High-Dose Ionizing Radiation Accelerates Atherosclerotic Plaque Progression by Regulating P38/NCOA4-Mediated Ferritinophagy/Ferroptosis of Endothelial Cells

Abstract

Purpose: Radiation therapy (RT) significantly increased the incidence of coronary artery diseases, especially atherosclerosis. Endothelial dysfunction has been the major side effect of RT among tumor patients who received RT. However, the involvement between endothelial dysfunction and radiation-induced atherosclerosis (RIA) remains unclear. Here, we constructed a murine model of RIA, aiming to uncover its underlying mechanisms and identify novel strategies for RIA prevention and treatment.

Methods and materials: Eight-week-old ApoE^-/- mice that were fed a Western diet were subjected to partial carotid ligation (PCL). Four weeks later, ionizing radiation (IR) of 10 Gy was performed to verify the detrimental role of IR on atherogenesis. Ultrasound imaging, RT quantitative polymerase chain reaction, histopathology and immunofluorescence, and biochemical analysis were performed 4 weeks after IR. To study the involvement of endothelial ferroptosis induced by IR in RIA, mice after IR were administrated with ferroptosis agonist (cisplatin) or antagonist (ferrostatin-1) intraperitoneally. Western blotting, autophagic flux measurement, reactive oxygen species level detection, and coimmunoprecipitation assay were carried out in vitro. Furthermore, to determine the effect of ferritinophagy inhibition on RIA, in vivo knockdown of NCOA4 was carried out by pluronic gel.

Results: We verified that accelerated plaque progression was concomitant with endothelial cell (EC) ferroptosis after IR induction, as suggested by a higher level of lipid peroxidation and changes in ferroptosis-associated genes in the PCL + IR group than in the PCL group within vasculature. In vitro experiments further validated the devastating effects of IR on oxidative stress and ferritinophagy in ECs. Mechanistic experiments revealed that IR induced EC ferritinophagy and subsequent ferroptosis in a P38/NCOA4-dependent manner. Both in vitro and in vivo experiments confirmed the therapeutic effect of NCOA4 knockdown in alleviating IR-induced ferritinophagy/ferroptosis of EC and RIA.

Conclusions: Our findings provide novel insights into the regulatory mechanisms of RIA and first prove that IR accelerates atherosclerotic plaque progression by regulating ferritinophagy/ferroptosis of ECs in a P38/NCOA4-dependent manner.