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 existing 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 increased 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-inflammatory (IL-10, TGF-beta 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.