Enrichment of calcifying extracellular vesicles using density‐based ultracentrifugation protocol

Abstract

Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular morbidity and mortality. However, little is known about the difference between this pathologic vesicle population and other EVs that contribute to physiological cellular processes. One major challenge that hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation. We show that after 10 min of ultracentrifugation at 100,000×g, calcifying EVs are depleted from the conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted portion. We utilized mass spectrometry to establish functional proteomic differences between the calcifying EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations preferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle differences between calcifying EVs and other vesicle populations that may be translated into therapeutic targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times required to pellet the vesicle populations can also be used to estimate physical differences between the vesicles.

Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

“…TNAP activity converts pyrophosphate into phosphate (34) and is necessary for matrix vesicle-dependent bone mineralization (35), while its role in EV-dependent vascular calcification remains unclear (36). TNAP-negative vesicles derived from exosomal origins may participate in hyperphosphatemicinduced SMC calcification associated with CRD (21, 37), whereas TNAP-positive SMC-derived vesicles may exert the osteogenic actions observed in atherosclerosis (38). The regulation of TNAP activity in calcifying SMC-derived EVs, however, remains poorly understood.…”

Emerging roles for vascular smooth muscle cell exosomes in calcification and coagulation

“…; Hutcheson et al . a ). These data suggest that osteogenic conditions may affect the VSMC EV repertoire and/or composition in a cell and context‐specific manner and the exact role of osteogenic transcription factors in the production of calcifying EVs by VSMCs is yet to be determined.…”

Conserved and Divergent Modulation of Calcification in Atherosclerosis and Aortic Valve Disease by Tissue Extracellular Vesicles

“…In fractions 1-4, NTA identified particles ~200 nm in diameter with a size distribution characteristic of EVs, both of which agree with our previous reports of EVs released in vitro from cultured macrophages, vascular SMCs, and valve interstitial cells (VICs) under calcifying conditions. (6, 16, 23) While vesicles were enriched in fractions 1-4 (demonstrated by proteomics, TEM, and NTA), mean particle size remained relatively consistent (~100-250 nm) across all fractions (Figure 3B,D), likely due to the presence of digested collagen fragments in denser fractions.…”