Verification of the predictive value of EV-associated biomarkers MMP9 and CEACAM1 in rehabilitation of ischemic stroke

<b>Aim:</b> Extracellular vesicles (EVs) contribute to stroke rehabilitation by mediating intercellular signaling during inflammation and tissue repair. Here we report EV-associated surface proteins as potential biomarkers for predicting recovery of activities of daily living (ADL) during the subacute phase of ischemic stroke (IS). <b>Methods:</b> IS patients and healthy controls (HCs) were recruited for this study, with serum samples analyzed across three study stages. In the discovery subset (10 IS, 6 HCs), serum proteomics was used to identify differentially expressed proteins (DEPros) and perform Gene Ontology (GO) enrichment analysis. In the exploration subset (7 IS, 12 HCs), a proximity-dependent barcoding assay (PBA) was employed to profile surface proteins on individual EVs and screen for biomarker candidates. In a validation cohort, patients were grouped by ADL improvement (little-effect recovery, LE, <i>n</i> = 30; obvious-effect recovery group, OE, <i>n</i> = 22) based on Longshi Scale and Barthel Index assessments at admission and at 3 months follow-up. Targeted biomarker validation was performed with enzyme-linked immunosorbent assay (ELISA) and receiver operating characteristic (ROC) analysis. <b>Results:</b> A total of 113 DEPros were identified, with GO term enrichment in EV-related pathways. PBA profiling revealed matrix metalloproteinase 9 (MMP9), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), melanoma cell adhesion molecule (MCAM), and gelsolin (GSN) as candidate biomarkers. In the validation cohort, MMP9 and CEACAM1 were significantly elevated in the LE group. ROC analysis showed area under the curve (AUC) of 0.726 for MMP9 and 0.700 for CEACAM1 in distinguishing LE from OE. <b>Conclusion:</b> Elevated serum levels of EV-associated biomarkers MMP9 and CEACAM1 were associated with poor ADL recovery, supporting their potential as prognostic biomarkers for stroke rehabilitation outcomes.