An Update on Isolation Methods for Proteomic Studies of Extracellular Vesicles in Biofluids

Li, Jing; He, Xianqing; Deng, Yuanyuan; Yang, Chenxi

doi:10.3390/molecules24193516

Cited by 62 publications

(54 citation statements)

References 107 publications

(154 reference statements)

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“…Differential ultracentrifugation is the most commonly used method for the isolation of EVs from biological fluids or cell culture media [ 78 ]. A dilution of biological fluids with PBS before centrifugation can increase the EV purity and recovery by decreasing the viscosity, even if this procedure is not always required for each fluid [ 79 ].…”

Section: Proteomic Methodsmentioning

confidence: 99%

“…Size exclusion chromatography is another type of size-based isolation strategy that consists of moving the sample through a column packed with heterogeneous polymeric beads (e.g., Sepharose and Sephadex) with diverse pore size [ 78 ]. SEC separates proteins in solution based on their sizes, thus larger sized species are eluted earlier than the smaller molecules of interest that can penetrate all the stationary phase pore system and elute later.…”

Section: Proteomic Methodsmentioning

confidence: 99%

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Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

et al. 2020

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Extracellular vesicles (EVs) are lipid-bound vesicles released from cells under physiological and pathological conditions. Basing on biogenesis, dimension, content and route of secretion, they can be classified into exosomes, microvesicles (MVs) and apoptotic bodies. EVs have a key role as bioactive mediators in intercellular communication, but they are also involved in other physiological processes like immune response, blood coagulation, and tissue repair. The interest in studying EVs has increased over the years due to their involvement in several diseases, such as cardiovascular diseases (CVDs), and their potential role as biomarkers in diagnosis, therapy, and in drug delivery system development. Nowadays, the improvement of mass spectrometry (MS)-based techniques allows the characterization of the EV protein composition to deeply understand their role in several diseases. In this review, a critical overview is provided on the EV’s origin and physical properties, as well as their emerging functional role in both physiological and disease conditions, focusing attention on the role of exosomes in CVDs. The most important cardiac exosome proteomic studies will be discussed giving a qualitative and quantitative characterization of the exosomal proteins that could be used in future as new potential diagnostic markers or targets for specific therapies.

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Section: Proteomic Methodsmentioning

confidence: 99%

Section: Proteomic Methodsmentioning

confidence: 99%

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

et al. 2020

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“…Stirred ultrafiltration is a simple and fast way to isolate EVs based on their size [121,122]. The pressure generated by the externally supplied nitrogen causes the sample to be passed through the ultrafiltration membrane resulting in EVs isolation.…”

Section: Isolating Protocols and Their Impact On Ev Biologymentioning

confidence: 99%

Message in a Bottle: Upgrading Cardiac Repair into Rejuvenation

Balbi

Costa

Barile

2020

Cells

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Ischaemic cardiac disease is associated with a loss of cardiomyocytes and an intrinsic lack of myocardial renewal. Recent work has shown that the heart retains limited cardiomyocyte proliferation, which remains inefficient when facing pathological conditions. While broadly active in the neonatal mammalian heart, this mechanism becomes quiescent soon after birth, suggesting loss of regenerative potential with maturation into adulthood. A key question is whether this temporary regenerative window can be enhanced via appropriate stimulation and further extended. Recently the search for novel therapeutic approaches for heart disease has centred on stem cell biology. The "paracrine effect" has been proposed as a promising strategy to boost endogenous reparative and regenerative mechanisms from within the cardiac tissue by exploiting the modulatory potential of soluble stem cell-secreted factors. As such, growing interest has been specifically addressed towards stem/progenitor cell-secreted extracellular vesicles (EVs), which can be easily isolated in vitro from cell-conditioned medium. This review will provide a comprehensive overview of the current paradigm on cardiac repair and regeneration, with a specific focus on the role and mechanism(s) of paracrine action of EVs from cardiac stromal progenitors as compared to exogenous stem cells in order to discuss the optimal choice for future therapy. In addition, the challenges to overcoming translational EV biology from bench to bedside for future cardiac regenerative medicine will be discussed.

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“…Briefly, the harvested medium was collected and subjected to ultracentrifugation [17,21]. Subsequently, the cells, cell debris, and apoptotic bodies were removed by centrifugation at 300×g for 10 min, 2000×g for 15 min, and 5000×g for 15 min, sequentially.…”

Section: Evs Isolationmentioning

confidence: 99%

M1 But Not M0 Extracellular Vesicles Induce Polarization of RAW264.7 Macrophages Via the TLR4-NFκB Pathway In Vitro

et al. 2020

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In response to different stimuli (e.g., infections), naive macrophages polarize into M1 macrophages, which have the potential to secrete numerous proinflammatory cytokines and extracellular vesicles (EVs). EVs are important mediators of intercellular communication. Via horizontal transfer, EVs transport various molecules (e.g., proteins, DNA, and RNA) to target cells. This in vitro study elucidated that M1-EVs from macrophages induced by interferon-γ (IFN-γ) and lipopolysaccharide (LPS) 24 h (M1), but not M0-EVs from untreated macrophages (M0), shifted M0 into M1 phenotype via activating the nuclear factor-κB pathway. The characteristics of these EVs were assessed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and a western blot assay. RAW 264.7 cells were incubated with M1-EVs (experimental group) or PBS (sham group) or M0-EVs (control group) for 24 h. The viability, change of shape, and phenotype differentiation of the macrophages were identified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and immunofluorescence staining. The TLR4-NFκB pathway of RAW264.7 macrophages was assessed by a western blot assay. M1-EVs but not M0-EVs were incorporated by the RAW264.7 cells and directly induced polarization of RAW264.7 macrophages to M1 macrophages. This polarization was demonstrated by significant upregulation of the M1 macrophage marker CD86 in the experimental group (49.93 ± 5.0%) as compared with that in the control and sham groups (1.22% and 1.46%, respectively) and significant upregulation of iNOS in the experimental group (75 ± 5.0%) as compared with that in the control and sham groups (0%). Furthermore, cell viability was higher (1.3 times) in the experimental group as compared with that in both the sham and control groups. The regulatory mechanism of M1-EVs on RAW 264.7 macrophages polarization and activation was triggered by the

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An Update on Isolation Methods for Proteomic Studies of Extracellular Vesicles in Biofluids

Cited by 62 publications

References 107 publications

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

Message in a Bottle: Upgrading Cardiac Repair into Rejuvenation

M1 But Not M0 Extracellular Vesicles Induce Polarization of RAW264.7 Macrophages Via the TLR4-NFκB Pathway In Vitro

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