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.</p

Assessment of the absolute pixel size of the CryoEM maps.

<p>Assessment of the absolute pixel size of the CryoEM maps.</p

EV71 difference map interpretation.

<p>Central sections of calculated difference density maps and close-ups of relevant features are shown to indicate the position of the N-terminus of VP1. (A) The empty capsid density map was subtracted from 9 Å A-particle density map. The difference density (red) is shown superimposed on the A-particle map. The difference density corresponds to the RNA in the particle center, and does not fill the pillars that extend inward from the capsid shell. This can be seen clearly in the close-up of the right upper quadrant of the central section. (B) The calculated procapsid density map was subtracted from the 6.3 Å A-particle density map. The difference density (red) is shown in relation to the A-particle map (grey). The difference density fills the pillars that surround the 5-fold axis and also corresponds to the viral genome in the interior of the particle. The close-up of the upper right quadrant shows that the pillar is mostly filled with difference density (red). (C) The calculated procapsid map was subtracted from the empty capsid density map. The resulting difference density (red) is shown on the empty capsid map (grey). The close-up view shows that the difference density is localized to the pillar (solid arrow head), base of the canyon (unfilled arrow head), and capsid exterior near the 5-fold axis. These same densities are seen in their symmetry related positions with a small noisy density at the tip of the five-fold plug also present.</p

Capsid-RNA interactions in the EV71 A-particle.

<p>Stereogram of the EV71 A-particle showing two columns of protein density extending from the capsid shell to interact with the viral RNA genome. The fitted procapsid crystal structure (3VBU) is depicted as a ribbon diagram with VP0, VP1, and VP3 in green, blue, and red, respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Wang1" target="_blank">[30]</a>. The A-particle cryoEM density is depicted as grey mesh. The first ordered N-terminal residue of VP1 in the crystal structure, His73, is shown as cyan spheres and extends into the protein density. The unfilled density beneath His 73 is attributed to the N-terminus of VP1, which is disordered and therefore absent in the procapsid crystal structure, but present in the A-particle.</p

Model for EV71 uncoating.

<p>(A) Binding of a cellular receptor to the capsid of the mature virus (or heating) results in an expanded A-particle. This capsid conformation is characterized by the openings at each two-fold axis of symmetry, the externalization of the N-terminus of the VP1 protein (residues 1–72) from the base of the canyon, and the expulsion of the VP4 protein. (B) If the A-particle forms in proximity to a cellular membrane the VP4 proteins form a channel through which the RNA can enter the host cell cytoplasm. The exposed N-terminii of VP1 proteins anchor the A-particle to the membrane via amphipathic helices.</p

Lifecycle of a human enterovirus.

<p>Three viral structural proteins (VP0, VP1, and VP3) function as a single structural subunit, the protomer. Five protomers then form a pentamer, twelve of which can self-associate to form a naturally occurring empty capsid (often referred to as procapsid). In pathway one, after the twelve pentamers form an empty procapsid (1A) the genome is inserted (1B). In the second proposed pathway 12 pentamers assemble around a genome to form a provirion (2). In this pathway pentamers that associate in the absence of genome would serve as a reservoir of capsid components in infected cells, and procapsid would be an off-pathway assembly byproduct. The provirion is a short-lived intermediate, as the autocatalytic cleavage of VP0 to form VP2 and VP4 is rapid and efficient. This cleavage results in the native virus, which upon binding to a cellular receptor transitions to an A-particle, which has expelled VP4 and exposed the N-terminus of VP1. An unknown secondary trigger causes the genome to egress, leaving behind an empty capsid shell. Note: The procapsid in this figure is shown in an expanded form, like that of EV71. Other picornavirus procapsids are the same diameter as their native virion (poliovirus and foot and mouth disease virus <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Basavappa1" target="_blank">[20]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Curry1" target="_blank">[21]</a>).</p

Cryo-electron microscopy image reconstruction data.

<p>Cryo-electron microscopy image reconstruction data.</p

Coulombic surface coloring of the two-fold and five-fold axis.

<p>(A) The EV71 A-particle two-fold axis of symmetry has a high concentration of negatively charged residues (red) with minimal regions of positive charge (blue). This charge distribution will likely serve to aid in the egress of viral RNA by providing a “slippery” surface at the site of genome release. (B) The EV71 five-fold vertex is composed of neutral (white) and positive (blue) charges. In addition to the presence of a density plug at the base of the five-fold, these charges would repel the RNA suggesting the five-fold is not the site of genome expulsion.</p

Cryo-EM reconstructions of the EV71 A-particle and empty capsid.

<p>(A, C) Surface rendered cryo-EM maps of the 6.3 Å EV71 A-particle and the 9.2 Å EV71 empty capsid are shown at a contour level of 1σ colored radially as indicated by the scale bar that depicts the most internal buried density in red gradating to the most external density in dark blue). The five-fold, three-fold, and two-fold axes are labeled. (B, D) Central sections of the EV71 A-particle and empty capsid with density shown in grey. The A-particle contains density corresponding to viral RNA (grey interior), whereas the empty capsid is completely devoid of interior density, indicating the genome has been released. Symmetry axes are labeled and the plug of density that closes the pore through the five-fold vertex is indicated on the central sections of the A-particle and empty capsid.</p

Genome maps of Mycobacteriophage BAKA.

<p>The annotated map of BAKA is represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#pone-0069273-g003" target="_blank">Figure 3</a>.</p