Targeting Sphingosine-1-Phosphate Signaling Remodels Extracellular Vesicles During Vascular Calcification

Vascular calcification (VC), a critical predictor of cardiovascular mortality in chronic kidney disease (CKD), is driven by the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) and the concomitant release of calcifying extracellular vesicles (EVs), including matrix vesicles (MVs) that serve as nucleation sites for mineral deposition. While we have previously established that sphingosine-1-phosphate (S1P) signaling contributes to VC and that its modulator Fingolimod reduces calcification in experimental CKD models, the impact of this compound on EV biogenesis and cargo during VSMC transdifferentiation remained unexplored. In this study, VSMCs were induced to calcify over 21 days with or without Fingolimod treatment, and EVs were isolated from both the culture medium and the extracellular matrix for comprehensive characterization. Our results demonstrate that EVs released from calcifying VSMCs powerfully stimulate further transdifferentiation, confirming their pro-calcifying role. Notably, while Fingolimod did not alter the total number of EVs secreted, it induced a significant redistribution towards smaller and more heterogeneous vesicle populations, and specifically decreased the secretion of MVs. Furthermore, protein analysis revealed that Fingolimod modifies exosomal biogenesis and protein cargo sorting, effectively reducing MV protein markers and features associated with calcification. Collectively, these findings uncover a novel mechanism of action for Fingolimod, showing that it functionally remodels the EVs landscape, altering size distribution, vesicle subpopulation release, and protein composition, thereby interfering with the EV-mediated propagation of calcification. This work positions the modulation of S1P-dependent EVs communication as a promising therapeutic strategy to counteract vascular pathology in CKD.