A comparative study unraveling the effects of TNF-α stimulation on endothelial cells between 2D and 3D culture

Endothelial cell (EC) dysfunction is an important predictor of and contributor to the pathobiology of cardiovascular diseases. However, most in vitro studies are performed using monolayer cultures of ECs on 2D tissue polystyrene plates (TCPs), which cannot reflect the physiological characteristics of cells in vivo. Here, we used 2D TCPs and a 3D culture model to investigate the effects of dimensionality and cardiovascular risk factors in regulating endothelial dysfunction. Cell morphology, oxidative stress, inflammatory cytokines and endothelial function were investigated in human umbilical vein endothelial cells (HUVECs) cultured in 2D/3D. The differentially expressed genes in 2D/3D-cultured HUVECs were analysed using Enrichr, Cytoscape and STRING services. Finally, we validated the proteins of interest and confirmed their relevance to TNF-α and the culture microenvironment. Compared with 2D TCPs, 3D culture increased TNF-α-stimulated oxidative stress and the inflammatory response and changed the mediators secreted by ECs. In addition, the functional characteristics, important pathways and key proteins were determined by bioinformatics analysis. Furthermore, we found that some key proteins, notably ACE, CD40, Sirt1 and Sirt6, represent a critical link between endothelial dysfunction and dimensionality, and these proteins were screened by bioinformatics analysis and verified by western blotting. Our observations provide insight into the interdependence between endothelial dysfunction and the complex microenvironment, which enhances our understanding of endothelial biology or provides a therapeutic strategy for cardiovascular-related diseases.