyne cm⁻², oscillating at 1 Hz) for 72 h. Apoptosis and necrosis rates were measured ± incubation with NMVs (1 × 10³ µL⁻¹) for 2 and 4 h using a flow cytometry-based assay that measured Annexin V binding and propidium iodide uptake. The percentage of cells that were positive for fluorescence for Annexin V or propidium iodide was quantified and it was found that NMVs did not alter HCAEC apoptosis or viability, respectively (Supplementary Fig. ^{14a, b}).