The CXCL4-induced Monocyte/Macrophage Phenotype Promotes Extracellular Matrix Calcification through MMP7+S100A8+Annexin V+ Extracellular Vesicles

Introduction: The CXCL4-induced monocyte/macrophage phenotype has recently emerged as a novel player in the vascular inflammatory response driving atherosclerotic plaque development. Specifically, CXCL4-polarized CD68⁺ cell abundance has been associated with plaque instability, thus potentially increasing the risk of plaque rupture with its sequelae heart attack or ischemic stroke. Peripheral blood-derived monocytes (PBMCs) differentiated by CXCL4 exhibit a unique transcriptome characterized by expression of calcium nucleation factor S100A8 and matrix metalloproteinase 7 (MMP7). However, the mechanisms involved in the CXCL4-induced vascular inflammatory response causing plaque extracellular matrix modulation are unknown.

Methods: Single-cell RNA sequencing data obtained from coronary plaque tissue was examined for CXCL4-dependent transcriptional signatures in plaque macrophages. Human PBMCs were differentiated with CXCL4 in vitro and characterized in terms of osteogenic gene expression, extracellular vesicle (EV) release and EV secretome. CXCL4-induced PBMC-derived EV were incubated in three-dimensional extracellular matrix hydrogels, and their potential to nucleate calcium mineral was examined by near-infrared calcium tracer and visualized using scanning electron microscopy. Dependency of calcifying EV release and EV calcification potential on the Wnt5a-Ca²⁺ signaling pathway was investigated in CXCL4-induced PBMCs. CD68⁺S100A8⁺MMP7⁺ cell abundance was correlated with plaque calcification in human carotid plaque tissue.

Results: Single-cell sequencing of human coronary plaques identified a CXCL4-susceptible macrophage population with a distinct transcriptome functionally related to EV secretion. CXCL4-differentiated PBMCs exhibited increased expression of S100A8, MMP7, ALP and OPN, suggesting osteogenic phenotype transition, concomitant with elevated release of annexin V⁺ MMP7⁺ S100A8⁺ EV. Moreover, secreted EV showed increased alkaline phosphatase activity as a constituent of EV calcification potential, resulting in enhanced extracellular matrix calcification. Selective inhibition of ALP, MMP7, or EV release abrogated calcification by CXCL4-induced PBMCs. In extracellular matrix hydrogels, CXCL4-induced PBMC-derived EV prompted markedly increased calcium mineral nucleation compared to M-CSF control. Specific upregulation of the Wnt5a-pCaMKII signaling axis was appreciated upon CXCL4 differentiation. Conditioning of CXCL4-induced PBMCs with Wnt5a or Wnt5a-specific inhibitor Box5 revealed a stimulatory effect of paracrine Wnt5a on quantitative EV release and pro-calcific EV cargo, resulting in increased EV calcification potential and extracellular matrix calcification. Co-incubation of EV released by pre-conditioned CXCL4-induced PBMCs activated inflammatory and calcifying gene expression programs in vascular smooth muscle cells. Histological analysis of human carotid artery plaques revealed a significant correlation between CD68⁺S100A8⁺MMP7⁺ cell abundance and Wnt5a-pCaMKII pathway activation with medial fibro-calcification as a histological feature of the advanced atherosclerotic plaque.

Conclusion: This study unravels the mechanism of the CXCL4-induced monocyte/macrophage phenotype in vascular inflammatory responses, propagating extracellular matrix calcification via secretion of calcifying annexin V⁺ MMP7⁺ S100A8⁺ EV in a Wnt5a-dependent manner.