Proteomic profile of extracellular vesicles in anaphylaxis and their role in vascular permeability.
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ABSTRACT:
SUBMITTER: Nunez-Borque E
PROVIDER: S-EPMC8360103 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. METHODS:Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. RESULTS:Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. CONCLUSION:Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.
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Project description:Periodontitis is increasingly associated with increased risk of cardiovascular and other systemic diseases. The Gram-negative anaerobe, Porphyromonas gingivalis, is a key periodontal pathogen, and several lines of evidence link the presence of this bacterium in the circulation with vascular disease. The outer membrane vesicles (OMVs) produced by P. gingivalis have been shown to play a role in periodontitis, although, to date, little is known about their interaction with the vasculature; therefore, this study assessed the effects of P. gingivalis OMVs on the endothelium. OMVs were isolated from wild-type strain W83 and the gingipain-deficient strain ΔK/R-ab. Immunoblotting along with cryo-EM showed gingipain expression in W83 but not ΔK/R-ab-derived OMVs, where gingipains were localized to the cell wall surface. Confluent endothelial cell monolayers infected with either W83 or W83-derived OMV displayed significantly increased dextran permeability over those infected with ΔK/R-ab or its OMV. Moreover, W83-derived OMVs induced significantly more vascular disease in a zebrafish larvae systemic infection model over 72 h compared to those injected with gingipain-deficient OMVs or controls. In line with these data, human microvascular endothelial cells (HMEC-1) displayed an OMV-associated, gingipain-dependent decrease in cell surface levels of the intercellular adhesion molecule PECAM-1 (CD31) when examined by flow cytometry. These data show, for the first time, that OMVs from P. gingivalis mediate increased vascular permeability, leading to a diseased phenotype both in vitro and in vivo. Moreover, these data strongly implicate gingipains present on the OMV surface in mediating these vascular events, most likely via a mechanism that involves proteolytic cleavage of endothelial cell-cell adhesins such as PECAM-1. These data provide important evidence for the role of bacterial-derived OMVs in mediating systemic disease.
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Project description:Purpose/Aim: 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 in vitro 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.Materials and methodsVesicles 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.ResultsSignificant 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.ConclusionsResults of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.
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Project description:Extracellular vesicles (EVs), the lipid bilayer membranous structures of particles, are produced and released from almost all cells, including eukaryotes and prokaryotes. The versatility of EVs has been investigated in various pathologies, including development, coagulation, inflammation, immune response modulation, and cell-cell communication. Proteomics technologies have revolutionized EV studies by enabling high-throughput analysis of their biomolecules to deliver comprehensive identification and quantification with rich structural information (PTMs, proteoforms). Extensive research has highlighted variations in EV cargo depending on vesicle size, origin, disease, and other features. This fact has sparked activities to use EVs for diagnosis and treatment to ultimately achieve clinical translation with recent endeavors summarized and critically reviewed in this publication. Notably, successful application and translation require a constant improvement of methods for sample preparation and analysis and their standardization, both of which are areas of active research. This review summarizes the characteristics, isolation, and identification approaches for EVs and the recent advances in EVs for clinical biofluid analysis to gain novel knowledge by employing proteomics. In addition, the current and predicted future challenges and technical barriers are also reviewed and discussed.
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