The Effect of TNF and VEGF on the Properties of Ea.hy926 Endothelial Cells in a Model of Multi-Cellular Spheroids

Endothelial cells play a major role in the development of inflammation and neoangiogenesis in cancer and chronic inflammatory diseases. In 3D cultures, cells are under conditions that closely resemble those exist-ing in healthy and disease-stricken human organs and tissues. Therefore, the development of a 3D model based on the Ea.hy926 endothelial cell line is an urgent need in molecular and cellular biology. Cell cultivation on an anti-adhesive substrate under static conditions was shown to lead to the formation of spheroids (3D cultures). Expression of ICAM-1 and VEGFR-2 and production of cytokines were screened in 2D and 3D cultures in the presence of TNF and VEGF. According to flow cytometry and confocal microscopy data, TNF significantly in-creased the expression of the cell adhesion molecule ICAM-1 in both 2D and 3D cultures but did not affect the expression level of VEGFR-2. Increased production of pro-inflammatory (IL-8, IL-6, IP-10) and anti-inflam-matory (IL-10, TGF-β 1–3) factors was observed in spontaneous 3D cultures but not in 2D cultures, which was confirmed by flow cytometry and qPCR. TNF-induced secretion of IL-10, GM-CSF, and IL-6 was 11-, 4.7-, and 1.6-fold higher, respectively, in 3D cultures compared to 2D cultures. Thus, the use of a Ea.hy926 3D cell culture is a promising approach in studying the effects of anti- and pro-inflammatory agents on endothelial cells.