Rapamycin attenuates ferroptotic stress and improves macrophage efferocytosis in experimental atherosclerosis.
TL;DR
Rapamycin attenuates ferroptotic and mitochondrial injury, restores macrophage efferocytosis, and reduces atherosclerotic plaque burden, supporting its therapeutic potential in experimental atherosclerosis.
Abstract
Background
Defective macrophage efferocytosis contributes to necrotic core formation and plaque instability in atherosclerosis. Ferroptosis and mitochondrial dysfunction have been implicated in macrophage injury, but whether ferroptosis-associated mitochondrial dysfunction contributes to impaired efferocytosis and whether rapamycin can ameliorate these abnormalities remain unclear.
Objectives
This study aimed to investigate whether rapamycin improves macrophage efferocytosis under conditions of ferroptosis-associated mitochondrial dysfunction in atherosclerosis.
Methods
Human carotid atherosclerotic plaques from three donors were analyzed. In vitro, THP-1-derived macrophages were treated with FIN-56 (5 μM, 48 h) with or without rapamycin (5 μM), and ferroptosis-associated injury, mitochondrial function, inflammatory phenotype, and efferocytosis were evaluated. Ferrostatin-1 and MitoTEMPO were used in pharmacological rescue experiments to assess the contributions of ferroptotic stress and mitochondrial oxidative stress, respectively. RNA sequencing was performed with three biological replicates per group. In vivo, ApoE-/- mice were fed a high-fat diet for 12 weeks and subsequently treated with vehicle or rapamycin (1 mg/kg, twice weekly for 4 weeks; n = 6 allocated per group), with endpoint-specific sample sizes specified for individual analyses.
Results
FIN-56 increased lipid peroxidation and mitochondrial ROS, reduced GPX4 expression, mitochondrial membrane potential, intracellular ATP, and respiration, and impaired efferocytosis. FIN-56-induced ferroptotic stress also reduced MERTK and MFG-E8 protein expression. Ferrostatin-1 partially restored macrophage efferocytosis under FIN-56 treatment, whereas MitoTEMPO reduced mitochondrial ROS and improved efferocytosis, supporting a contributory role of mitochondrial oxidative stress. Rapamycin partially reversed these functional and molecular abnormalities. In vivo, rapamycin increased the plaque efferocytosis index from 0.36 ± 0.02 to 1.37 ± 0.15 and reduced whole-aorta lipid-positive area from 25.32 ± 0.98% to 12.28 ± 1.49%.
Conclusion
Our findings indicate that ferroptosis-associated mitochondrial dysfunction may contribute to impaired macrophage efferocytosis in atherosclerosis. Rapamycin attenuates ferroptotic and mitochondrial injury, restores macrophage efferocytosis, and reduces atherosclerotic plaque burden, supporting its therapeutic potential in experimental atherosclerosis.