Deciphering the role of CCL4-CCR5 in coronary artery disease pathogenesis: insights from Mendelian randomization, bulk RNA sequencing, single-cell RNA, and clinical validation.

Background: Alterations in circulating CCL4 levels have been implicated in coronary artery disease (CAD), but the causal relationship and underlying mechanisms remain unclear. Objective: This study aims to analyse the role of CCL4 and its receptor (CCR5) in CAD using Mendelian randomisation (MR) analysis, bulk RNA and single cell RNA sequencing (scRNA-seq). Methods: The MR analysis was used to determine the causal relationship between 91 circulating inflammatory proteins and CAD. Bulk RNA sequencing data was used to demonstrate the expression profile of CCL4/CCR5. The localisation of CCL4/CCR5 was determined using scRNA-seq data. Functional enrichment analyses were used to infer the potential role of CCL4 in CAD. Additional clinical samples were utilized to validate the results of MR. Results: We identified six circulating inflammatory proteins associated with CAD. Of these, CCL4 was identified as a key inflammatory cytokine associated with CAD risk for MR analysis.The bulk RNA sequencing data from the Gene Expression Omnibus (GEO) datasets showed that CCR4 receptor(CCR5) expression was significantly higher in human atherosclerotic plaques compared to controls. Notably, scRNA-seq analysis revealed CCL4 was highly expressed in T cells, monocytes and macrophages. Clinical specimens confirmed high levels of serum CCL4 expression in CAD patients by ELISA.Functional enrichment analysis revealed that CCL4 was primarily enriched in the cytokines and cytokine receptors, viral proteins with cytokines and cytokine receptors, and chemokine signaling pathways. Conclusion: Our study presented a genetic insight into the pathogenetic role of CCL4-CCR5 in CAD, which may provide new insights for further mechanistic and clinical investigations of inflammatory cytokine-mediated CAD.