CXCL4-induced PBMCs Modulate Vascular Extracellular Matrix through Wnt5a-CaMKII-dependent Release of Calcific Extracellular Vesicles and Matrix Metalloproteinase-7

Abstract Background Macrophage heterogeneity plays an increasing role in the study of vascular inflammatory responses. The CXCL4-induced monocyte/macrophage phenotype has previously been implicated with atherosclerotic plaque destabilization, a key process preceding plaque rupture. Monocyte-derived macrophages were found to exhibit a unique transcriptome in the presence of CXCL4 characterized by upregulation of S100A8 and MMP7. However, the mechanisms involved in CXCL4-induced monocyte-mediated vascular inflammation are unknown. Methods Single-cell RNA sequencing data were examined for CXCL4-dependent gene expression signatures in plaque macrophages. Human PBMCs were differentiated with CXCL4 and subsequently characterized in terms of osteogenic gene and protein expression signatures and calcific extracellular vesicle release. Association of the CXCL4-induced phenotype with the Wnt pathway was investigated, and CXCL4-induced PBMC-derived EV were analyzed for their potential to elicit an inflammatory response in vSMC. In-vitro findings were verified histologically in calcified human carotid artery plaques. Results In human plaque macrophages, single-cell sequencing revealed a CXCL4-susceptible subpopulation bearing a distinct proinflammatory gene expression profile. CXCL4-differentiated PBMCs exhibited a marked induction of S100A8, MMP7 and osteogenic marker transcription concomitant with augmented release of calcific EVs enriched with MMP7, S100A8 and alkaline phosphatase. Under osteogenic conditions, increased overt calcification of the extracellular matrix was observed in vitro . Analysis of inflammatory pathway activation identified the paracrine Wnt5a-CaMKII signaling axis to be causally linked to the CXCL4-induced osteogenic PBMC phenotype, S100A8 and MMP7 enrichment as well as calcific potential of secreted EV. Additionally, CXCL4-polarized PBMC-derived EV differentially stimulated osteogenic/inflammatory genotype transition in vSMC. In human carotid artery plaques, occurrence of CXCL4-induced mononuclear cells coincided with Wnt5a-CaMKII pathway activation and progressive plaque calcification. Conclusions This study introduces a novel mechanism driving monocyte/macrophage-mediated extracellular matrix remodeling in calcific inflammatory responses through Wnt5a-CaMKII-activated secretion of MMP7 + S100A8 + calcifying EV by CXCL4-induced pro-inflammatory monocytes. Graphical Abstract