Alamandine suppresses vascular calcification through inhibition of ferroptosis

Background and aims: Vascular calcification is commonly found in pathological processes of chronic kidney disease (CKD), diabetes and atherosclerosis, which increases the risk of adverse cardiac events. Recent studies have shown that Alamandine (ALA)/mas associated G protein coupled receptor D (MrgD), an axis of noncanonical renin-angiotensin system (Ras), exerts beneficial effects on cardiovascular systems. However, it is still unclear whether it protects against vascular calcification.

Methods: High phosphate and calcium were used to induce calcification of vascular smooth muscle cells (VSMCs) and mouse model of aortic calcification was induced by vitamin D₃. Alizarin red staining and calcium content assay were used to assess calcification. Western blot analysis was used to examine the protein expression levels.

Results: ALA serum levels were significantly lower in patients with thoracic calcification compared to healthy controls. High calcium and phosphate induced calcification of VSMCs. ALA treatment inhibited VSMC calcification and blockage of receptor MrgD abrogated the inhibitory effect of ALA on VSMC calcification. Consistently, ALA/MrgD significantly attenuated calcification of rat and human arterial rings ex vivo, and inhibited mouse aortic calcification in vivo. Mechanistically, VSMC calcification was accompanied by the occurrence of Ferroptosis as indicated by increased cell death, increased levels of Reactive Oxygen Species (ROS) and malondialdehyde (MDA), and decreased expression of Ferroptosis inhibition signaling molecules SLC7A11 and Glutathione Peroxidase 4 (GPX4). Furthermore, inhibition of GPX4 by RAS-selective lethal 3 (RSL3) exacerbated calcification of VSMCs under osteogenic conditions. Of note, ALA antagonized RSL3-induced VSMC calcification, suggesting ALA attenuated VSMCs calcification through inhibiting Ferroptosis.

Conclusions: our study for the first time revealed that ALA/MrgD suppressed VSMC calcification under osteogenic condition and aortic calcification in VitD₃-overloaded mice. Moreover, we unveiled that ALA/MrgD inhibited vascular calcification via modulation of Ferroptosis. These findings present a novel targeting strategy for the treatment of vascular calcification.

Alamandine; Ferroptosis; GPX4; Oxidative stress; Vascular calcification; Vascular smooth muscle cell.