Altered Inflammatory Protein Release by Vascular Smooth Muscle Cells in 3D-Culture

Aortic smooth muscle cells (AoSMCs) are widely used in cardiovascular research for studies on inflammatory processes in atherosclerotic lesions. Usually, AoSMCs are cultured in two-dimensional (2D) cell culture models, although limitations exist compared to the three-dimensional (3D) environment, and differences between 2D and 3D conditions on inflammatory markers have not been explored. This study aims to elucidate differences in inflammatory biomarkers in 2D versus 3D cultured AoSMCs. After inflammatory treatment of 2D and 3D cultured cells, protein release and gene expression were measured by Olink proteomics/enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. The 2D cultured cells showed generally higher release of inflammatory proteins, such as C-C motif chemokine 20 (CCL20), transforming growth factor alpha (TGF-α) and TNF superfamily member 12 (TWEAK), compared to the 3D model. In contrast, more matrix metallopeptidase-10 (MMP-10) and interleukin-8 (IL-8) were released from 3D cultured cells compared to 2D cultured cells. Elevated release of interleukin-1β in addition to transcriptional activation of inflammasome components was also observed in the 2D condition compared to the 3D condition. In conclusion, release of inflammatory proteins differs depending on the cell culture model, where 2D cultured AoSMCs release higher levels of several pro-inflammatory proteins compared to 3D cultured AoSMCs.