Plasma Proteins Identify Increased Number of Carotid Plaques and Predict Occurring Atherosclerotic Events

Background The number of carotid plaques independently predicts incident atherosclerotic cardiovascular disease (ACVD). However, performing vascular imaging in apparently healthy subjects is challenging, owing organizational/economical barriers. Plasma proteomics can offer an alternative approach to identify individuals with carotid plaques, at high risk of eventually developing ACVD. Methods In this observational study, we studied by Normalized Protein eXpression (NPX; OlinkTM), the plasma levels of 368 proteins in 664 subjects from the PLIC study, who were screened by ultrasound for the presence of carotid plaques. We clustered, by artificial intelligence, the proteins that more accurately identified subjects, stratifying them according to the number of plaques they presented with. We also study prediction of occurring events over 22 years. Results 299/664 subjects had at least 1 carotid plaque. Among those, 77 subjects presented with only one plaque, 101 with 2 plaques and 121 with ≥3 plaques (3+). The remaining 365 subjects with no plaques acted as controls. The proteins differently expressed versus controls increased as a function of the number of plaques. 32 proteins were shared among the groups of subjects with plaques, but 87, significantly associated with the presence of 3+ plaques, improved the AUC of the ROC, together with the ACVD risk factors, to discriminate subjects with 3+ plaques versus the AUC of the ROC considering the ACVD risk factors only (AUC= 0.918 (0.887-0.943) vs AUC= 0.760 (0.716-0.801) respectively, p<0.001). The ACVD risk factors barely predicted the 198 occurring events (AUC= 0.559 (0.521-0.598)), but proteomics associated with plaques improved the prediction (AUC= 0.739 (0.704-0.773), p<0.001). By analyzing the biological processes, we identified that chemotaxis/migration of leukocytes and the signaling of interleukins/cytokines were the top pathways involved. Conclusions Plasma proteomics helps to identify apparently healthy subjects with higher number of carotid plaques more accurately and to predict occurring ACVDs in those individuals. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The work of A.B and A.L.C. is supported by a grant Ricerca Corrente from the Ministry of Health to IRCCS Multimedica; PRIN 2017H5F943; ERANET ER-2017-2364981 and a grant A.L.C. RF-2019-12370896 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The PLIC Study has been approved by the Ethics Committee of the University of Milan (SEFAP/Pr.0003). Written informed consent was obtained from subjects (all over 18 years-old), in accordance with the Declaration of Helsinki. This work is an observational study and it was conducted following the standards of the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) initiative. 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. Yes 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). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The work includes data analyzed from an array of proteomics measured in plasma. All raw data can be provided upon reasonable written request to ALC.