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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Several proteomics studies have been conducted to identify new cerebrospinal fluid (CSF) biomarkers in Multiple Sclerosis (MuS). However, the complexity of CSF and its invasive collection, limits its ...use. Therefore, the goal of biomarker research in MuS is to identify novel distinctive targets in CSF or in easily accessible biofluids. Tears represent an interesting matrix for this purpose, because (1) tears are related to the central nervous system (CNS) and (2) the CNS contains Extracellular Vesicles (EVs) derived from brain cells. These EVs are emerging new biomarkers associated to several neurological disorders. Here we applied an optimized flow cytometer for the identification and subtyping of EVs from CSF and tears. We found, for the first time, microglia-derived and neural-derived EVs in tears. The flow cytometer was used to sort and purify 106 EVs from untouched CSF and tears of MuS patients and healthy subjects. Purified EVs were analyzed with shotgun proteomics analysis, revealing that EVs from both CSF and tears of MuS patients conveyed similar proteins. Our data demonstrated a specific EVs-mediated molecular cross talk between CSF and tears, which opens the door to new diagnostic perspectives for MuS.
Data are available via ProteomeXchange with identifier PXD013794.
Proteomics characterization of released Extracellular Vesicles (EVs) in CSF and tears of Multiple Sclerosis patients represents a pioneering application that helped in recognizing information about the biologically relevant molecules. We found, for the first time, microglia-derived and neural-derived EVs in tears. Moreover, purified EVs revealed that both CSF and tears of Multiple Sclerosis patients conveyed similar proteins involved in inflammation, angiogenesis and immune response signalling. We think that our data will contribute to enhance knowledge in Multiple Sclerosis mechanisms and help in biomarker discovery. Moreover tears represent one of the most convenient body fluid for biomarker discovery in Multiple Sclerosis, since it is an high informative and easy accessible. The opportunity to export such a platform to a territory monitoring plan opens the door to new diagnostic perspectives for Multiple Sclerosis.
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•Extracellular vesicles (EVs) FACS- purified have been used for proteomics.•Microglia-derived and neural-derived EVs were identified in tears.•Tears and CSF EVs convey similar proteins, in Multiple Sclerosis.•EV proteins deliver nuclear information and the program to insert it into the target cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Multiple Sclerosis (MuS) is a complex multifactorial neuropathology, resulting in heterogeneous clinical presentation. A very active MuS research field concerns the discovery of biomarkers helpful to ...make an early and definite diagnosis. The sphingomyelin pathway has emerged as a molecular mechanism involved in MuS, since high levels of ceramides in cerebrospinal fluid (CSF) were related to axonal damage and neuronal dysfunction. Ceramides are the hydrolysis products of sphingomyelins through a reaction catalyzed by a family of enzymes named sphingomyelinases, which were recently related to myelin repair in MuS. Here, using a lipidomic approach, we observed low levels of several sphingomyelins in CSF of MuS patients compared to other inflammatory and non-inflammatory, central or peripheral neurological diseases. Starting by this result, we investigated the sphingomyelinase activity in CSF, showing a significantly higher enzyme activity in MuS. In support of these results we found high number of total exosomes in CSF of MuS patients and a high number of acid sphingomyelinase-enriched exosomes correlated to enzymatic activity and to disease severity. These data are of diagnostic relevance and show, for the first time, high number of acid sphingomyelinase-enriched exosomes in MuS, opening a new window for therapeutic approaches/targets in the treatment of MuS.
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, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate ...and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-
activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of
CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative
CA inhibitors or to be used in association with current drugs for the management of
infection and limiting the spread of antibiotic resistance.
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Abstract
Protease proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of LDL cholesterol clearance and has been associated with cardiovascular risk. PCSK9 inhibitors increase in vivo ...circulating endothelial progenitor cells (EPCs), a subtype of immature cells involved in ongoing endothelial repair. We hypothesized that the effect of PCSK9 on vascular homeostasis may be mediated by EPCs in patients with or without type 2 diabetes mellitus (T2DM). Eighty-two patients (45 with, 37 without T2DM) at high cardiovascular risk were enrolled in this observational study. Statin treatment was associated with higher circulating levels of PCSK9 in patients with and without T2DM (p < 0.001 and p = 0.036) and with reduced CD45
neg
/CD34
bright
(total EPC compartment) (p = 0.016) and CD45
neg
/CD34
bright
/CD146
neg
(early EPC) (p = 0.040) only among patients with T2DM. In the whole group of patients, statin treatment was the only independent predictor of low number of CD45
neg
/CD34
bright
(β = − 0.230; p = 0.038, adjusted R
2
= 0.041). Among T2DM patients, PCSK9 circulating levels were inversely related and predicted both the number of CD45
neg
/CD34
bright
(β = − 0.438; p = 0.003, adjusted R
2
= 0.173), and CD45
neg
/CD34
bright
/CD146
neg
(β = − 0.458; p = 0.002, adjusted R
2
= 0.191) independently of age, gender, BMI and statin treatment. In high-risk T2DM patients, high endogenous levels of PCSK9 may have a detrimental effect on EPCs by reducing the endothelial repair and worsening the progression of atherothrombosis.
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Extracellular Vesicles (EVs) are circulating particles surrounded by a plasma membrane carrying a cargo consisting of proteins, lipids, RNAs, and DNA fragments, stemming from the cells from which ...they originated. EV factors (i.e., miRNAs) play relevant roles in intercellular crosstalk, both locally and systemically. As EVs increasingly gained attention as potential carriers for targeted genes, the study of EV effects on the host immune response became more relevant. It has been demonstrated that EVs regulate the host immune response, executing both pro- and anti-inflammatory functions. It is also known that physical exercise triggers anti-inflammatory effects. This review underlines the role of circulating EVs as players in the anti-inflammatory events associated with the regulation of the host’s immune response to physical exercise.
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Neurotrophic Keratopathy (NK), classified as an orphan disease (ORPHA137596), is a rare degenerative corneal disease characterized by epithelial instability and decreased corneal sensitivity caused ...by the damage to the corneal nerves. The administration of human recombinant nerve growth factor (rhNGF) eye drops, as a licensed-in-Europe specific medication for treatment of moderate and severe NK, has added promising perspectives to the management of this disorder by providing a valid alternative to the neurotization surgery. However, few studies have been conducted to the molecular mechanism underlying the response to the treatment. Here, we carried out tears proteomics to highlight the protein expression during pharmacological treatment of NK (Data are available via ProteomeXchange with identifier PXD025408).Our data emphasized a proteome modulation during rhNGF treatment related to an increase in DNA synthesis, an activation of both BDNF signal and IL6 receptor. Furthermore, the amount of neuronal Extracellular Vesicles EVs (CD171+) correlated with the EVs carrying IL6R (CD126+) together associated to the inflammatory EVs (CD45+) in tears. Such scenario determined drug response, confirmed by an in vivo confocal microscopy analysis, showing an increase in length, density and number of nerve fiber branches during treatment. In summary, rhNGF treatment seems to determine an inflammatory micro-environment, mediated by functionalized EVs, defining the drug response by stimulating protein synthesis and fiber regeneration.
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Primary open-angle glaucoma (POAG) represents the leading cause of irreversible blindness worldwide and is a multifactorial, chronic neurodegenerative disease characterized by retinal ganglion cell ...and visual field loss. There are many factors that are associated with the risk of developing POAG, with increased intraocular pressure being one of the most prevalent. Due to the asymptomatic nature of the disease, the diagnosis of POAG often occurs too late, which necessitates development of new effective screening strategies for early diagnosis of the disease. However, this task still remains unfulfilled. In order to provide further insights into the pathophysiology of POAG, we applied a targeted metabolomics strategy based on a high-throughput screening method for the determination of tear amino acids, free carnitine, acylcarnitines, succinylacetone, nucleosides, and lysophospholipids in naïve to therapy glaucomatous patients and normal controls. Also, we conducted proteomic analyses of the whole lacrimal fluid and purified extracellular vesicles obtained from POAG patients and healthy subjects. This multi-omics approach allowed us to conclude that POAG patients had lower levels of certain tear amino acids and lysophospholipids compared with controls. These targeted analyses also highlighted the low amount of acetylcarnitine (C2) in POAG patient which correlated well with proteomics data. Moreover, POAG tear proteins seemed to derive from extracellular vesicles, which carried a specific pro-inflammatory protein cargo.
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Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical ...processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.
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Extracellular vesicles (EVs) actively participate in inter-cellular crosstalk and have progressively emerged as key players of organized communities of cells within multicellular organisms in health ...and disease. For these reasons, EVs are attracting the attention of many investigators across different biomedical fields. In this scenario, the possibility to study specific placental-derived EVs in the maternal peripheral blood may open novel perspectives in the development of new early biomarkers for major obstetric pathological conditions. Here we reviewed the involvement of EVs in feto-maternal crosstalk mechanisms, both in physiological and pathological conditions (preeclampsia, fetal growth restriction, preterm labor, gestational diabetes mellitus), also underlining the usefulness of EV characterization in maternal-fetal medicine.
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