Fibroproliferative Remodeling of Immune‐Vascular Co‐Culture Spheroid Models in Response to Atherogenic Stimulation

ABSTRACT

Cell‐based models are highly valuable platforms for preclinical studies of atherosclerosis, offering controllable environments to investigate disease mechanisms. Three‐dimensional (3D) spheroids, a class of cell‐based models, enable the study of atherosclerotic disease phenomena under physiologically relevant yet controlled conditions. In this study, we examined the lipid‐induced changes in a co‐culture spheroid model comprising THP‐1‐derived macrophages and primary human coronary artery smooth muscle cells (HCASMCs) following treatment with 20 or 40 µg/mL oxidized LDL (ox‐LDL), a known atherogenic agent. Over a 10‐day period, we assessed changes in spheroid morphology (size and compactness), cell viability, metabolic activity, intracellular composition, gene expression, pro‐inflammatory cytokine release and extracellular matrix deposition, relative to the untreated control. Lipid exposure increased the abundance of macrophages in ox‐LDL‐treated spheroids (LS20‐3 h: 35.5 ± 21.9%; LS40‐3 h: 38.3 ± 8.7%) compared with controls (21.8 ± 0.7%) as early as day 3. Upregulation of fibronectin (LS20‐3 h: ~1.8‐fold; LS40‐3 h: ~2.1‐fold), CD36 (~1.8‐fold), and ABCA‐1 (~1.9‐fold) was also observed. Moreover, spheroids exposed to 40 µg/mL ox‐LDL exhibited reduced viability and compactness compared with those treated with 20 µg/mL by day 10. While lipid‐treated spheroids showed non‐statistically significantly higher levels of pro‐inflammatory IL‐1β cytokine expression at day 0 which declined (> 85% decrease) by day 3, all spheroid groups showed comparable global fibronectin ECM secretion indices. Collectively, these findings reveal lipid‐induced proliferative changes to spheroid composition & functionality and show their potential as biomimetic models for the study of atherosclerotic disease phenomena.