Proteomic differences between microvascular endothelial cells and the EA.hy926 cell line forming three‐dimensional structures

Proteomic changes of two types of human endothelial cells (ECs) were determined and compared to morphological alterations occurring during the scaffold‐free in vitro formation of 3D structures resembling vascular intimas. The EA.hy926 cell line and human microvascular ECs (HMVECs) were cultured on a random positioning machine or static on ground (normal gravity) for 5 and 7 days, before their morphology was examined and their protein content was analysed by MS after free‐flow electrophoretic separation. A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVEC forming 3D structures faster than the EA.hy926 cells. Five hundred and eighty‐four of these kinds of proteins were present in both types of cells. They included a number of metabolic enzymes, of structure‐related and stress proteins. Comparing proteins of EA.hy926 cells growing either adherently on ground or in 3D aggregates on the random positioning machine revealed that ribosomal proteins were enhanced, while tubes are formed and various components of 26S proteasomes remained prevalent in static normal gravity control cells only. The fast developing tube‐like 3D structures of HMVEC suggested a transient augmentation of ribosomal proteins during the 3D assembling of ECs.