Macrophage P2Y12 regulates iron transport and its inhibition protects against atherosclerosis

Iron retention is commonly observed in atherosclerotic plaques and is believed to be detrimental to atherosclerosis. Platelet P2Y12 is a target of antiplatelet therapy in preventing thrombotic complications of atherosclerosis. The protective effect of P2Y12 on hematopoiesis reported by our previous work implies the involvement of P2Y12 in iron metabolism.

This study further investigated the role of P2Y12 in the iron metabolism of macrophages, the key player in systemic iron homeostasis and atherosclerosis.

The association between serum iron and the use of P2Y12 inhibitors was evaluated by a case-control study in human. Secondary iron overload and atherosclerosis animal models were established in P2Y12-deficient zebrafish to explore the role of P2Y12 in macrophage iron metabolism in vivo. Both iron-overloaded murine primary peritoneal macrophages (PMs) and ox-LDL-treated PMs with P2Y12 knockdown were used for in vitro studies. RNA sequencing and pharmacological approaches were performed to investigate the downstream mechanisms.

Increased serum iron level was positively associated with P2Y12 inhibitor usage [odds ratio (OR) = 10.333 (1.281-83.370)]. Elevated serum iron level and transferrin saturation, reduced hepatic and splenic iron content, and decreased iron staining in macrophages were observed in secondary iron overload P2Y12-deficient zebrafish. Deficiency of P2Y12 in ApoEb^-/- zebrafish fed a high-fat diet reduced atherosclerosis progression and intraplaque iron retention. Furthermore, reduced ferritin, restored cell viability and expression of ferroptosis marker proteins, and decreased ROS formation and inflammatory cytokines were observed in both iron-overloaded and ox-LDL-treated PMs with P2Y12 knockdown in vitro, while reversed phenotypes were observed after agonist-induced P2Y12 activation. Mechanistically, P2Y12 inhibition in iron-overloaded or ox-LDL-treated PMs suppressed NF-κB p65 phosphorylation and hepcidin expression, both of which were reversed by P2Y12 activation.

P2Y12 inhibition decreased hepcidin autocrine through repressing NF-κB p65 phosphorylation in macrophages, preventing intracellular iron retention and atherosclerosis.