The contributions of increased reactive oxygen species and inflammatory macrophages to the development of aortic valve calcification

Abstract

Calcific aortic valve disease (CAVD), an active cardiovascular disease, is the most common cause of aortic valve stenosis. The risk of developing CAVD increases with age. In this study, a three-dimensional (3D) model of the aortic valve fibrosa layer was created to evaluate the role of monocytes/macrophages in the development of CAVD in the presence of reactive oxygen species (ROS). The 3D model includes porcine aortic valve interstitial cells (PAVIC), porcine aortic valve endothelial cells (PAVECs), and THP-1 monocytes that have differentiated into M1-like macrophages. Hydrogen peroxide (H₂O₂) was used to induce ROS at a concentration of 100 μM to mimic pathophysiological ROS conditions and promote calcification while maintaining cell viability. Prolonged exposure to H₂O₂ (up to 14 days) caused calcification in the in vitro 3D model. The introduction of THP-1 monocytes or M1-like macrophages after pretreatment with H₂O₂ accelerated the calcification process. The M1-tri-culture model formed nodules with increased calcium and phosphate when compared with controls and co-culture models. Except in the M1-tri-culture model, H₂O₂ treatment inhibited hydrogel contraction, indicating a connection between ROS and cellular contraction. These findings provide information on the pathogenesis of CAVD, demonstrating the crucial role of monocytes/macrophages and ROS in the calcification of the aortic valve fibrosa layer. This 3D model offers a robust platform for additional study on CAVD-specific therapeutic strategies.