Primary human umbilical vein endothelial cells

Primary human umbilical vein endothelial cells from pooled donors were purchased from Lonza (Walkersville, Maryland, USA) and cultured according to the manufacturer’s instructions in 5% CO₂ at 37°C in humidified air. Cells were cultured in endothelial cell basal medium-2 (EBM-2) supplemented with EBM-2 SingleQuot (Lonza), pH 7.6–8.0. Media was changed every other day until cells were 70%–80% confluent. HUVECs were subcultured using TryPLE Express, pH 8.0 (ThermoFisher Scientific, Waltham, Massachusetts, USA). Cell growth was limited to 12 population doublings, and all experiments were carried out using cells between passages 3–5. For serum experiments, cells were cultured with 20%–50% serum for 6 or 24 hours prior to further analysis.

Primary human umbilical vein endothelial cells (Lonza Biologics PLC, Slough, UK) were cultured in EGM-2 medium and passaged according to the manufacturers' recommendations. Cells (4×10⁴/well) between passage 2 and 5 were plated in 0.33 cm² wells and transfected with 50 nM non-targeting small interfering RNA (siRNA, siCtrl, D-001810-01, Dharmacon, Inc., Lafayette, CO, USA), or with siRNA targeting egfl7 [siEgfl7, J-015668-10, Dharmacon, Inc., (15 (link),16 (link))] or ICAM-1 (siICAM-1, L-003502-00 Dharmacon, Inc.) using Lipofectamine RNAiMax (Life Technologies, ThermoFisher Scientific, Inc., Waltham, MA, USA). Three days later, the cells were lysed and total RNA isolated using the Nucleospin RNA kit and reagents (Macherey-Nagel, Düren, Germany). RNA were quantified using a Nanodrop (ThermoFisher Scientific, Inc.) and RT was performed using the High Capacity Reverse Transcription kit (Life Technologies, ThermoFisher Scientific, Inc.). qPCR was performed in duplex reactions, mixing TaqMan FAM-labelled probes for human egfl7 (Hs00211952_m1) or for ICAM-1 (Hs00164932.m1) with the normalizing β2-microglobulin (B2M) VIC-labelled probe in the same tube and processed in a StepOne machine. Data were expressed as 2^−ΔΔCq where ΔCq=Cq of gene-Cq of B2M, and ΔΔCq=ΔCq sample-ΔCq control.

Primary human umbilical vein endothelial cells were purchased from Lonza. Mouse brain endothelial cells (bEnd.3) were obtained from American Type Culture Collection (Manassas, VA). Human umbilical vein endothelial cell and bEnd.3 cells were cultured as described earlier.^{45 (link)} Human peripheral blood mononuclear cells (PBMCs) were isolated from blood from healthy donors by density gradient centrifugation using Ficoll Paque (GE Healthcare, Pittsburg, PA). The Institutional Review Board at the UT Health Science Center at Tyler approved the blood donation protocol, and the participants gave written informed consent.

Primary human umbilical vein endothelial cells (Lonza) were maintained in endothelial growth medium EGM-2 (Lonza) and used at passages 4–8. Human HEK-293T cells (Clonetech) were cultured in high-glucose DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin (Life Technologies) and 2 mM l-glutamine (Life Technologies). All cell types were maintained at 37°C in 5% CO₂ in a humidified incubator.