Dataset Information

Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-dot Based Nanoparticle Tracking Analysis.

ABSTRACT: Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker). The methods were further validated by their ability to efficiently recover the known amount of EC-MVs and EPC-MVs from particle-depleted plasma, and to detect the dynamical changes of plasma MVs in ischemic stroke patients, as compared with traditional flow cytometry. These novel methods provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs.

SUBMITTER: Wang J

PROVIDER: S-EPMC4837394 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-dot Based Nanoparticle Tracking Analysis.

Wang Jinju J Zhong Yun Y Ma Xiaotang X Xiao Xiang X Cheng Chuanfang C Chen Yusen Y Iwuchukwu Ifeanyi I Gaines Kenneth J KJ Zhao Bin B Liu Shiming S Travers Jeffrey B JB Bihl Ji C JC Chen Yanfang Y

Scientific reports 20160420

Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and sp ...[more]

PMID: 27094208

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Project description:BackgroundMicrovesicles are vesicles shed by plasma membranes following cell activation and apoptosis. The role of lymphocyte-derived microvesicles in endothelial function remains poorly understood.MethodsCD4+ T cells isolated from peripheral blood of healthy human donors were stimulated using anti-CD3/anti-CD28-coated beads. Proteomic profiling of microvesicles was performed using linear discriminant analysis (LDA) from activated T cells (MV.Act) and nonactivated T cells (MV.NAct). In addition, data processing analysis was performed using MaxQUANT workflow. Differentially expressed proteins found in MV.Act or MV.NAct samples with identification frequency = 100%, which were selected by both LDA (p < .01) and MaxQUANT (p < .01) workflows, were defined as "high-confidence" differentially expressed proteins. Functional effects of MV.Act on human primary microvascular endothelial cells were analysed.ResultsT cells released large amounts of microvesicles upon stimulation. Proteomic profiling of microvesicles using LDA identified 2279 proteins (n = 2110 and n = 851 proteins in MV.Act and MV.NAct, respectively). Protein-protein interaction network models reconstructed from both differentially expressed proteins (n = 594; LDA p ≤ .01) and "high-confidence" differentially expressed proteins (n = 98; p ≤ .01) revealed that MV.Act were enriched with proteins related to immune responses, protein translation, cytoskeleton organisation and TNFα-induced apoptosis. For instance, MV.Act were highly enriched with IFN-γ, a key proinflammatory pathway related to effector CD4+ T cells. Endothelial cell incubation with MV.Act induced superoxide generation, apoptosis, endothelial wound healing impairment and endothelial monolayer barrier disruption.ConclusionsT cell receptor-mediated activation of CD4+ T cells stimulates the release of microvesicles enriched with proteins involved in immune responses, inflammation and apoptosis. T cell-derived microvesicles alter microvascular endothelial function and barrier permeability, potentially promoting tissue inflammation.

Project description:BackgroundThis study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs).MethodsGene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry.ResultsA total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206.ConclusionBy upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.

Dataset Information

Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-dot Based Nanoparticle Tracking Analysis.

Publications

Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-dot Based Nanoparticle Tracking Analysis.

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