Proteomics characterization of extracellular vesicles sorted by flow cytometry reveals a disease-specific molecular cross-talk from cerebrospinal fluid and tears in multiple sclerosis

Pieragostino, Damiana; Lanuti, Paola; Cicalini, Ilaria; Cufaro, Maria Concetta; Ciccocioppo, Fausta; Ronci, Maurizio; Simeone, Pasquale; Onofrj, Marco; Pol, Edwin van der; Fontana, Antonella; Marchisio, Marco; Boccio, Piero Del

doi:10.1016/j.jprot.2019.103403

Cited by 108 publications

(134 citation statements)

References 31 publications

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“…Last but not least, flow cytometry (FC), commonly used for the analysis of cells, is being actively developed for EV analysis [146,147] and is adopted by an increasing number of research groups, mainly to study the larger EVs [148,149]. Direct FC analysis using tracers that stain the whole vital EV compartment, such as lipophilic carbocyanine dyes, combined with phalloidin, that selectively binds to F-actin, accurately discriminates EVs from artifacts [42][43][44]150]. Standardized FC has a high potential for the detection of EVs in body fluids and, when combined with specific antibodies, concurrently allows EV immunophenotypic characterization [42,44,84,131,151,152] ( Figure 2).…”

Section: Methodological Approachesmentioning

confidence: 99%

“…Direct FC analysis using tracers that stain the whole vital EV compartment, such as lipophilic carbocyanine dyes, combined with phalloidin, that selectively binds to F-actin, accurately discriminates EVs from artifacts [42][43][44]150]. Standardized FC has a high potential for the detection of EVs in body fluids and, when combined with specific antibodies, concurrently allows EV immunophenotypic characterization [42,44,84,131,151,152] ( Figure 2). Imaging flow cytometers combining conventional FC with fluorescence provide new, highly sensitive tools for EV studies [153,154].…”

Section: Methodological Approachesmentioning

confidence: 99%

“…Due to their substantial stability, EVs circulate systemically and have been detected in basically all body fluids, including blood, urine, cerebrospinal fluid, saliva, milk, and tears [16,[42][43][44][45][46][47][48]. Moreover, there is clear indication that EVs can cross multiple biological barriers, as demonstrated by the finding of glial/neuronal EVs in the cerebrospinal fluid, blood, tears, and urine [44]. Specific delivery to distant targets is ensured by cell/tissue tropisms depending on surface markers [49,50].…”

Section: General Characteristics and Biological Significance Of Evsmentioning

confidence: 99%

“…It is evident that EVs can cross the BBB in both directions, propagating inflammation across the blood brain barrier (BBB), mediating neuroprotection, and modulating regenerative processes [45] (Figure 3). In fact, EVs shed from CNS neurons and glia can be found in peripheral blood and in tears [44,205], particularly when inflammatory mediators activate CNS endothelia, enhancing BBB permeability [206,207]. In this regard, it has been observed that the permeability of the BBB is increased in neurodegenerative conditions such as Alzheimer's disease, vascular dementia, and multiple sclerosis [45,[208][209][210].…”

Section: Evs and The Blood-brain Barriermentioning

confidence: 99%

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Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

Simeone

Bologna

Lanuti

et al. 2020

IJMS

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147

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Extracellular vesicles act as shuttle vectors or signal transducers that can deliver specific biological information and have progressively emerged as key regulators of organized communities of cells within multicellular organisms in health and disease. Here, we survey the evolutionary origin, general characteristics, and biological significance of extracellular vesicles as mediators of intercellular signaling, discuss the various subtypes of extracellular vesicles thus far described and the principal methodological approaches to their study, and review the role of extracellular vesicles in tumorigenesis, immunity, non-synaptic neural communication, vascular-neural communication through the blood-brain barrier, renal pathophysiology, and embryo-fetal/maternal communication through the placenta.

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Section: Methodological Approachesmentioning

confidence: 99%

Section: Methodological Approachesmentioning

confidence: 99%

Section: General Characteristics and Biological Significance Of Evsmentioning

confidence: 99%

Section: Evs and The Blood-brain Barriermentioning

confidence: 99%

See 2 more Smart Citations

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

Simeone

Bologna

Lanuti

et al. 2020

IJMS

Self Cite

147

117

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“…Next, 50 µg of proteins was digested for each treatment by using trypsin (Promega, Madison, WI, USA). For protein label free identification and quantification, tryptic peptides from each sample were analyzed in triplicate with LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer as already described [20].…”

Section: Label Free Proteomicsmentioning

confidence: 99%

Ponatinib Induces Vascular Toxicity through the Notch-1 Signaling Pathway

Madonna

Pieragostino

Cufaro

et al. 2020

JCM

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Ponatinib, a third-generation tyrosine kinase inhibitor (TKI), is the only approved TKI that is effective against T315I mutations in patients with chronic myeloid leukemia (CML). Specific activation of Notch signaling in CML cells by ponatinib can be considered as the "on-target effect" on the tumor and represents a therapeutic approach for CML. Nevertheless, ponatinib-induced vascular toxicity remains a serious concern, with underlying mechanisms being poorly understood. We aimed to determine the mechanisms of ponatinib-induced vascular toxicity, defining associated signaling pathways and identifying potential rescue strategies. We exposed human umbilical endothelial cells (HUVECs) to ponatinib or vehicle in the presence or absence of the neutralizing factor anti-Notch-1 antibody for exposure times of 0-72 h. Label-free proteomics and network analysis showed that protein cargo of HUVECs treated with ponatinib triggered apoptosis and inhibited vasculature development. We validated the proteomic data showing the inhibition of matrigel tube formation, an up-regulation of cleaved caspase-3 and a downregulation of phosphorylated AKT and phosphorylated eNOS. We delineated the signaling of ponatinib-induced vascular toxicity, demonstrating that ponatinib inhibits endothelial survival, reduces angiogenesis and induces endothelial senescence and apoptosis via the Notch-1 pathway. Ponatinib induced endothelial toxicity in vitro. Hyperactivation of Notch-1 in the vessels can lead to abnormal vascular development and vascular dysfunction. By hyperactivating Notch-1 in the vessels, ponatinib exerts an "on-target off tumor effect", which leads to deleterious effects and may explain the drug's vasculotoxicity. Selective blockade of Notch-1 prevented ponatinib-induced vascular toxicity.

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Experimental and Biological Insights from Proteomic Analyses of Extracellular Vesicle Cargos in Normalcy and Disease

Charest

2020

Adv. Biosys.

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Proteomics characterization of extracellular vesicles sorted by flow cytometry reveals a disease-specific molecular cross-talk from cerebrospinal fluid and tears in multiple sclerosis

Cited by 108 publications

References 31 publications

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

Ponatinib Induces Vascular Toxicity through the Notch-1 Signaling Pathway

Experimental and Biological Insights from Proteomic Analyses of Extracellular Vesicle Cargos in Normalcy and Disease

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