Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization

<p><b>Purpose/Aim:</b> Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an <i>in vitro</i> model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. <b>Materials and Methods:</b> Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. <b>Results:</b> Significant increase of the number of extracellular vesicles was detected after 72 h of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. <b>Conclusions:</b> Results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.