Atherosclerotic plaque epigenetic age acceleration is characterized by mesenchymal reprogramming and poor prognosis

Epigenetic age estimators (clocks) are known to be predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques can be used for predicting secondary events. Here we estimated an age adjusted measure of epigenetic age, epigenetic age acceleration (EAA), using DNA methylation of human atherosclerotic plaques and of blood. EAA of plaque, but not blood, independently predicted secondary events in a 3-year follow-up (HR=1.3, p= 0.018). Plaque EAA concurred with a high metabolic epigenetic and transcriptional state in plaques. Patients with diabetes and a high body mass index had a higher plaque EAA. EAA was lower in female plaques compared to male plaques by approximately 2 years. Single-cell RNA-seq revealed mesenchymal smooth muscle cells and endothelial cells as main drivers of EAA. Plaque-specific ageing may help identify processes that explain poor health outcomes. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the European Union project European Research Council consolidator grant 866478 (UCARE), and ERA-CVD 2017T099 ENDLESS, Leducq PlaqOmics and AtheroGEN. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Not Applicable The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Athero-Express is an ongoing, prospective biobank study, collecting atherosclerotic plaques from patients undergoing carotid endarterectomy (CEA) in two Dutch tertiary referral hospitals: University Medical Centre Utrecht and St Antonius Hospital, Nieuwegein. Ethical approval was granted by the METC at the University Medical Center Utrecht. Indication for surgery was based on international guidelines for carotid and iliofemoral atherosclerotic disease20-23, and standardized treatment protocols and operative techniques were applied. The medical ethics committees in both participating centres approved the study. All patients provided written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Not Applicable I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Not Applicable I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Not Applicable All data and scripts for replication of this study could be provided upon reasonable request. * EAA : Epigenetic age acceleration EC : Endothelial cell SMC : Smooth muscle cell