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BackgroundIn addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.MethodsTo study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.ResultsA total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.ConclusionsThe methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.

KEY WORDSproteomics; matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF); liquid chromatography tandem mass spectrometry (LC-MS-MS); glioblastoma; U87MG; major vault protein; cystatin B; Hsp27; tissue transglutaminase; epidermal growth factor receptor ABSTRACT An in-frame deletion of 801 bp in exons 2-7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MG⌬EGFR) that confers high malignancy to tumor cells. Two-dimensional gel electrophoresis followed by in-gel digestion of separated spots and protein identification by LC-MS-MS and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MG⌬EGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT-PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low-grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors. GLIA 42:194 -208, 2003.

Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions? Contact the NRC Publications Archive team atPublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=33302487-9987-42d0-bb7f-3b59445d5f72 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=33302487-9987-42d0-bb7f-3b59445d5f72 AbstractCurrent methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody 'tracking' in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody 'redistribution' from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.

Abstract-Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na ϩ ]/[K ϩ ] ratio after the sustained blockage of the Na ϩ ,K ϩ -ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na Key Words: apoptosis Ⅲ vascular smooth muscle Ⅲ proteome Ⅲ ion transport Ⅲ ouabain R emodeling of the blood vessel plays an important role in a variety of human vascular disorders, including hypertension, 1-3 atherosclerosis, 4 arterial injury, and restenosis after angioplasty. 5-7 Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as the main factor contributing to the regulation of their number during remodeling, 8 -12 which inspired numerous studies of the mechanisms of the induction and progression of VSMC apoptosis. The execution phase of apoptosis in VSMCs is triggered similarly to that in the other cell types by activation of the caspase cascade, cleavage of intracellular proteins, and final disintegration of the cell. In contrast, the induction phase is specific for different subtypes of remodeling and involves the integration of multiple pro-and antiapoptotic signals, including the expression of death receptors, protooncogenes, and tumor suppressor genes. [13][14][15][16][17][18][19] Our recent studies showed that inhibition of the VSMC Na ϩ ,K ϩ pump with ouabain, or in K ϩ -free medium, rescues cells from apoptosis triggered by a number of factors including serum deprivation. 20 Equimolar substitution of extracellular Na ϩ with K ϩ completely abolished the effect of ouabain 20 showing that antiapoptotic action was indeed caused by the inversion of [NaThe development of cell death was blocked upstream of caspase-3 activati...

This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 l total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C 18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of ϳ5 nM (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2-1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gelisolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system. Molecular & Cellular Proteomics 1:157-168, 2002.Proteomics research entails the global characterization of proteins expressed in cells under defined conditions. Such studies are particularly important in view of the conflicting evidence regarding the correlation between the abundance of expressed proteins and gene-expression levels obtained from mRNA microarrays (1, 2). The monitoring of protein expression profiles remains a very challenging task because of the wide dynamic range of expressed proteins and the variability of gene products (splicing variants, N-and C-terminal truncations, co-and post-translational modifications, etc.), which may change between and within the tissues of an organism (3). The traditional approach to isolating and characterizing proteins from biological samples has been separation by twodimensional gel electrophoresis (2-D 1 gel), followed by identification of protein spots using sensitive mass spectrometry techniques and data base searching (3-5). An alternate approach to 2-D gel includes a comprehensive chromatographic separation of the proteolytic fragments derive...

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An integrated microsystem providing rapid analyses of trace-level tryptic digests for proteomics application is presented. This modular microsystem includes an autosampler and a microfabricated device comprising a sample introduction port and an array of separation channels together with a low dead-volume facilitating the interface to nanoelectrospray mass spectrometry. Sequential injection and separation of peptide standards and tryptic digests was achieved with a throughput of up to 30 samples per hour with less than 3% sample carryover. Replicate injections of peptide mixtures indicated that reproducibility of migration time was typically better than 2.3% relative standard deviation (RSD) whereas RSD values of 3.7-11.8% were observed on peak height. Mass spectral detection of submicromolar protein digests (< 7 femtomoles/injection) was achieved using a quadrupole/time of flight instrument in less than 2 min/per sample with peak widths of 1.8-7.0 s. The analytical potential of this integrated device for the identification of gel isolated proteins from Neisseria meningitidis immunotype L3 has been demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.