Effect of iron overload on endothelial cell calcification and its mechanism

Vascular calcification is related to many diseases. Iron has a certain relationship with endothelial cells and vascular calcification. The purpose of this study was to assess the effect of iron overload on endothelial cell calcification and related mechanisms through cell experiments.

Human umbilical vein endothelial cells were treated with different concentrations of FeSO₄ (50, 100, 150, and 200 µM), and deferoxamine (DFO) and ferrostatin. Alkaline phosphatase activity, malondialdehyde (MDA) level, reactive oxygen species (ROS) level, and lipid superoxidation after FeSO₄ treatment were assessed. Alizarin red staining was used to observe calcium deposition. Quantitative polymerase chain reaction (qPCR) and western blot were adopted to examine the expression of calcification markers, iron metabolism-related factors, apoptosis pathway-related factors and ferroptosis markers. The TUNEL method was employed to detect cell apoptosis.

FeSO₄ of 100 µM significantly promoted the occurrence of cell ferroptosis, increased the levels of MDA and ROS, and decreased the ratio of glutathione (GSH) or glutathione disulfide (GSSG) and the expression level of glutathione peroxidase (GPX4). The addition of DFO and ferrostatin significantly modified the effects of FeSO₄. Calcium deposition was most obvious in the cells treated with 100 µM FeSO₄. FeSO₄ significantly upregulated Runt-related transcription factor 2 (RUNX2) and Bone morphogenetic protein 2 (BMP2), ferritin heavy chain (FTH) and ferritin light chain (FTL), and downregulated Matrix Gla Protein (MGP) and divalent metal transporter 1 (DMT1). The results also showed that FeSO₄ induced cell apoptosis by TUNEL method. The elevated Bcl2-associated death protein (Bad) and Bcl2-associated X protein (Bax) and the reduction in Bcl-2, p-Bad, p-AKT, and t-AKT were found. DFO and ferrostatin significantly reduced the iron-induced calcification and apoptosis of endothelial cells. DFO significantly increased the expression level of Bcl-2, and reduced the expression level of Bad.

Iron overload contributes to the process of endothelial cell calcification by inducing apoptosis and ferroptosis. Iron chelators and ferroptosis inhibitors alleviate endothelial cell apoptosis, ferroptosis, and calcification induced by iron overload.