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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.

SUBMITTER: Hutcheson JD

PROVIDER: S-EPMC4261240 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol.

Hutcheson Joshua D JD Goettsch Claudia C Pham Tan T Iwashita Masaya M Aikawa Masanori M Singh Sasha A SA Aikawa Elena E

Journal of extracellular vesicles 20141205

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 c ...[more]

PMID: 25491249

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Project description:Tissue factor (TF) is the main initiator of coagulation and procoagulant phospholipids (PPL) are key components in promoting coagulation activity in blood. Both TF and PPL may be presented on the surface of extracellular vesicles (EVs), thus contributing to their procoagulant activity. These EVs may constitute a substantial part of pathological hypercoagulability that is responsible for triggering a higher risk of thrombosis in certain patients. The aim of this study was to describe a model system for the isolation of EVs required for investigating their effect on coagulation. Differential ultracentrifugation (DUC) with and without a single washing step was used to isolate and evaluate the procoagulant capacity of EVs from healthy volunteers through analysis of thrombin generation and PPL activity. Ultracentrifugation at 20,000 × g and 100,000 × g resulted in pellets containing larger vesicles and smaller vesicles, respectively. Isolation yield of particle concentration was assessed by nanoparticle tracking analysis. Immunoelectron microscopy and western blotting revealed vesicles positive for the commonly used EV-marker CD9. Plasma proteins and lipoproteins were co-isolated with the EVs; however, application of a washing step clearly diminished the amount of contaminants. The isolated EVs were capable of enhancing thrombin generation, mainly due to PPL predominantly present in pellets from 20,000 × g centrifugation, and correlated with the activity measured by a PPL activity assay. Thus, DUC was proficient for the isolation of EVs with minimal contamination from plasma proteins and lipoproteins, and the setup can be used to study EV-associated procoagulant activity. This may be useful in determining the procoagulant activity of EVs in patients at potentially increased risk of developing thrombosis, e.g. cancer patients. Abbreviations: TF: Tissue factor; PL: Phospholipids; EVs: Extracellular vesicles; FXa: Activated coagulation factor X; TGA: Thrombin generation assay; PPL: Procoagulant phospholipids; DUC: Differential ultracentrifugation; NTA: Nanoparticle tracking analysis; TEM: Transmission electron microscopy; SPP: Standard pool plasma; CTI: Corn trypsin inhibitor; 20K: 20,000 × g; 100K: 100,000 × g; FVIII: Coagulation factor VIII.

Project description:Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 ×g for 24 h. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4%; 48.1 ± 5.8%; 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield.

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Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol.

Publications

Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol.

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