Showing papers in "Journal of extracellular vesicles in 2012"

Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes

Abstract: Background: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that 20 exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia. Methods: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-a-induced 25 activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach. Results: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis. 30 Conclusion: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition. Keywords: extracellular vesicles; exosomes; RNA; proteomics; hypoxia; tumor necrosis factor alpha. (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18396 - DOI: 10.3402/jev.v1i0.18396 To access the supplementary material to this article: Tables SI-SIV and Figures S1-2, please see Supplementary files in the column to the right (under Article Tools)

Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation.

Abstract: Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8) By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 115 g/ml) but also in fractions of densities over 120 g/ml, indicating the presence of heterogenous vesicle populations CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles

ExoCarta as a resource for exosomal research

Abstract: Exosomes are a class of extracellular vesicles that are secreted by various cell types. Unlike other extracellular vesicles (ectosomes and apoptotic blebs), exosomes are of endocytic origin. The roles of exosomes in vaccine/ drug delivery, intercellular communication and as a possible source of disease biomarkers have sparked immense interest in them, resulting in a plethora of studies. Whilst multidimensional datasets are continuously generated, it is difficult to harness the true potential of the data until they are compiled and made accessible to the biomedical researchers. Here, we describe ExoCarta ( http://www.exocarta.org ), a manually curated database of exosomal proteins, RNA and lipids. Datasets currently present in ExoCarta are integrated from both published and unpublished exosomal studies. Since its launch in 2009, ExoCarta has been accessed by more than 16,000 unique users. In this article, we discuss the utility of ExoCarta for exosomal research and urge biomedical researchers in the field to deposit their datasets directly to ExoCarta. Keywords: exosome resource; microvesicle database; exosome tools; extracellular organelles; Vesiclepedia (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18374 - http://dx.doi.org/10.3402/jev.v1i0.18374

Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells

Abstract: Background: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of 20 this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 + progenitor cells. Methods: The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of 25 these exosomes and the shuttle of the RNA to other mast cells and CD34 + progenitor cells. Results: In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of 30 the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34 + hematopoietic progenitor cells. Conclusion: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34 + progenitor cells, implying a role in cells maturation process. 35 Keywords: exosomes; extracellular vesicles; human mast cells; RNA; esRNA (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18389 - DOI: 10.3402/jev.v1i0.18389 To access the supplementary material to this article: Tables S1-S6, please see Supplementary files in the column to the right (under Article Tools)

Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

Abstract: The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.

Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells

Abstract: Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis.

CD4 + T cell activation promotes the differential release of distinct populations of nanosized vesicles

Abstract: Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the plasma membrane. Vesicles actively released by CD4 T cells have immune-modulatory characteristics. Using our recently developed high-resolution flow cytometry-based method for the analysis of individual nanosized vesicles, we here investigated how T cell receptor (TCR)-triggering and co-stimulatory signals influence the quantity and characteristics of nanosized vesicles released by CD4 T cells. We found that the number of released nanosized vesicles within the buoyant density range characteristic for exosomes (1.101.19 g/ml) was increased by TCR-triggering and that additional co-stimulatory signals had a potentiating effect on vesicle release. However, the increase in the number of released vesicles varied substantially between density fractions within the 1.101.19 g/ml range and was highest for the vesicle populations in 1.14 and 1.17 g/ml fractions. Heterogeneity was also observed within the individual density fractions. Based on lipid bilayer fluorescent labelling intensity and light scattering, 3 distinct vesicle subpopulations were identified. One vesicle subpopulation increased significantly more upon T cell activation than the other subpopulations, and this was dependent on high levels of co-stimulation. These data show that T cells release a heterogeneous population of nanosized vesicles and indicate that T cells differentially regulate the release of distinct vesicle subpopulations depending on their activation status.

Liver extracellular vesicles in health and disease

Abstract: Extracellular vesicles (EVs) play an important role in cell-to-cell communication. Although there are different kinds of vesicles, each with their own secretion and capture biology, all of them carry a cargo of proteins, lipids, metabolites and nucleic acids. They act as vehicles for exchange of biological materials and signals and are involved in the regulation of various physiological processes. Liver is an essential organ containing different cell populations fulfilling various functions, which need to be strictly controlled and coordinated. There are a few articles showing the role of liver-derived EVs. On the basis of them, we present here a hypothesis of the implication of such vesicles in the physiology of the liver. Different liver cell types, including hepatocytes, cholangiocytes and stellate cells, secrete and capture EVs and interact with them. Liver injury changes the abundance and cargo of EVs; these changes are likely to be important for the outcome of stress response. Although a substantial effort has been put into the characterization of EVs in isolated populations, it is only recently that some more comprehensive information has begun to appear. In this article, we hypothesize about the role of EVs in liver microenvironment and their possible function using published data from both hepatic and non-hepatic systems.

Stable cell fate changes in marrow cells induced by lung-derived microvesicles

Abstract: Background: Interest has been generated in the capacity of cellular-derived microvesicles to alter the fate of different target cells. Lung, liver, heart and brain-derived vesicles can alter the genetic phenotype of murine marrow cells; however, the stability of such changes and the mechanism of these changes remain unclear. In the present work, we show that lung-derived microvesicles (LDMV) alter the transcriptome and proteome of target marrow cells initially by mRNA and regulator(s) of transcription transfer, but that long term phenotype change is due solely to transfer of a transcriptional regulator with target cell. Methods/results: In vivo studies: Whole bone marrow cells (WBM) were co-cultured with LDMV (both isolated from male C57BL/6 mice) or cultured alone (control). One week later, cultured WBM was transplanted into lethally-irradiated female C57BL/6 mice. Recipient mice were sacrificed 6 weeks later and WBM, spleens and livers were examined for the presence of lung-specific gene expression, including surfactants A, B, C and D, aquaporin-5, and clara cell specific protein, via real-time RT-PCR. Immunohistochemistry was also performed on lungs to determine the number of transplanted marrowderived (Y chromosome+) type II pneumocytes (prosurfactant C+). Mice transplanted with LDMV cocultured WBM expressed pulmonary epithelial cell genes in the cells of their bone marrow, livers and spleens and over fivefold more transplanted marrow-derived Y+/prosurfactant C+cells could be found in their lungs (vs. control mice). In vitro studies: WBM (from mice or rats) was cultured with or without LDMV (from mice or rats) for 1 week then washed and cultured alone. WBM was harvested at 2-week intervals for real-time RT-PCR analysis, using species-specific surfactant primers, and for Western Blot analysis. Proteomic and microRNA microarray analyses were also performed on cells. LDMV co-cultured WBM maintained expression of pulmonary epithelial cell genes and proteins for up to 12 weeks in culture. Surfactant produced at later time points was specific only to the species of the marrow cell in culture indicating de novo mRNA transcription. These findings, in addition to the altered protein and microRNA profiles of LDMV co-cultured WBM, support a stable transcriptional mechanism for these changes. Conclusions: These data indicate that microvesicle alteration of cell fate is robust and long-term and represents an important new aspect of cellular biology. Keywords: bone marrow cells; lung; microvesicles; bone marrow transplant; transcription factor (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18163 - http://dx.doi.org/10.3402/jev.v1i0.18163

International Society for Extracellular Vesicles: first annual meeting, April 17–21, 2012: ISEV-2012

Abstract: Extracellular micro- and nano-scale membrane vesicles produced by different cells are recognised as an essential entity of physiological fluids in a variety of organisms and function as mediators of intercellular communication employed for the regulation of multiple systemic and local processes. In the last decade, an exponential amount of experimental work was dedicated to exploring the biogenesis and secretion mechanisms, physiological and pathological functions and potential applications of the extracellular vesicles (EVs). Noteworthy is the large heterogeneity of in vitro and in vivo models applied, technical approaches developed in these studies and the diversity of designations assigned to different or similar types of EVs. Hence, there is a clear necessity for a uniform nomenclature and standardisation of methods to isolate and characterise these vesicles. In April 2012, the first meeting of the International Society for Extracellular Vesicles (ISEV) took place bringing together this exponentially grown scientific community. The University of Gothenburg (Krefting Research Centre) together with the Interim Board of the Society created in September 2011 (Jan Lotvall, Clotilde Thery, Xandra Breakefield, Marca Wauben, Yong Song Gho, Lawrence Rajendran, Graca Raposo, Douglas Taylor, Margareta Sjostrand and Esbjorn Telemo) organised this fantastic event that counted 488 registered and contributing participants. This meeting report provides a retrospective summary of the broad spectrum of ISEV-2012 sessions. Again, we emphasise novel findings, discussions and decisions met by the community during the meeting.

The launch of Journal of Extracellular Vesicles (JEV), the official journal of the International Society for Extracellular Vesicles - about microvesicles, exosomes, ectosomes and other extracellular vesicles

Abstract: In 2011, researchers around the world interested in extracellular vesicles (EV) joined forces and founded the International Society for Extracellular Vesicles (ISEV). Membership has grown to approximately 750 in eight months, and the Society’s first meeting will take place in Gothenburg, Sweden, on 18-21 April 2012. Already approximately 500 participants have been attracted to this event. These are signs of rapid expansion in global research in the field of EV. (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18514 - http://dx.doi.org/10.3402/jev.v1i0.18514

ISEV RNA Workshop-New York City, October 1-2, 2012.

Abstract: The workshop focused on extracellular vesicle RNA (evRNA). It was organized by the International Society for Extracellular Vesicles (ISEV: www.isev. org) in New York on 1-2 October 2012 and was a resounding success. Jan Lotvall (President of ISEV, Gothenburg University, Sweden) and Marca Wauben (ISEV Board Member, Utrecht University, The Netherlands) organized it in a very friendly way in a beautiful venue, all of which was ideal in promoting open and successful scientific exchanges. In total, 50 participants were selected from over 100 applications, based on their expertise in the field of extracellular RNA and/or vesicles (microvesicles, exosomes, ectosomes and others) over several years. Ten percent of the participants came from Australia/Asia, with the remainder equally represented from Europe and the Americas. In addition, a WebEx access to the PowerPoint presentations was set up. This allowed for approximately 50 additional ISEV members to participate remotely in the seminar. (Published: 19 October 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 19857 - http://dx.doi.org/10.3402/jev.v1i0.19857

Micro-Vesiculation and Disease, London, 13–14 September 2012

Abstract: The Biochemical Society (London, UK) held a “Focused meeting” on “Micro-Vesiculation and Disease” on 13–14 September 2012 within the London Metropolitan University, London, UK. Focused meetings are dedicated to emerging life science topics. This meeting was organised by Jameel Inal, Una Fairbrother, Sheelagh Heugh and Annie Bligh and supported by Niamh Mangan and Caron Barter. The organisers aimed to bridge the findings on extracellular vesicles at the cellular level with organismal research on therapy and pathophysiology. The meeting was centred around a selected set of longer talks touching upon different subjects and specifically allowed longer times for discussion. Finally, speakers were asked to write short review articles based on their talks, to be published in a special issue of Biochemical Society Transactions . Note that this short report summarises the author's understanding of the different presentations. The author apologises in advance to the presenters for any unwilling inaccuracy. (Published: 30 October 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 19768 - http://dx.doi.org/10.3402/jev.v1i0.19768