Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification

Abstract

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD). Cell death such as apoptosis plays a critical role in vascular calcification. Ferroptosis is a type of iron-catalyzed and regulated cell death resulting from excessive iron-dependent reactive oxygen species and lipid peroxidation. However, it is unclear whether ferroptosis of vascular smooth muscle cells (VSMCs) regulates vascular calcification in CKD. Our results showed that high calcium and phosphate concentrations induced ferroptosis in rat VSMCs in vitro. Inhibition of ferroptosis by ferrostatin-1 dose-dependently reduced mineral deposition in rat VSMCs under pro-osteogenic conditions, as indicated by alizarin red staining and quantification of calcium content. In addition, gene expression analysis revealed that ferrostatin-1 inhibited osteogenic differentiation of rat VSMCs. Similarly, ferrostatin-1 remarkably attenuated calcification of rat and human arterial rings ex vivo and aortic calcification in vitamin D₃-overloaded mice in vivo. Moreover, inhibition of ferroptosis by either ferrostatin-1 or deferoxamine attenuated aortic calcification in rats with CKD. Mechanistically, high calcium and phosphate downregulated expression of SLC7A11 (a cystine-glutamate antiporter), and reduced glutathione (GSH) content in VSMCs. Additionally, GSH depletion induced by erastin (a small molecule initiating ferroptotic cell death) significantly promoted calcification of VSMCs under pro-osteogenic conditions, whereas GSH supplement by N-acetylcysteine reduced calcification of VSMCs. Consistently, knockdown of SLC7A11 by siRNA markedly promoted VSMC calcification. Furthermore, high calcium and phosphate downregulated glutathione peroxidase 4 (GPX4) expression, and reduced glutathione peroxidase activity. Inhibition of GPX4 by RSL3 promoted VSMC calcification. Thus, repression of the SLC7A11/GSH/GPX4 axis triggers ferroptosis of VSMCs to promote vascular calcification under CKD conditions, providing a novel targeting strategy for vascular calcification.

Keywords: SLC7A11; chronic kidney disease; ferroptosis; oxidative stress; vascular calcification; vascular smooth muscle cell.