Introduction: CXCL4-induced monocytes/macrophages have recently emerged as novel players in the vascular inflammatory response driving atherosclerotic plaque development. Specifically, CXCL4-differentiated CD68⁺ mononuclear cells have been implicated in plaque destabilization, a key process preceding plaque rupture with its potentially fatal sequelae such as heart attack or stroke. Human PBMCs differentiated by CXCL4 exhibited a unique transcriptome characterized by differential expression of calcium nucleation factor S100A8 and matrix metalloproteinase 7 (MMP7). However, the mechanisms involved in the CXCL4-induced phenotype and its potential effects in vascular inflammatory processes are unknown.

Methods: Human PBMCs obtained from healthy donors were differentiated in vitro with either M-CSF or CXCL4 and subsequently characterized in terms of osteogenic gene expression signatures and calcific extracellular vesicle (EV) release. Both differentiated PBMCs and PBMC-derived EVs were analyzed for osteogenic protein markers, and EVs were used in coculture experiments to elicit osteogenic or proinflammatory gene signature in vascular smooth muscle cells. Wnt pathway activation and corresponding downstream signaling pathways were investigated in the CXCL4-induced osteogenic PBMC phenotype. Ex-vivo punch specimen from diseased human carotid arteries were stained for CD68⁺ S100A8⁺ MMP7⁺ cells and Wnt pathway activation, and correlative analysis with plaque calcification was performed.

Results: CXCL4-differentiated monocytes exhibited a markedly increased expression of S100A8, MMP7 and genes associated with the osteogenic genotype, concomitant with an elevated release of annexin5⁺ S100A8⁺ EVs selectively carrying alkaline phosphatase as a constituent of EV calcific potential. Moreover, MMP7 was selectively enriched in CXCL4-differentiated PBMC-derived EVs. Under osteogenic conditions, increased overt calcification by CXCL4-differentiated PBMCs was induced in vitro. The Wnt signaling pathway, which has previously been implicated in vascular inflammation and calcification, showed specific upregulation of Wnt5a-dependent CaMKII phosphorylation upon CXCL4 differentiation. Stimulation of CXCL4-induced PBMCs with exogenous Wnt5a or Wnt5a-specific inhibitor Box5 resulted in activation and reciprocal inhibition of all features of the osteogenic phenotype in CXCL4-induced PBMCs, including calcific EV release, overt calcification, as well as osteogenic gene and protein expression signatures. Moreover, activation of the Wnt5a-CaMKII axis in CXCL4-differentiated PBMCs was causally associated with MMP7 expression on RNA and protein levels. As paracrine signaling mediators, EVs released by CXCL4-induced PBMCs with or without prior Wnt5a stimulation activated expression of alkaline phosphatase and pro-inflammatory IL6 genes in vascular smooth muscle cells. In carotid artery specimen from patients undergoing carotid thromboendarterectomy surgery, immunohistochemical analysis revealed a significant correlation between the abundance of CD68⁺ S100A8⁺ MMP7⁺ cells, activation of the Wnt5a-CaMKII pathway and medial fibro-calcification as a histological feature of the advanced atherosclerotic plaque.

Conclusion: This study introduces a novel mechanism driving vascular calcific and inflammatory responses through propagation of calcific S100A8⁺ MMP7⁺ EVs by Wnt5a-Ca²⁺ pathway activation in CXCL4-differentiated pro-inflammatory mononuclear cells.