Cell Lines and Virus Infection

Primary human aortic endothelial cells (HAECs) were obtained from PromoCell (Heidelberg, Germany) and cultured or passaged in the endothelial cell growth medium MV2 (PromoCell, C-22022, Germany) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Cell detachment was performed with 0.05% trypsin in ethylene diamine tetra acetic acid (EDTA; Thermo Fisher, 25300120, United States). All cells used were between passages 3 and 8.

The human cytomegalovirus (HCMV) VHL/E strain was used in this study. The virus was propagated in Retinal Pigment epithelial cells (RPE-1). The RPE-1 cells were harvested after HCMV infection and underwent a freeze /thaw process to release the virus. Infection of HAECs was conducted at a multiplicity of infection (MOI) of 5 as determined by titration on RPE-1 cells. Then, the HAECs were incubated at 37°C for 2 h to absorb the virus, washed three times with phosphate buffer solution (PBS), and added with fresh complete medium. Cell fractions or supernatants were harvested at various postinfectious times.