PEAR1/EAF1 deficiency impairs aspirin responsiveness in atherosclerotic endothelium: A novel mechanism of atypical aspirin resistance.

BACKGROUND: Aspirin resistance (AR) severely limits the secondary prevention of atherosclerotic cardiovascular disease (ASCVD). Traditionally, aspirin resistance is attributed to intrinsic defects in blood cells, such as platelets or erythrocytes. However, the failure of aspirin to suppress endothelial inflammation, a key driver of residual cardiovascular risk, remains overlooked. We hypothesize that AR is partly an endothelial pathology mediated by the Platelet Endothelial Aggregation Receptor 1 (PEAR1) / ELL-associated factor 1 (EAF1) interaction.

METHODS: We integrated bioinformatic analyses, including weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, and support vector machine-recursive feature elimination (SVM-RFE), using bulk RNA sequencing (RNA-seq) data (GSE38511) and single-cell RNA sequencing (scRNA-seq) data (GSE159677) to identify and localize AR-related hub genes. An in vitro AR-like endothelial model was established in human umbilical vein endothelial cells (HUVECs) using oxidized low-density lipoprotein (ox-LDL), tumor necrosis factor-alpha (TNF-α), and aspirin. Key inflammatory and signaling changes were further validated in human aortic endothelial cells (HAECs). The effects of PEAR1 and EAF1 overexpression on endothelial function and the nuclear factor kappa B (NF-κB) / NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling pathway were assessed by Western blotting, quantitative real-time polymerase chain reaction (qPCR), immunofluorescence, and functional assays.

RESULTS: Bioinformatics identified PEAR1 and EAF1 as core hub genes significantly associated with AR and primarily localized to endothelial cells (ECs). In the in vitro AR model, PEAR1 and EAF1 expression was significantly decreased, which correlated with activation of the NF-κB/NLRP3 pathway. Similar inflammatory and signaling changes were confirmed in HAECs, supporting the reproducibility of the endothelial hyporesponsive phenotype in an alternative arterial endothelial model. Conversely, co-overexpression of PEAR1 and EAF1 synergistically suppressed phosphorylation of NF-κB p65 and expression of NLRP3. This interaction also inhibited endothelial migration and invasion while promoting apoptosis.

CONCLUSION: The PEAR1/EAF1 interaction ameliorates aspirin resistance in atherosclerosis. This effect is associated with suppression of the endothelial NF-κB/NLRP3 inflammatory signaling pathway and altered endothelial cell fate under inflammatory stress. Targeting the PEAR1/EAF1 interaction represents a novel potential therapeutic strategy for overcoming AR.