MEDIUM GROWTH MEDIA CHANGES BIOPHYSICAL AND BIOCHEMICAL PROPERTIES OF EXTRACELLULAR VESICLES FROM MOVAS

Vascular calcification is a multifactorial pathology and it occurs mainly in elderly ages. Also, a contributing factor is elevated serum phosphate levels. The vascular smooth muscle cells (VSMC) suffer transdifferentiation to osteoblast-like cells by expression of osteogenic genes, elevated tissue-nonspecific alkaline phosphatase (TNAP) and increased production of extracellular vesicles (EV).

To understand if the extracellular vesicles’ properties change in different culture media, MOVAS cell line (mouse aortic smooth muscle cells) was used as an in vitro model. The EVs released from cells are distinguished by where it resides: in culture medium (mEVs) or in extracellular matrices (MVs). The cells were cultured in basal medium (DMEM with 10% FBS) and in osteogenic media (basal media plus ascorbic acid 50µL/mL and β-glycerophosphate 10mM). To isolate the EVs, a differential centrifugation protocol was used: mEVs were directly purified from the extracellular medium, whereas MVs were isolated from the extracellular matrix by collagenase treatment. The vesicles were characterized by NTA, TNAP activity and protein concentration. Those analyses were performed in the interval of 7, 14 and 21 days of culture to each culture media.

NTA analysis of mEVs in basal medium revealed 1.23×10¹⁴ particles/mL after 7 days of culture, gradually decreasing to 8.6×10¹³ in 21d. In contrast, a peak of 2.11×10¹⁴ was observed in osteogenic medium at 14d. Meanwhile, the behavior of MVs was similar in both basal and osteogenic media, showing a peak of around 2×10¹⁴ at 7d, followed by a reduction to 6×10¹³ by day 21.Protein concentration follows according to the number of vesicles varying from 2 to 7 ng/mL. Nonetheless, when it comes to specific TNAP activity, for both osteogenic EVs (in 14d) TNAP activity were approximately to 30 U/mg presenting higher values than the basal EVs, which were ~20 U/mg. 

Even though this work represents just preliminary, it is notable that mEVs and MVs exhibit different behaviors, suggesting that each plays a distinct role and acts synergistically in the process of ectopic calcification. Understanding these mechanisms and distinct properties could lead to the identification of specific molecular targets for potential treatments.